Вы находитесь на странице: 1из 36

Physiol Rev 95: 1205–1240, 2015

Published September 2, 2015; doi:10.1152/physrev.00037.2014

MEMBRANE REPAIR: MECHANISMS AND


PATHOPHYSIOLOGY
Sandra T. Cooper and Paul L. McNeil

Institute for Neuroscience and Muscle Research, Kids Research Institute, The Children’s Hospital at Westmead,
Sydney, New South Wales, Australia; Discipline of Paediatrics and Child Health, Faculty of Medicine, University
of Sydney, Sydney, New South Wales, Australia; and Department of Cellular Biology and Anatomy, Institute of
Molecular Medicine and Genetics, Georgia Regents University, Augusta, Georgia

Cooper ST, McNeil PL. Membrane Repair: Mechanisms and Pathophysio-

L
logy. Physiol Rev 95: 1205–1240, 2015. Published September 2, 2015;
doi:10.1152/physrev.00037.2014.—Eukaryotic cells have been confronted through-
out their evolution with potentially lethal plasma membrane injuries, including those
caused by osmotic stress, by infection from bacterial toxins and parasites, and by
mechanical and ischemic stress. The wounded cell can survive if a rapid repair response is
mounted that restores boundary integrity. Calcium has been identified as the key trigger to activate
an effective membrane repair response that utilizes exocytosis and endocytosis to repair a mem-
brane tear, or remove a membrane pore. We here review what is known about the cellular and
molecular mechanisms of membrane repair, with particular emphasis on the relevance of repair as
it relates to disease pathologies. Collective evidence reveals membrane repair employs primitive yet
robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily
honed for simplicity and success. Yet to be fully understood is whether core membrane repair
machinery exists in all cells, or whether evolutionary adaptation has resulted in multiple compen-
satory repair pathways that specialize in different tissues and cells within our body.

I. INTRODUCTION 1205 tion is a common type of cellular injury in eukaryotic


II. PHYSIOLOGIES OF MEMBRANE INJURY 1208 cells, and effective membrane repair mechanisms have
III. THE PROTEIN MACHINERY OF... 1215 evolved to rapidly reseal a membrane breach to ensure
IV. LITERATURE OVERVIEW: A cell survival. These repair mechanisms utilized the newly
COLLECTIVE... 1231 evolved endomembrane and cytoskeletal systems. Within
V. CONCLUDING REMARKS 1232 this review we outline the subcellular and molecular
events that restore bilayer integrity after a membrane
disruption injury, highlighting the protein families impli-
I. INTRODUCTION cated in membrane repair, and the ancient biology that
underpins membrane resealing and cell survival from a
A. The Vulnerability of a Single Membrane membrane breach.
Bilayer
B. Membrane Injury Underlies Many Human
Unlike bacterial cells, eukaryotic cells are not protected Pathologies
by a hardened and impermeant cell wall. The “naked”
membrane bilayer covering early eukaryotes permitted
the evolution of phagocytic vesicles for the uptake of Many human pathologies are characterized by mem-
nutrients, and secretory vesicles for the extrusion of brane injury, and modulation of membrane repair path-
waste products, enzymes, and signaling factors. The loss ways holds tremendous therapeutic potential. Plasma
of a cell wall also led to the development of a new internal membrane disruptions have been documented under
protective skeleton, the cytoskeleton. Together, cytoskel- physiological conditions in many mechanically active tis-
etal networks working in concert with internal mem- sues, such as in the stratified epithelium that covers our
branes led to the development of the eukaryotic endo- body, the endothelia that line our blood vessels, and the
membrane system. epithelial barrier of our gastrointestinal tract (178). Dis-
ruptions are especially frequent in skeletal muscle, espe-
However, an unprotected bilayer member renders eu- cially when it undergoes high-force, eccentric contrac-
karyotic cells more vulnerable to mechanical and chemi- tions (91, 180, 199). In certain forms of muscular dys-
cal stressors. Consequently, plasma membrane disrup- trophy, the frequency of disruption initiated by

0031-9333/15 Copyright © 2015 the American Physiological Society 1205


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

physiological contractions is far higher than in normal an innate ability to repair very large wounds. Microin-
muscle (54, 180). jection of human oocytes for in vitro fertilization creates
an enormous lesion encompassing hundreds of square
Membrane disruptions are also caused by bacterial pore- micrometers, but is readily survivable. Indeed, sea urchin
forming toxins (PFTs) that are potent virulence factors oocytes can repeatedly reseal sequential wounds encom-
secreted by most pathogenic bacteria (120). As the name passing a thousand square micrometers of surface,
suggests, PFTs form stable membrane pores that perfo- though they cannot survive a single insult if extracellular
rate the plasma membrane of host cells. Pore formation calcium is removed (181). Cells can also repair abundant
by bacterial pathogens is thought to serve many pur- smaller wounds (nanometer diameter), such as those in-
poses, the most obvious being lysis and induction of cell duced by bacterial PFTs, using both calcium-dependent
death programs in immune cells, to mute immune cell (123, 129) and calcium-independent processes (164).
activity and thus facilitate bacterial infection. Pores may
also serve as channels for the bacteria to deliver other
virulence factors and to access cellular nutrients from D. Size Matters With Membrane Injury
infected cells for their own metabolic growth, such as
amino acids, ions, and ATP (165). Large pores formed by 1. Spontaneous resealing
the cholesterol-dependent cytolysins can span 40 nM
(257) and are also permeable to cellular proteins. How- Tiny membrane injuries (less than a nanometer), such as
ever, in moderate doses, cells and organisms survive the those created by electroporation or proteins that induce
onslaught of PFT perforation, and we will discuss recent lipid disorder, may repair spontaneously. Exposure of
developments regarding membrane repair mechanisms hydrophobic domains of lipids rapidly results in diffu-
mobilized for survival from bacterial pores. sion of lipids around the break site to form a curved edge.
If the membrane consists only of a phospholipid bilayer,
Cells within our vital organs also suffer membrane dam- lipid disorder present on the curved edges of a disruption
age with ischemia-reperfusion injury, as occurs following provides a driving force for resealing, and is a function of
heart attack and stroke. Ischemic membrane injury rep- disruption diameter squared (FIGURE 1, A1– 4). However,
resents a complex cascade of events that results from an when the injured membrane belongs to a eukaryotic cell
interruption to the circulation that feeds an organ oxygen and the phospholipid bilayer is tethered to an underlying
and nutrients. A lack of oxygen causes depletion of ATP. cytoskeleton (FIGURE 1, B1– 4), the opposing force of
ATP-dependent pumps begin to fail, resulting in disequi- membrane tension, a function of disruption diameter
librium in the potassium-sodium gradient, acidosis, and cubed, prevents spontaneous repair of biological mem-
an inability to extrude or sequester calcium. Sodium in- branes when the disruption exceeds a certain diameter.
flux causes cell swelling, and calcium influx induces pro- Based on experimental and theoretical data, that diame-
teolysis and triggers mitochondrial dysfunction, produc- ter is in the nanometer range (106).
tion of free radicals, and apoptosis. Cell swelling, acido-
sis, and oxidation compromises the plasma membrane. 2. Active resealing pathways
Membranes become leaky, with breaches sufficiently
large to allow the release of cellular enzymes (125). Par- Injuries larger than a few nanometers in diameter require
ticularly in the case of contractile cells of the heart, con- the help of an active membrane repair mechanism. For these
traction with reperfusion exacerbates membrane injury, larger membrane disruptions, the opposing forces of mem-
and a cascade of necrosis follows. Indeed, traumatic brane tension preclude spontaneous membrane resealing,
brain injury is also characterized by widespread disrup- and in the case of bacterial pore-forming toxins, the stable
tion of neuronal plasma membranes. It has been pro- proteinaceous structure of the pores cannot be simply re-
posed that these membrane disruption events initiate a sealed.
“death cascade” that is a major contributor to patient
morbidity (39). Repair of very large disruptions, hundreds to thousands of
nanometers in diameter, requires calcium-dependent exocy-
tosis (27, 267), involving both vesicle-vesicle and vesicle-
C. The Influx of Calcium Through a plasma membrane fusion to crudely “patch” the compro-
Membrane Breach Is the Key Trigger for mised plasma membrane (285). The sea urchin egg can
Membrane Repair replace over 2,000 square microns of surface membrane
ripped from its surface in ⬍5 s (285), and neurons and
Universally accepted within the membrane repair field is muscle cells can survive complete transections (45, 101,
the critical role of calcium as an activating trigger for the 146). In the sea urchin egg, large secretory granules form a
rapid membrane repair of large lesions. Indeed, initiation membrane patch at large injuries. However, which vesicle
of membrane repair may represent one of the most prim- population(s) are utilized for membrane patching in differ-
itive forms of calcium signaling. Eukaryotic cells possess ent mammalian cells and tissues is not yet clear (see sect.

1206 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

A1 Membrane Disruption B1 Membrane Disruption

Cytoskeleton
A2 B2
H 2O H2O

A3 Lipid Disorder B3 Lipid Disorder

Membrane Tension

A4 B4

FIGURE 1. Spontaneous resealing of plasma membrane injuries in the nanometer range is opposed by the
forces of the underlying membrane cytoskeleton. For an injury to a phospholipid bilayer alone (A1-4), the lipid
disorder present on the curved edges of the disruption provides the driving force to spontaneously reseal the
injury and is a function of disruption diameter squared. However, if the injured phospholipid bilayer is tethered
to underlying cytoskeleton (B1-4), the membrane tension from adhesion to the cytoskeleton confers an
opposing force for resealing, a function of disruption diameter cubed, and prevents spontaneous repair of
membrane disruptions that exceed diameters in the nanometer range (106).

IIIA). Calcium-triggered exocytosis is also thought to re- tant unanswered question. The answers likely relate both to
duce membrane tension, which facilitates resealing driven the delivery of membrane lipids to reduce lipid packing, as
by lipid disorder (FIGURES 2 AND 3) (292). How exactly the well as the associated remodeling of the submembraneous
exocytic addition reduces membrane tension is an impor- cytoskeletal that is required for exocytic delivery of vesicles.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1207


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

Membrane Disruption
1

Lipid Bilayer

Cytoskeleton

FIGURE 2. Calcium-activated exocytosis reduces mem-


brane tension and promotes spontaneous repair driven by
2 lipid disorder for injuries hundreds of nanometers in diam-
Lipid Disorder Lipid Disorder eter. With larger injuries, the opposing force of mem-
brane tension exceeds the resealing forces of lipid disor-
der at the edges of the disruption, negating the driving
forces of spontaneous membrane resealing. Eukaryotic
cells have been shown to utilize calcium-activated exocy-
tosis to reduce membrane tension and promote repair via
lipid-disorder driven attractions. The reduction in mem-
Membrane Tension brane tension is likely due directly to the addition of phos-
pholipids to reduced lipid packing, as well as due in part to
the cytoskeletal remodeling associated with vesicular
transport at the plasma membrane.
3
Lipid Disorder Lipid Disorder

Membrane Tension

Smaller injuries such as those caused by bacterial PFTs (nano- rich plasma membrane microdomains. These pathogens
meter range) do not appear to utilize “membrane patching” release pore-forming proteins, named for their capacity
and in fact cannot be repaired but must be removed. Thus to perforate the plasma membrane of their target cell.
exosomal secretion and endosomal uptake are employed for Pore-forming proteins are best characterized in bacteria
removal of bacterial-lined pores (14, 58, 123, 129, 280) (see (for detailed reviews, see Refs. 30, 105, 120, 213), but
sects. IIA and III, D and E). These membrane repair mecha- are also produced by many higher organisms such as sea
nisms are likely also important to remove and remodel hastily anemones and jellyfish (8, 318), earthworms (260), and
repaired larger lesions as part of the membrane repair process plants (70). Interestingly, the mammalian immune sys-
(FIGURE 3). tem has reciprocally adopted a pore-forming strategy to
lyse invading pathogens. Several members of comple-
II. PHYSIOLOGIES OF MEMBRANE INJURY ment membrane attack complex function as pore-form-
ing proteins to lyse bacteria and other pathogens (167),
and natural killer cells and cytotoxic T-cells expressing
A. Pore-Forming Toxins the pore-forming protein perforin to provide passage for
granzymes into target pathogens to initiate a cell death
We begin with what was likely an early ancestral challenge cascade (19, 149, 239). Mammalian cells also use a pore-
requiring membrane repair, eukaryotic cell survival from forming strategy to activate apoptosis via the Bcl2 pro-
bacterial PFTs. tein Bax (145, 217, 251).

1. Evolution and structure PFTs effectively induce lytic death of many cell types in
vitro, although the role of pore formation in vivo during
The plasma membrane of a cell represents a remarkable infection is more complex (for a comprehensive recent re-
landscape of ordered and partitioned proteins and lipids, view, see Ref. 165). PFTs specifically target an organism’s
working together to maximize cellular signaling and immune defense by inducing lytic death of immune cells (via
communication. Pathogens have evolved that exploit the pores) as well as inducing cell death programs triggered by
predictable clustering of receptors within cholesterol- potassium efflux, calcium influx, ATP depletion, mitochon-

1208 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

Large membrane injuries result


in rapid influx of Ca2+.

Ca2+ influx triggers exocytosis.


FIGURE 3. Very large plasma membrane
disruptions (micron diameter) require mem-
brane patching. The calcium influx of a mem-
brane injury activates vesicular exocytosis and
homo- and heterotypic fusion of cytoplasmic
vesicles. Exocytic fusion reduces membrane
tension, and vesicle-vesicle fusion events pro-
vide a patch as a replacement for the mem-
Vesicular delivery of extra membrane Exocytosis also provides small membrane
reduces membrane tension and facilitates patches to repair lesions. brane barrier missing at the disruption site.
resealing driven by lipid disorder. The membrane patch may serve only tempo-
rarily as a surface barrier replacement that is
subsequently remodeled and removed via exo-
cytic and/or endocytic machinery.

Membrane integrity must be rapidly


restored for cell survival

Hastily-repaired large injuries may require remodeling via exosomal shedding


or endocytosis. These pathways are used for removal of bacterial pores,
though their role in repair of larger lesions is not yet clear.

drial damage, disrupted ion homeostasis, and swelling. and play a major role in their virulence. Pathogenic bacteria
Pores are thought to also provide an entry point for other such as Escherichia coli, Pneumococcus, Streptococcus,
bacterial virulence factors that aide their infectivity, repli- Staphylococcus, Vibrio cholera, Clostridia, Listeria, and
cation, and ability to escape immune detection. One of the Diphtheria, to name but a few, each produce pore-forming
main causes of patient morbidity relates to the deleterious proteins that contribute worldwide to infection-related
overstimulation of inflammatory pathways that eventually morbidity and mortality. Pore-forming proteins from dif-
compromises the integrity of epithelial and endothelial bar- ferent species typically do not share high sequence homol-
riers, allowing the infection to spread, and disrupting the ogy, but are known to assume similar tertiary structures
fidelity of the vasculature (165). (93, 111, 301, 312) and are thought to function using the
same mode of action. There are two main classes of PFTs:
A) PFTS ARE THE LARGEST CLASS OF VIRULENCE-RELATED BACTERIAL the ␣-PFTs that adopt an ␣-helical fold for membrane in-
TOXINS. PFTs represent the largest class of bacterial toxins sertion (for example, colicins from Escherichia coli) and

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1209


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

␤-PFTs that adopt a ␤-barrel conformation for membrane A) SURVIVAL FROM PFT UTILIZES BOTH EXOCYTIC AND ENDOCYTIC
insertion and pore formation (the most common form of RESPONSES. SLO treatment can induce cell blebbing and re-
PFTs) (213). PFTs are released as soluble proteins by bac- lease of microvesicles containing pores and SLO protein
teria and bind to target membranes, recognizing specific (14, 118, 136) (see FIGURE 4). However, perforin (287,
GPI-anchored proteins (1, 60, 198) or lipid compartments, 288) and SLO (122, 280) have also been shown to induce
such as the cholesterol-dependent cytolysins (240, 257, endocytosis into an abnormally enlarged endosomal com-
296). Binding to specific receptors or lipid raft regions on partment. Following pore insertion into the plasma mem-
the plasma membrane increases their local concentration, brane, a local elevation in intracellular calcium induces ly-
facilitating their oligomerization, which is an essential step sosome fusion with the plasma membrane (123, 229) and
in pore formation. The capacity of PFTs to metamorphosize release of acid sphingomyelinase (280). It is proposed that
from their soluble form into oligomers, capable of trans- hydrolysis of sphingomyelin head groups leads to the for-
membrane insertion and subsequent formation of a pore, is mation of ceramide-enriched plasma membrane micro-do-
a truly remarkable feature. mains that activate endocytosis, and rapid removal of the
SLO pores into the degradative endosomal pathway (280)
2. The membrane repair response to PFT infection
(FIGURE 4). Endocytic removal of toxin pores presents its
own set of problems, as these pores may remain conductive
The response of an intoxicated cell to PFTs depends on the
in the early endosomal pathway and release acid hydrolases
type of toxin, the levels of toxin, and the period of exposure
into the cellular cytoplasm. However, several PFTs induce
to toxin (for reviews, see Refs. 30, 46). Interestingly, cells
require much longer to recover from infection by small- endocytic removal from the plasma membrane as part of
diameter pores (⬃2 nm) such as Staphylococcus ␣-toxin (6 their pathogenic entry into the cell (96). Thus how endo-
h or more) (118), compared with large-diameter pores (40 somal permeability contributes to PFT toxicity is only be-
nm) such as Streptococcus streptolysin-O (SLO), that are ginning to be teased out.
removed by microvesicular shedding and/or endocytosis
within minutes (14, 123, 129, 219). The differences in In vivo studies in C. elegans have addressed the question
membrane repair response between small and large pores of how cells escape PFT attack, and also emphasize the
are most likely due to the fact that small pores are not unique interplay between exocytic and endocytic aspects
permeable to calcium (305, 321), and thus do not activate of the repair response (118, 164) (FIGURE 4). PFT infec-
rapid calcium-activated exocytic and endocytic responses. tion of C. elegans gut epithelial cells by S. aureus ␣-toxin
Recovery of plasma membrane integrity following infection (118), B. thuringiensis Cry5B, and V. cholerae cytolysin
by bacterial streptolysin-O (SLO) is perhaps one of the most (VCC) (164) induces rapid endocytosis and also exo-
well-characterized examples of how cells are able to utilize somal shedding of pore-containing vesicles. Studies by
both exocytic and endocytic responses to survive a mem- Los et al. (164) demonstrated these responses were de-
brane injury. pendent on Rab-5 and Rab-11 (164), master regulators

Exosomal
shedding FIGURE 4. Survival from bacterial pore-
Annexins are co-shed forming toxins utilizes both exocytic and en-
with pores in exosomes
docytic responses. Bacterial pore-forming
Bacterial toxins oligomerize and insert in the plasma
pore-forming toxins Exosomal membrane of target cells forming a diffus-
shedding
ible pore. Evidence suggests these pores
are removed both by endosomal degrada-
tive pathways (123, 164, 280) and exo-
? somal shedding (14, 118, 136). Shed mi-
ESCRT-mediated Endosomal crovesicles containing streptolysin-O have
shedding recycling been shown to also contain annexins A1
and A6 (219). In C. elegans, Rab 5 endo-
cytic and Rab 11 recycling pathways are
implicated in pore removal (164). ESCRT
Rab 11 machinery (pink helix) has been implicated
Rab 5 in exosomal shedding (129), although
other exosomal machinery may also be in-
volved (indicated by a question mark).

Endosomal
degradation

1210 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

of, respectively, endosome formation and exocytic recy- taneously among all of the bundles of myofibers within a
cling. RNAi knockdown of Rab-5 or Rab-11 resulted in muscle group. However, eccentric stretch can result in
decreased endocytosis and worm hypersensitivity to disruption of the sarcomeric apparatus (5), and because
PFTs. Importantly, Rab-11 depletion specifically pre- the t-tubule network is firmly anchored to these contrac-
vented microvillar shedding. The authors propose that tile units, it too suffers a lateral stretch and gets pulled
the worm’s epithelial cells use both routes of PFT elimi- out of position (221). The fine longitudinal tubules that
nation. Toxins are directed via Rab-5-based endocytosis connect adjacent transverse tubules (80) rapidly and re-
into the lysosome and are also shed via Rab-11-based versibly swell with eccentric stretch, reducing the effi-
exosomal shedding of microvillar membrane vesicles into ciency of electrical conductivity and contraction.
the gut lumen (FIGURE 4).
A) INTENSE ECCENTRIC EXERCISE IN UNTRAINED SUBJECTS CAN KILL
Data suggest that endocytic and exosomal pathways to re- MUSCLE FIBERS. When untrained normal healthy controls
move toxin-lined pores can occur via both calcium-depen- are subject to shorts bouts of repeated eccentric stretch,
dent and calcium-independent pathways. ␣-Toxin, Cry5B, for example, 20 min of stepping up and down a stair
and VCC form small-diameter pores (1-2 nm) (213, 320) (stepping down the stair is the eccentric stretch), the mus-
and are thought not to be permeable to calcium (305, 321). cle membrane is injured and allows the release of the
Moreover, endocytosis and exosomal shedding of Cry5B muscle enzyme creatine kinase into the serum over the
and VCC following in vivo infection of C. elegans were following hours and days (199). Eccentric muscle dam-
shown to occur independently of extracellular calcium in age is characterized by a feeling of weakness and wobbli-
the medium (164). In contrast, removal of large calcium- ness immediately after the exercise, with muscles becom-
conductive SLO pores in mammalian cells occurs by rapid ing tender, sore, and stiff 1 or 2 days after the injurious
calcium-dependent endocytosis (123) and calcium-depen- event (254). Ultrastructural analysis of muscle biopsies
dent exosomal shedding (14). Additionally, the ESCRT ma- taken from control subjects who have undergone a pro-
chinery has been recently implicated in the calcium-depen- tocol of eccentric stretch reveals marked disruption in the
dent exosomal shedding of bacterial pore-forming toxins organization of the skeletal muscle contractile apparatus
(129), and this is discussed in greater detail in section IIIF. immediately after the exercise (91, 221). Indeed, the ec-
Therefore, whether rapid calcium-dependent or slower cal- centric stretch injury induces such significant damage
cium-independent pathways are utilized, likely relates to that over the following days and weeks, the pool of mus-
the size of the pores and whether they are conductive to cle stem cells, called satellite cells, are activated and pro-
calcium. liferate to repair and rebuild lethally injured myofibers
(91, 131).

B. Muscle Injury and Muscular Dystrophy 2. Persistent membrane injury in muscular dystrophy

1. Eccentric injuries and t-tubules In patients with muscular dystrophy, creatine kinase is
persistently elevated (regularly hundreds of times higher
Muscle fibers are particularly prone to injury when sub- than control levels) and muscle biopsy samples show his-
jected to lengthening contractions, referred to as eccen- topathological signs of ongoing degeneration and regen-
tric damage (180). Eccentric injury occurs because whilst eration of muscle fibers. The most common form of mus-
lengthening a muscle, for example, your quadriceps as cular dystrophy is Duchenne’s muscular dystrophy
you stride down hill, you simultaneously ask the muscle (DMD), an X-linked inherited disorder affecting ⬃1:
to contract against a lengthening stretch, when the mem- 3,000 boys, due to mutations in the cytoskeletal protein
brane tension is significantly increased. The muscle dystrophin (113).
plasma membrane transverse tubule (t-tubule) network is
particularly sensitive to eccentric stretch, as this network Dystrophin is part of the spectrin superfamily of cytoskel-
of small-diameter tubules runs perpendicular to the long etal proteins and is thus proposed to lend elasticity to the
axis of contraction. T-tubules are invaginations of the muscle plasma membrane, as well as provide the structural
muscle plasma membrane, ⬃20 – 40 nM in diameter (size cornerstone of the muscle costamere. The costamere is a
differs slightly amongst species) (90), that penetrate deep focal adhesion-like complex assembled at regular intervals
into the interior of the myofiber. T-tubules are anchored along the sarcomere, providing a stabilizing connection be-
at precise intervals along the sarcomere (the contractile tween the cytoskeletal apparatus via the intermediate fila-
unit of muscle) and contain the voltage-gated channels ment network, through the plasma membrane, and out to
responsible for initiating the wave of calcium release that the extracellular matrix (85). At costameres, dystrophin
activates muscle contraction. T-tubules are essential to forms part of a large transmembrane glycoprotein complex
rapidly conduct the electric impulse from the nerve into (86, 121). The lack of dystrophin, or other components of
the myofiber interior, such that all of the voltage-gated the dystrophin-glycoprotein complex, causes different
channels open in unison, and contraction occurs simul- forms of inherited muscular dystrophies (55) and renders

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1211


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

muscle much more susceptible to injury with eccentric However, these were single fibers studied in isolation. Stud-
stretch (216) (for a review, see Ref. 56). ies of whole muscles derived from mdx and control mice
concurred that calcium appears to “leak” into dystrophin-
In 1999, a new gene was identified as the cause of a form deficient fibers after bouts of contraction, inducing local
of inherited muscular dystrophy, dysferlin (22, 161). hypercontraction. This region of local hypercontraction ex-
Dysferlin did not sediment with the dystrophin-glycopro- erts a lateral strain on adjacent lengthened regions (see FIG-
tein complex and instead was shown to be required for URE 5). These regions then incurred a more significant in-
acute resealing of laser-injured myofibers (18). Thus, jury that caused global hypercontraction and “concertina-
rather than playing a structural role in the sarcolemmal like” retraction of fibers within the muscle bundle (53). This
stability, dysferlin was proposed as a key mediator of sort of fiber retraction, ripping away from adjacent muscle
calcium-dependent muscle membrane repair and is dis- fibers, would reasonably cause large membrane injuries,
cussed within section IIIB. A primary defect in skeletal sufficient to explain the large egress of creatine kinase in
patients with muscular dystrophy (FIGURE 5). Even in
muscle membrane repair characterizes dysferlinopathy,
healthy muscles, an in vivo model of a large strain injury
but is also a feature of muscle fibers from diabetic mice
caused membrane injuries of sufficient magnitude to allow
(116) and cells from patients with lysosomal storage dis-
uptake of large dextran macromolecules, which are ex-
ease (49, 119, 280) (discussed further in sect. IIIF). Fur-
cluded at later time points, but does not activate satellite cell
thermore, evidence for t-tubule injury and repair is also a
repair (235, 236). This provides direct evidence that an
feature in statin myopathy (77, 302). Thus an imbalance injurious strain to healthy muscle causes membrane injuries
in susceptibility to membrane injury, and capacity for that are repaired in a regeneration-independent manner (see
membrane repair, may be common in many forms of sect. IIIB).
myopathy.

A) WHAT DOES MUSCLE MEMBRANE INJURY LOOK LIKE? We do C. Ischemic Membrane Injury
not really know the precise nature of membrane disrup-
tions caused by repeated eccentric stretch, by lengthening 1. Ischemia-reperfusion injury is a leading killer in the
strain with a sporting injury, or suffered with more rou- Western world
tine physical activity in a patient with muscular dystro-
phy. What we do know is that the egress of creatine Disorders associated with ischemia-reperfusion injury,
kinase (199) and the uptake of albumin into eccentrically such as heart attack, stroke, and vascular disease, encom-
injured muscle fibers (180) indicates sites of membrane pass the largest causes of mortality and morbidity in the
permeability are sufficiently large to allow the transfer of Western world. Disruption in the blood flow to a tissue
large macromolecules. The dimensions of serum albumin or organ can result in large regions of cell death (infarct),
are ⬃80 Å ⫻ 80 Å ⫻ 30 Å (272) (roughly 8 nM ⫻ 8 nM and often occur as a consequence of a clot within a major
⫻ 3 nM). Therefore, for significant levels of albumin to artery delivering circulation. The size of the infarct is
primarily determined by the length of time blood flow is
enter an injured muscle fiber, there must either be multi-
obstructed and the nature of the blood vessel affected.
ple large lesions encompassing hundreds to thousands of
Blood delivers oxygen and glucose to tissues, both of
square nanometers, a multitude of smaller lesions hun-
which are required for the production of ATP by the
dreds of square nanometers, or perhaps a mixture of
mitochondrial respiratory chain. Thus ischemia induces
both. All possibilities are consistent with the notion that
a rapid loss of cellular ATP, and a complex cascade of
mechanical stretch leads to physical disruption of the events ensues from ATP depletion. For comprehensive
plasma membrane and t-tubule network (180). reviews in the area of ischemia-reperfusion injury, please
refer to References 132, 193, and 247 and references
Studies of dystrophin-deficient fibers isolated from the therein.
mdx murine model of DMD and loaded with various
sodium- and calcium-sensitive dyes could find no evi- 2. Cells need ATP to maintain ion homeostasis
dence for overt membrane tears following eccentric
stretch (316). Rather, Yeung et al. (316) provide evidence ATP drives the sodium and potassium exchange pumps
that dystrophin deficiency alters the properties of stretch- that maintain plasma membrane potential, and vitally,
activated calcium channels that leech calcium into the the F1/F0-ATPase used to generate mitochondrial mem-
cell after a bout of eccentric injury. The dysregulation in brane potential (⌬␺). ATP also drives the pumps that
calcium homeostasis leads to calpain overactivity, mito- extrude calcium from the cell (sodium-calcium ex-
chondrial dysfunction, and oxidative damage (for re- changer) and the pumps that drive cytoplasmic calcium
view, see Ref. 6). Oxidation of membrane lipids and back into the endoplasmic or sarcoplasmic reticulum af-
proteins is thought to render muscle fibers more suscep- ter each contraction in heart and muscle (via sarcoplas-
tible to injury with subsequent challenges. mic endoplasmic reticulum calcium ATPase, SERCA).

1212 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

Muscle Fiber
plasma membrane t-tubules

myofibrils

satellite cell

Eccentric Stretch Injury

hypercontraction
lateral strain

Stretch-induced injury causes Ca2+ influx and local hypercontraction

lateral strain

Lateral strain on adjacent regions of the plasma membrane increases vulnerability


to a stretch-induced secondary rupture

Increased membrane tension results in a more severe


membrane wound and global hypercontraction.
Fiber deformation from hypercontraction may also
cause membrane ruptures by ripping away from the
tight connections between adjacent myofibers

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1213


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

During ischemia, cells are obliged to utilize anaerobic ablating mitochondrial membrane potential, uncoupling
metabolism to produce ATP. This results in a rapid in- the electron transport chain, and inhibiting ATP produc-
crease in lactate and protons (acidosis), and from here, tion. Water also flows through the MPT pore, causing mi-
there is a downhill spiral of compensatory mechanisms tochondria to swell and rupture. If a large number of mito-
that conspire to result in calcium overload and toxicity. chondria within a cell activate the MPT pore, the cell will
not be able to synthesize sufficient ATP, ion homeostasis
To try and reneutralize normal cytosolic pH, cells furi- will be lost, the cell will swell, the plasma membrane will
ously pump out the protons in exchange for sodium using rupture, and in many cases the cell will initiate cell death
the Na⫹/H⫹ exchanger (191). These pumps cannot keep cascades through either necrotic, apoptotic, or autophagic
up with demand, particularly with low cellular ATP, and pathways (103, 104).
the massive influx of sodium ions results in cell swelling.
Attempts are also made to extrude the sodium in ex- 4. Brain is particularly sensitive to ischemia
change for calcium using the sodium/calcium exchanger
that begins to operate in reverse, allowing more lethal Different tissues differ in their sensitivity to ischemic injury,
calcium into the cell. Unfortunately, with ATP in short with brain being the most sensitive, then heart, kidney,
supply, a large problem for contractile heart cells is re- liver, and lastly skeletal muscle, which is remarkably resis-
uptake of calcium following contractions, whereby ATP- tant to ischemia-reperfusion injury. Sensitivity to ischemic
dependent SERCA is compromised, and calcium release injury seems to relate to the capacity of a tissue to transition
by the ryanodine receptor is enhanced (279). successfully to anaerobic metabolism, the capacity of its
fuel stores, and its intrinsic resistance to oxidative stress
(132). Brain is utterly dependent on oxidative metabolism
3. Calcium toxicity, cell swelling, and oxidative
and has very low levels of stored glycogen, in contrast to
damage conspire to injure membranes
liver and skeletal muscle, for example. Brain also has lower
levels of antioxidants such as superoxide dismutase, gluta-
Reperfusion adds fuel to the fire. Although reestablishing thione peroxidase (4), and heme oxygenase (68). Collec-
the oxygen supply is essential for cell survival, restora- tively, these attributes render the brain particularly sensitive
tion of extracellular pH to neutrality increases the proton to ischemic injury, with stroke being a leading cause of
gradient for cells with an acidic intracellular pH; the worldwide morbidity and mortality.
Na⫹/H⫹ exchanger works busily and sodium levels in-
crease, followed by an even larger influx of calcium via 5. What does ischemic membrane injury look like?
the sodium/calcium exchanger (192). Elevated levels of
submembraneous calcium activate calpains that cleave The precise structural form of ischemic membrane injury
focal adhesion complexes and cytoskeletal elements (see is not known. Membrane permeability most likely occurs
sect. IIIC), compromising the fidelity of the plasma mem- as a combination of effects related to calcium toxicity,
brane. Also with this flush of oxygen, reactive oxygen calpain activation, cell swelling due to altered sodium/
species (ROS) are produced by the mitochondria, due to potassium homeostasis, oxidative damage, and in the
damage to the electron transport chain incurred during case of cardiac myocytes, contraction-induced mechani-
ischemia, defective transport of electrons, and produc- cal injury of a damaged plasma membrane. The heart
tion of superoxide (10, 94, 322). ROS cause widespread cannot stop beating. Like skeletal muscle injury, ischemic
oxidation of both protein and lipids, another insult for membrane permeability is inferred by the egress and up-
the plasma membrane. take of large macromolecules such as lactate dehydroge-
nase and albumin that are unable to cross an intact lipid
Increased levels of cytosolic calcium and ROS can activate a bilayer. Thus we know the membrane is permeable, and
large mitochondrial conductance channel called the mito- permeable enough to allow high levels of macromolecu-
chondrial permeability transition pore (MPT) (16). The lar passage, but we do not know exactly what is the size,
MPT pore allows protons into the mitochondrial matrix, number, or disposition of the membrane breaches.

FIGURE 5. What might membrane injury to muscle fibers look like? Muscle fibers have a complex plasma membrane network with a repeating register
of deep plasma membrane invaginations called the t-tubule network. Muscle fibers are subject to huge variations in membrane tension, due to their
contractile activity. Repeated eccentric exercise in healthy subjects (i.e., stepping down for 20 min) is known to induce damage so severe that muscle fibers
degenerate over the following days and weeks (91, 131, 199). Patients with muscular dystrophy are more susceptible to injury from eccentric stretch (216),
with studies in mouse models suggesting susceptibility to injury can escalate with multiple insults (53). One model explaining membrane injury in dystrophin-
deficient muscle fibers proposes that an initial injury causes a local influx of calcium and a local region of hypercontraction. These shortened sarcomeres
induce a concomitant lengthening of adjacent sarcomeres and increased lateral strain to the plasma membrane. Subsequent insult(s) of eccentric stretch
result in a more severe wound and global hypercontraction, producing fiber retraction within the muscle bundle (53). As muscle fibers have strong interfiber
connections, muscle injuries may manifest both as shearing of the membrane from increased membrane tension and strain, as well as ripping of plasma
membrane regions from fiber retraction or hypercontraction.

1214 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

III. THE PROTEIN MACHINERY OF ACUTE before the transmembrane domain. This ␣-helical sequence is
MEMBRANE REPAIR referred to as the SNARE motif and facilitates interaction of the
three SNARE proteins into a parallel coil-coiled structure (275).
The quaternary ␣-helical complex formed by assembly of syn-
A. SNAREs
taxin, SNAP, and VAMP is extremely stable and resistant to
heating (but not boiling) in sodium dodecyl sulphate (SDS) lysis
1. Evolution and structure
buffer. These ternary complexes self-assemble to form a ring (50),
The SNAREs (soluble N-ethylmaleimide-sensitive factor at- zippering from the NH2 terminus to the COOH terminus to
tachment protein receptors) comprise three families of pro- draw the opposing membranes of the vesicle and target mem-
teins that interact to form the core machinery of vesicle branes together for fusion (for recent reviews, see Refs. 210, 234).
fusion: syntaxins, SNAPs (soluble N-ethylmaleamide at-
tachment proteins), and VAMPs (vesicle associated mem- The surface of exocytic vesicles is negatively charged due to polar
brane proteins). SNARE proteins have ancient eukaryotic phosphate headgroups, providing a natural vesicle-vesicle and
origins and are present in protozoans, early unicellular eu- vesicle-plasma membrane repulsion that must be overcome for
karyotes, fungi, yeast, and animals, and are central players membrane fusion. Thus membrane fusion is thought to be driven
in the evolution of the eukaryotic endomembrane system by the strong association of the SNARE components, the binding
(65– 67). of positively charged calcium to neutralize the negative repulsion
and repel water between the fusing membranes (128), and local
A) SNARE COMPLEX AND HOW VESICLE FUSION HAPPENS. Three disruption of the membrane curvature of the opposing bilayers by
proteins form a SNARE complex: two proteins from the target the transmembrane domains of the SNARE proteins (266; for
membrane, syntaxin (26) and SNAP (207), and one from the recent reviews, see Refs. 127, 133).
vesicle membrane, VAMP (295). Most SNARE proteins are tail-
anchored transmembrane proteins, with the “business side” of B) SYNAPTOTAGMINS ARE LATE EVOLUTIONARY ARRIVALS TO THE
the protein facing the cytoplasm, and only a short luminal or SNARE COMPLEX. At neural synapses, synaptotagmins 1 and 2
extracellular tail (see FIGURE 6). A common feature of all trigger the calcium-activated fusion of neurotransmitter-
SNAREs is the presence of a cytoplasmic ␣-helical heptad repeat containing vesicles (35, 38, 81) through interaction with
domain, consisting of ⬃67 amino acids, often positioned just SNARE vesicle fusion machinery (253). Synaptotagmins 1

SNARE VAMP
TM

Habc SNARE Syntaxin


TM

SNARE SNARE SNAP

TM
C2A C2B Synaptotagmin

TM
C2A C2B C2C DysF C2D C2DE C2E C2F Dysferlin
FIGURE 6. Schematic representation of the
Mini-dysferlinC72 structural features of the protein families impli-
C2E C2F
cated in membrane repair.

Cys His Asn

Calpain
N Cysteine protease C2-like Penta-EF hand

Superhelical domains
N-term Annexin

B-box PRY-SPRY MG53


Ring coil-coil

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1215


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

and 2 localize to secretory vesicles and are type I transmem- SNAREs, or inhibitory antibodies recognizing synaptotag-
brane proteins with two, tandem cytoplasmic C2 domains mins or SNAREs, collectively implicated SNARE proteins
and show calcium-regulated interaction with members of in the vesicle fusion of membrane repair (72, 291).
the SNARE complex. However, the precise mechanism by
which synaptotagmins facilitate or accelerate calcium-reg- B) A ROLE FOR SYNAPTOTAGMIN VII AND LYSOSOMAL EXOCYTOSIS
ulated exocytic fusion remains the topic of intense debate. It FOR MEMBRANE REPAIR? In 2001, Reddy et al. (229) reported
is thought that calcium-binding to synaptotagmin triggers that membrane repair in nonsecretory skin fibroblasts was
phospholipid binding of its C2 domains to the vesicle and mediated by synaptotagmin VII (SytVII) via calcium-acti-
plasma membrane, as well as binding to the SNARE com- vated lysosomal exocytosis (229). Membrane injury in-
plex, releasing complexins, which negatively regulate duced the calcium-dependent appearance of the luminal
SNARE assembly (282). epitope of lysosomal associated membrane protein
(LAMP-1) on the cell surface of wounded skin fibroblasts,
Interestingly, phylogenetic analyses reveal that synaptotag- and treatment with anti-SytVII antibodies, recombinant
mins originated in multicellular eukaryotes (61) evolution- SytVII C2A, or anti-LAMP-1 antibodies impaired lyso-
arily postdating syntaxins (65, 66), suggesting core SNARE somal exocytosis and membrane repair (229). A SytVII
machinery could once function independently of synap- knockout mouse was then generated and showed normal
totagmin in primitive cells, or alternately worked in concert growth and development, but developed an inflammatory
with a more evolutionary ancient predecessor. The synap- myopathy with elevated creatine kinase and muscle weak-
totagmin gene family rapidly expanded in metazoans, with ness with other autoimmune symptoms, such as dermato-
correlation between multicellular complexity and the num- myositis and an antinuclear antibody response (47). Embry-
ber of synaptotagmin paralogs particularly apparent in the onic fibroblasts from SytVII knockout mice were shown to
evolution of green plants (248). In humans, there are 16 be defective in lysosomal exocytosis and membrane reseal-
synaptotagmins (61) and 15 syntaxins (284), each display- ing after wounding (47). The authors later showed that loss
ing a distinct pattern of subcellular localization to mediate of SytVII resulted in fewer lysosomal fusion events overall
the trafficking of vesicular cargo between different intracel- and the properties of the fusion events were different (126).
lular destinations. Synaptotagmins-1 and -2 are essential In wild-type cells, calcium influx triggers lysosomal fusion
for rapid, synchronous neurotransmission at vertebrate events characterized by a small fusion pore and minimal
synapses and induce neonatal lethality in knockout mice diffusion of lysosomal transmembrane proteins into the
(97, 209). plasma membrane. In contrast, SytVII knockout fibroblasts
showed more complete lysosomal fusion events with merg-
2. Role in membrane repair ing of lysosome membrane contents with the plasma mem-
brane, suggesting SytVII regulates and restricts fusion pore
A) PARALLELS BETWEEN MEMBRANE REPAIR AND SYNAPTIC EXOCY- formation (126). Further studies are required to reconcile
TOSIS. Steinhardt et al. (267) significantly advanced our un- how these aberrations in lysosomal exocytosis relate to
derstanding of how membrane repair works, when they failed repair in SytVII null cells.
showed that membrane resealing utilized a process with
parallels to synaptic neurotransmission. Confocal micros- More detailed studies of the effect of recombinant domains
copy of damaged sea urchin embryos or unfertilized eggs of synaptotagmin I and VII on membrane repair showed
showed that a membrane puncture induced the exocytic that the C2A domain of SytVII had no effect on membrane
fusion of secretory yolk granules (27, 267). This injury- resealing, but did impair the facilitated response to a second
induced exocytic fusion was wholly dependent on calcium, injury at the same site (258). Interestingly, treatment with
not activated by other cations, and antagonized by magne- recombinant C2B of SytVII inhibited both initial membrane
sium, with each property also being a feature of synaptic resealing and the capacity of an injured cell to more readily
exocytosis. Larger membrane disruptions required vesicle- repair a second injury. Similar inhibition of membrane re-
vesicle fusion to form a patch, whereby steps of “vesicle-to- sealing was observed with recombinant C2B of synaptotag-
vesicle” fusion, and integration of fused “patches” into the min 1. Inhibitory effects were dependent on the calcium
plasma membrane, were both strictly dependent on extra- sensitivity of the C2 domains, with no effects elicited by
cellular calcium (179, 285). These studies also showed that calcium-binding mutants of each domain. However, the
successful membrane repair correlated with the degree of authors stress that treatment with recombinant C2 domains
exocytic fusion and that membrane repair was blocked or of synaptotagmins likely indirectly impacts membrane re-
severely inhibited by clostridial neurotoxins in sea urchin pair by promiscuously binding SNARE machinery and tar-
eggs (docked cortical granules), embryos (undocked corti- get phospholipids (258).
cal granules), and mammalian Swiss 3T3 cells (nonsecre-
tory cells) (27, 267). The clostridial neurotoxins are pro- 3. Perspectives
teases that specifically cleave SNARE proteins and induce
paralysis in infected victims (29, 252, 314). Further exper- Evidence suggests that lysosomes are not membrane repair
iments using recombinant fragments of synaptotagmins or organelles and do not directly contribute to the exocytic

1216 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

formation of “membrane patches” for lesion repair. Al- B. Dysferlin


though lysosomal exocytosis was shown to occur in re-
sponse to ballistics injuries in skeletal muscle cells, these 1. Evolution and structure
fusion events occurred distal to the injury site, and lyso-
somal markers did not demark vesicles recruited and en-
Dysferlin is a large (⬃240 kDa) tail-anchored transmem-
riched at injury sites labeled by dysferlin (150). Similarly, brane protein that bears the unique feature of seven tandem
zebrafish lysosomal membrane proteins did not accumulate C2 domains within its large cytoplasmic domain, the most
at injury sites in zebrafish larval muscle cells, although ly- of any protein family. C2 domains are independently fold-
sosomes were observed to fuse with the plasma membrane ing protein motifs comprised of ⬃110 –130 amino acids,
when near an injury site (238). However, there is an ex- originally identified in protein kinase C (202). C2 domains
panding body of evidence that lysosomal exocytosis occurs mediate lipid and protein binding, often regulated by coor-
in response to the acute elevation in intracellular calcium dination of calcium ions within a negatively charged bind-
caused by membrane injury and that machinery of the late ing pocket comprised of highly conserved acidic residues
endosomal pathway play key roles in membrane repair. (usually aspartate) (51). The crystal structures of many C2
Specific roles in membrane repair for late endosomal ma- domains have been solved and feature a folded sandwich of
chinery ESCRT (129, 250) and mucolipin-1 (49) are dis- two ␤-sheets, each containing four anti-parallel ␤-stands
cussed in section IIIF. Exocytic fusion of late endosomes/ (87, 274). Clustered at the end of the ␤-sheet sandwich
lysosomes may function to reduce membrane tension, to reside three variable connecting loops that, in the case of
deliver protein machinery required for vesicle formation calcium-sensitive C2 domains, contain the highly conserved
and exosomal shedding (129, 250), as well as to release acidic residues that form the binding pocket for multiple
enzymes such as acid sphingomyelinase to activate endocy- calcium ions. The binding of calcium within this pocket
tosis (280). directly facilitates membrane interaction (196), with the
amino acid composition of the loop region shown to influ-
Collectively, there is a strong body of evidence showing that ence phospholipid selectivity for targeting specific mem-
membrane repair is inhibited by clostridial neurotoxins, brane compartments (51).
recombinant protein fragments of SNARE proteins and
synaptotagmin C2 domains, and by antibodies that func- A) DYSFERLIN BELONGS TO AN ANCIENT FAMILY OF VESICLE FUSION
tionally inhibit SNAREs and synaptotagmins. Thus, al- PROTEINS. There are six mammalian ferlin proteins: dysferlin
though SNARE machinery is squarely implicated in the ves- (Fer1L1), otoferlin (Fer1L2), myoferlin (Fer1L3), Fer1L4,
icle fusion of membrane repair, it is yet to be determined Fer1L5, and Fer1L6, each characterized by a cytoplasmic
whether there is an ancient and preserved set of core domain bearing between five and seven tandem C2 domains
SNARE machinery for membrane repair, or whether there anchored by an extreme COOH-terminal transmembrane
have been evolutionary adaptations in the repair mecha- domain (for a recent review, see Ref. 151). Our phyloge-
nism to best suit the types of injury encountered, and the netic studies have revealed that ferlins have ancient origins
available endogenous vesicle fusion machinery, in different in eukaryotic biology (152). Ferlins are detected in all eu-
cell lineages. karyotic kingdoms, including unicellular phytoplankton
and in protozoans, indicating origins predating evolution-
While the extremely high concentration of local calcium ary branching. Ferlins have not yet been identified in fungi
around a wound site is not altogether dissimilar from an or plants, suggesting they may have been lost from these
active synaptic zone, or directly adjacent to the calcium- evolutionary lineages. Invertebrate and vertebrate animal
release channels of the triad junction in skeletal muscle, this models of ferlin deficiency are united by pathologies linked
level of intracellular calcium would be an unusual event in a to defective calcium-activated vesicle fusion (3, 18, 59, 205,
nonsecretory cell and would certainly create a unique and 242). Thus it is proposed that ferlins are a family of calci-
very active local environment. The flood of extracellular um-binding vesicle fusion proteins for regulated exocytosis,
calcium thought the breached plasma membrane will pro- with evidence suggesting more ancient evolutionary origins
miscuously initiate calpain-mediated proteolysis, calcium than the classical mediators of vesicle fusion, the synap-
second messenger signaling, and calcium-regulated fusion totagmins (see section IIIA) (151).
of any nearby primed or “primable” vesicles. In the setting
of a membrane injury where it is repair or die, the evolu- 2. Localization
tionary pressure on the effectiveness of membrane repair
means that each of these cascades must play an important Dysferlin is expressed ubiquitously in mammalian tissues,
and interrelated role. It is difficult to reason whether it is with high levels in skeletal muscle and heart (9). Dysferlin
more likely that a core, evolutionary ancient membrane localizes to the skeletal muscle plasma membrane, also
repair machine exists in all cells, or whether promiscuity in called the sarcolemma (9, 218), to the invaginating t-tubule
the fusion machinery and vesicle populations utilized for network (7, 135, 138, 163). Dysferlin has also been shown
membrane repair is central to the survival response itself. to localize to the apical plasma membrane of syncytiotro-

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1217


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

phoblasts (297), suggesting a role in polarized membrane A role for mammalian ferlins in calcium-activated vesicle
trafficking in the placenta. Studies of dysferlin trafficking in fusion was further strengthened by the discovery that
transfected C2C12 mouse myoblasts reveal that dysferlin otoferlin, the genetic basis of a form of inherited nonsyn-
shuttles between the plasma membrane and the endo-lyso- dromic deafness in humans (315), was due to defective cal-
somal pathway (88). cium-activated auditory neurotransmission at the cochlear
inner hair cell synapse (242). It remains debated whether
3. Role in human disease the role of otoferlin in the cochlear relates solely to func-
tions as a calcium-activated trigger for synaptic vesicle ex-
Dysferlin mutations underlie a form of autosomal recessive ocytosis, or whether its role may instead/also relate to en-
inherited muscular dystrophy, called limb girdle muscular docytosis and recycling of synaptic vesicles to restock the
dystrophy type 2B (LGMD2B) (22, 161). Dysferlinopathy ready releasable pool of neurotransmitter containing vesi-
is a late-onset form of muscular dystrophy, manifesting in cles required for sustained, high-frequency firing of the
older teenagers or adults, and is characterized by absence or highly demanding inner hair cell synapse (211).
marked reduction of dysferlin protein in the skeletal muscle
of affected patients. Curiously, prior to their presentation, Although dysferlin is expressed ubiquitously, the skeletal
dysferlinopathy patients show no evidence for subclinical muscles are particularly affected by loss of dysferlin. This
muscle weakness as children, with many patients reporting may relate to the specialized architecture of the skeletal
sporting distinction in their youth. This differs from other muscle t-tubule membrane network and its vulnerability to
later onset muscular dystrophies and myopathies, in which eccentric stretch. Dysferlin is abundantly expressed within
there is often a long history of poor sporting performance the t-tubule network, and dysferlin-deficient muscle fibers
and avoidance of strenuous physical activities. In dysferli- show major t-tubule abnormalities after a bout of in vivo
nopathy, physically able teenagers often suffer an injury lengthening strain injuries (135). Indeed, dysferlin-deficient
that is difficult to recover from, and begin to experience fibers suffer significant damage after long strain injury, re-
unexplained fatigue and muscle pain, followed by progres- quiring satellite cell-mediated repair pathways to rebuild
sive muscle weakness. Unfortunately, once dysferlin mus- necrosing muscle fibers following a stretch protocol (236).
cular dystrophy manifests, the physical decline can be rapid, In contrast, wild-type fibers readily survive a lengthening
from being able-bodied to nonambulant (unable to walk) in strain injury without evidence for necrosis, and without
4 – 8 yr (200). It is not understood why dysferlin deficiency requiring satellite-cell mediated repair (236). These data
does not clinically affect young muscles of affected children suggest dysferlin is particularly important for repair and
yet results in severe weakness and muscle degeneration in remodeling of t-tubule membranes.
adulthood, with some patients also presenting with mild
cardiac involvement (143, 310). B) DYSFERLIN TRANSLOCATION TO INJURY SITES. Bansal et al. (18)
studied the localization of dysferlin in myofibers injured via
4. Role in membrane repair aspiration through an 18-gauge needle, using fluorescent
dextran to demark injured fibers. Confocal microscopy re-
A) DYSFERLIN PLAYS A KEY ROLE IN CALCIUM-DEPENDENT MEM-
vealed intensely labeled “patches” of dysferlin that ap-
BRANE REPAIR. Dysferlin-deficient mice also exhibit a late- peared to correlate with potential sites of plasma membrane
onset progressive muscular dystrophy (18, 32, 112). A sem- disruption, inferred by brightfield images showing small
inal study by Bansal et al. in 2003 (18) revealed that muscle regions of thickened and nonuniform sarcolemma, visually
fibers lacking dysferlin did not show the same membrane discontinuous with an otherwise uniform section of myofi-
fragility as dystrophin-deficient fibers (the basis of Duch- ber sarcolemmal membrane, and consistent with a small
enne muscular dystrophy, the most common human mus- repair patch. These regions showed reduced or absent la-
cular dystrophy), and instead demonstrated defects in cal- beling for constitutively expressed sarcolemmal proteins
cium-activated membrane resealing following laser injury. caveolin-3 and ␦-sarcoglycan, consistent with recently re-
Control muscle fibers were shown to effectively reseal a paired injury “patches” derived from nonsarcolemmal
laser-induced plasma membrane injury and exclude the membrane sources. Dysferlin is also observed to intensely
styryl dye FM1-43 within 30 s, whereas dysferlin-null label the fine, meshlike network of longitudinal tubules of
mouse muscle fibers showed increased and prolonged entry the t-tubule network that become vacuolized and injured
of FM1-43 up to 2 min following the laser injury. Indeed, with over-stretch (304).
the kinetics and magnitude of FM1-43 dye entry in injured
dysferlin-null fibers resembled results from wild-type fibers Many groups have attempted to study the recruitment of
damaged in the absence of calcium. Given the homology of heterologously expressed dysferlin to sites of membrane
dysferlin to the C. elegans protein Fer-1, previously shown injury in cultured myotubes. Klinge et al. (138) suggested
to regulate calcium-activated vesicle fusion (3, 307), dysfer- that an NH2-terminal EGFP-dysferlin fusion protein
lin was therefore proposed to be a key mediator of calcium- showed calcium-dependent and injury-dependent plasma
activated vesicle fusion for muscle membrane repair. membrane enrichment in C2C12 myotubes injured by roll-

1218 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

ing glass beads. Dysferlin enrichment appeared broad and At later time points, dysferlin formed an intricate lattice
generalized over large areas of a myotube, in stark contrast (with MG53) labeling the broader surrounds of the mem-
to the tightly refined patches of endogenous dysferlin re- brane lesions (20 – 60 s post injury). One to two minutes
cruited to proposed injury sites in mature skeletal myofibers after injury, dysferlin labeled a bright arc of two closely
reported by Bansal et al. (18). This may reflect immaturity opposed membranes lying in a bed of dysferlin and
of the myogenic model employed by Klinge et al. (138), MG53 lattice, positioned parallel to the long axis of the
comparatively larger areas of membrane injury induced by cultured myotubes (FIGURE 7). These observations were
the rolling beads, or perhaps disruption of normal dysferlin consistent with a model whereby dysferlin initially labels
behavior through epitope-tagging. Arguably however, the the periphery of the lesion, forms a repair lattice that
precise site of membrane injury could not accurately be infiltrates the lesion surrounds as new membrane is de-
determined in either case, and therefore, it is difficult to livered, and cytoskeletal motors “zipper” the ballistics
qualify whether areas of dysferlin enrichment represent in- lesions together by drawing opposing membranes to-
jury sites, or not. gether lengthwise along the long axis of the myotube.

Live cell imaging experiments in transfected murine C) INJURY-ACTIVATED CALPAIN CLEAVAGE OF DYSFERLIN: EMER-
C2C12 myoblasts indicated that EGFP-dysferlin is re- GENCY PRODUCTION OF A SYNAPTOTAGMIN-LIKE VESICLE FUSION
cruited to sites of laser injury or needle microinjection MODULE FOR MEMBRANE REPAIR? Perplexingly, dysferlin
only when coexpressed with the muscle-specific mem- could only be detected at injury sites using an antibody
brane repair cofactor mitsigumin-53 (MG53, discussed recognizing the extreme COOH terminus of the dysferlin
in detail in sect. IIIE) (42). However, zebrafish dysferlin cytoplasmic domain, Hamlet-1, and not with three other
expressed as a COOH-terminal fusion with monomeric anti-dysferlin antibodies recognizing more NH2-terminal
teal fluorescent protein showed rapid recruitment to in- epitopes (150). Biochemical analyses subsequently re-
jury sites during in vivo imaging of laser-injured zebrafish
vealed that dysferlin was cleaved by activated calpains
myofibers, and zebrafish do not express a MG53 paralog
with membrane injury, releasing a COOH-terminal frag-
(238). Thus whether MG53 is required for injury recruit-
ment of 72 kDa, termed mini-dysferlinC72. Interestingly,
ment of dysferlin in muscle cells remains unclear. The
mini-dysferlinC72 bears the last two most ancestrally con-
sophisticated in vivo imaging experiments described by
served C2 domains and transmembrane domain (152),
Roostalu and Strahle (238) revealed that a short dysferlin
with structural parallels to the classical vesicle fusion
COOH-terminal fragment (the extracellular domain of
proteins, the synaptotagmins. Results therefore suggest
22 amino acids, transmembrane domain, and only 29 of
that it may not be full-length dysferlin that is recruited to
the ⬃2,000 amino acids of the cytoplasmic domain) was
injury sites in cultured human myotubes, but a COOH-
sufficient to confer targeting to recruited repair mem-
terminal fragment of dysferlin, mini-dysferlinC72.
branes. In contrast, truncated dysferlin NH2-terminal
domains were unable to be effectively recruited, suggest-
We have subsequently shown that calcium-dependent
ing cellular targeting of dysferlin by its transmembrane
domain to appropriate membrane compartments is vital cleavage of dysferlin is mediated by the ubiquitous cal-
for its mobilization and recruitment to injury sites. pains (calpains-1 and -2) via a cleavage motif encoded by
an alternately spliced exon, exon 40a (230). Thus not all
Lek et al. (150) recently developed a ballistics model of dysferlin isoforms may be cleaved by calpains in response
membrane injury in cultured human myotubes, producing to injury. Interestingly, other members of the ferlin fam-
readily identifiable enface injuries, suitable for high-resolu- ily are also cleaved by calpains to release similar COOH-
tion imaging. Three-dimensional structured illumination terminal modules (230). Evolutionary conservation of
microscopy (3D-SIM) was used to reconstruct the recruit- this feature implies that calpain cleavage of ferlins be-
ment of dysferlin to injury sites (150). Super-resolution im- stows an important functional modification in settings of
aging of injury sites resolved the rapid recruitment of dys- intense calcium signaling.
ferlin-containing cytoplasmic vesicles to sites of membrane
injury within 10 s, undergoing calcium-dependent integra- Interestingly, while dysferlin transcripts bearing exon 40a
tion into plasma membrane compartments decorated by are abundantly expressed in many human tissues (40 – 60%
MG53 (FIGURE 7). This process is surprisingly consistent of all transcripts in kidney, lung, liver, placenta, and pan-
with the mechanism proposed by Steinhardt et al. (267) creas) (230), somewhat counterintuitively, only ⬃15% of
nearly 20 years ago entitled: “membrane repair occurs via a dysferlin transcripts in skeletal muscle contain exon 40a
process analogous to synaptic exocytosis.” (220, 230). Thus not all dysferlin isoforms in skeletal mus-
cle can be cleaved by calpains. Further studies are needed to
Immediately following ballistics injury, dysferlin specifi- clarify the respective roles of full-length dysferlin (without
cally and intensely labeled the exposed phospholipids exon 40a) and cleaved mini-dysferlinC72 for membrane re-
encircling the ballistics injuries (10 s post injury) (150). pair of skeletal muscle and other tissues.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1219


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

Plasma membrane (PM) dysferlin

Ca2+ influx from membrane injury


activates local calpains

Intracellular compartment

MG53 arrives at injury


sites within 2 seconds.
?

Calpains cleave dysferlin, releasing a synaptotagmin-like


? C-terminal module with two C2 domains.

Which pool of dysferlin is recruited for


membrane repair?

Dysferlin vesicles undergo


Ca2+-regulated integration
at injury sites.

At 90 seconds post-injury, lesions are filled in


At 10 seconds post-injury, dysferlin and MG53 and are characterized by a dominant arc of
intensly label the edges of the lesion. dysferlin and MG53.

Confocal 3D-SIM Confocal 3D-SIM

1220 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

D) A PATIENT MINI-DYSFERLIN RESTORES MEMBRANE REPAIR, BUT 5. Perspectives


NOT DYSTROPHIC PATHOLOGY. Further evidence that cleaved
mini-dysferlinC72 plays a specialized role in membrane re- It is clear that dysferlin plays a key role in membrane repair.
pair has been provided serendipitously, through studies of a However, exactly what dysferlin does to mediate mem-
naturally occurring truncated dysferlin based on a patient brane repair is only beginning to be teased out. From the
with a genomic deletion within the dysferlin gene, that is perspective of the pathogenesis of dysferlinopathy, collec-
very similar to calpain-cleaved mini-dysferlinC72 (140). In tive evidence suggests defective membrane repair may be
this patient, a genomic region encompassing exons 2– 40 of only one contributing factor to disease pathology. Dysferlin
dysferlin are deleted, and the patient employs a cryptic deficiency also affects the trafficking and signaling of
splice site to express a truncated dysferlin with 13 amino growth factor receptors (71) and adhesion molecules (256).
Muscle injury may therefore present a “perfect storm” for
acids at the NH2 terminus derived from intronic sequences,
dysferlinopathy patients, where a defective response to an
followed by exons 41–55. Calpain-cleaved mini-dysfer-
acute membrane injury collides with a static defect in day-
linC72 bears approximately seven residues of exon 40a (al-
to-day trafficking of dysferlin-specific cargo, affecting mus-
though calpains do not strictly cleave at one site, and may
cle, vascular, and immune cells and producing a poor re-
cleave either side of their preferred site), followed by resi-
generative environment. Given the late presentation of dys-
dues encoded by exons 41–55. The patient presents with a
ferlin disease, one can only assume there are intrinsic
mild-moderate dysferlinopathy and transgenic expression
differences in requirements for dysferlin-dependent traf-
of the patient mini-dysferlin in dysferlin-null muscle fibers
ficking and membrane repair pathways between growing
restored normal membrane repair (140). These data sup-
muscle fibers of children versus fully mature adult myofi-
port our proposal that calpain-cleaved mini-dysferlinC72 is bers.
an important mediator of membrane repair. However,
transgenic expression of the patient mini-dysferlin did not Our recent discovery revealing activated calpains specifi-
prevent development of a dystrophic pathology in dysferlin- cally cleave dysferlin in response to membrane injury pro-
null mice (140, 166). Thus defective membrane repair does vides the first step linking membrane repair roles separately
not appear the sole factor underlying the pathology of dys- established for dysferlin and calpains (discussed in detail in
ferlinopathy. sect. IIIC). It remains to be experimentally determined
whether mini-dysferlinC72 possesses specialized vesicle fu-
E) WHICH POOL OF DYSFERLIN IS CLEAVED? A recent study created sion activity that is important for membrane repair, and
a transgenic mouse model expressing dysferlin (without whether dysferlin-laden cytoplasmic vesicles interact with
exon 40a) with an extracellular pHluorin tag (172). pHluo- classical SNARE fusion machinery. Also yet to be deter-
rin is a genetically modified form of GFP that is pH sensitive mined is the role of the cleaved dysferlin NH2-terminal
(184), in this case showing reduced fluorescence in acidic domain. Modal functions of different dysferlin C2 domains
compartments. McDade et al. (172) showed that immedi- makes sense in light of the evolutionary preservation of each
ately following membrane injury, dysferlin-pHluorin fluo- of the ferlin C2 domains, that are highly divergent from one
rescence diminished in regions surrounding, and distal to, another, but very similar to the analogous C2 domain in
the injury site, and cytoplasmic accumulations of dysferlin other ferlins. This tells us that each C2 domain is function-
formed in regions distal to the injury site. These results ally specialized.
suggest dysferlin may initially be endocytosed into acidic
compartments in response to membrane injury. Is it endo- Endocytosis, calpain cleavage, then reexocytosis seems a
cytosed dysferlin that is cleaved by calpains, then vesicles labored path for a rapid emergency response. However,
laden with cleaved mini-dysferlinC72 exocytosed at injury evidence that dysferlin may participate in both endocytic
sites (FIGURE 7)? and exocytic pathways during the membrane repair re-

FIGURE 7. A cartoon depicting the potential role of dysferlin-mediated vesicle fusion in membrane repair. Membrane injury causes a local influx of calcium
and activation of calpains. MG53 (40) shows diffuse enrichment at injury sites within 2 s of membrane injury in a calcium-independent manner (150). The
signal to activate recruitment of MG53 to injury sites is not clear, but may relate to its role as a ubiquitin ligase to target substrate(s) damaged as a
consequence of the membrane injury. Dysferlin is not detected at injury sites until 10 s postinjury, a delay we attribute to an intermediary step involving calpain
cleavage. Activated calpains cleave dysferlin within a motif specifically encoded by alternately spliced exon 40a (230). As dysferlin may only be detected at
injury sites with antibodies recognizing COOH-terminal epitopes, and not several antibodies to NH2-terminal or central domains (150), data suggest the
COOH-terminal cleaved fragment termed mini-dysferlinC72 is the form specifically recruited to injury sites. It remains uncertain whether full-length dysferlin is
also present at injury sites but is structurally precluded from antibody labeling and whether the dysferlin recruited for membrane repair is derived from plasma
membrane or intracellular membrane compartments. Super-resolution 3-dimensional structured illumination microscopy (3D-SIM) reveals dysferlin-laden
vesicles undergo calcium-dependent integration at injury sites (150). At 10 s postinjury, dysferlin and MG53 form a lattice that intensely labels the edges of
the lesions. Lesions expand and are filled in by a dysferlin and MG53 lattice. At 90 s postinjury, filled lesions are characterized by a dominant arc of dysferlin
and MG53 labeling we believe represents the original edges of the lesion that have been drawn together by cytoskeletal motors for resealing. [3D-SIM images
from Lek et al. (150), with permission from The Journal of Neuroscience.]

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1221


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

sponse is consistent with the dual exocytic and endocytic cium concentration for proteolytic activity: 10 –50 ␮M for
recycling roles proposed for otoferlin in auditory neu- ␮-calpain and 0.25– 0.35 mM for m-calpain. Many cal-
rotransmission (211, 242). Determination of the nature and pains show tissue-specific expression, for example, cal-
the cargo contained within dysferlin-laden vesicles re- pain-3a in skeletal muscle, calpain-6 in placenta, calpain-8
cruited to injury sites may provide valuable clues: are these and -9 in the gastrointestinal tract, and calpain-11 in testis
purely a source of membrane lipids to patch a hole, or are (see reviews in Refs. 102, 276).
soluble and transmembrane proteins codelivered that play
an integral role in the repair and remodeling process? The ubiquitously expressed calpain-1 and -2 are the best
studied of the calpains. In cultured cells, calpains show
cytosolic and membrane localization and are widely de-
C. Calpains
scribed to translocate to the plasma membrane in response
to cellular signaling by calcium (see, for example, Refs. 99,
1. Evolution and structure
100) and with growth factor receptor activation (153, 255).
The localization of calpains has been widely studied in skel-
Calpains are an ancient family of thiol-proteases, present in
protozoa, plantae, and eukaryota kingdoms (62, 84). The etal muscle fibers, with most labeling detected within the
ubiquitous calpains, calpain-1 and calpain-2, typically exist myofibrillar apparatus (142). More recent studies in
as heterodimers, with a large catalytic subunit of ⬃80 kDa skinned skeletal muscle fibers show that calpain-1 can freely
and a smaller regulatory subunit of 28 kDa (for comprehen- diffuse out of the myofibrillar compartment at resting cal-
sive review, see Ref. 102). The large catalytic subunit of cium levels, but becomes tightly bound within skinned fi-
calpain consists of four different domains (115, 270). Do- bers with increased cytosolic calcium (20 ␮M) (195). Mus-
main I is an ␣-helical domain comprised of 10 –20 amino cle cells present a complex environment when considering
acids and is divergent among calpain orthologs and paral- calpain activity; the calcium transients of muscle contrac-
ogs; with some isoforms bearing Zn-finger or transmem- tion would theoretically provide sufficient cytosolic calcium
brane helices within domain I (263). Proteolytic removal of to activate proteolytic activity of ␮-calpain but, fortunately
this NH2-terminal helix plays a role in the activation of for muscle fibers, does not. It is therefore proposed that
some calpains (137). Domain II forms the catalytic core, calpain activity in skeletal muscle is tightly regulated by
rich in essential catalytic residues cysteine, histidine, and calpastatin (an abundantly expressed natural and specific
asparagine. Domain III consists of an eight ␤-strand sand- inhibitor of calpain-1 and -2), their localization, and acces-
wich with structural resemblance to a C2 domain, and sibility to their discreet repertoire of protein substrates.
thought to confer calcium and phospholipid binding. Do-
main IV bears five tandem calcium-binding EF hand do- 3. Role in human disease
mains (33, 159). The presence of a calmodulin-like penta-
EF-hand module characterizes the “typical calpains” such
Calpains play a key role in development, with targeted
as ubiquitous calpain-1 and -2 and occurred late in calpain
knockout of calpain-2 resulting in embryonic lethality due
phylogeny, present only in metazoans (animal lineage). Pro-
tozoan, plant, and fungal calpains do not possess an EF- to an implantation defect (11, 79). Although calpain-1 and
hand containing domain IV; moreover, several “atypical” -2 cleave many of the same substrates in vitro, knockout of
mammalian calpains also lack this domain (62, 102). calpain-1 produces viable mice that are morphologically
normal but show defects in platelet function (13). This high-
A) CALPAINS SELECTIVELY MODIFY TARGET SUBSTRATES. Calpains lights different functional roles for calpain-1 and calpain-2,
target selective substrates and use complex substrate recog- whereby calpain-1 is unable to compensate for calpain-2
nition motifs dictated both by primary and secondary pro- deficiency during early embryonic development.
tein structure (162, 294). Calpains are not terminal degra-
dative enzymes but, rather, selectively modify their target Disturbances in calpain behavior are implicated in numer-
substrate. More than 100 proteins have been identified as ous human pathologies (see review and articles within Ref.
calpain substrates; many are cytoskeletal proteins, such as 317). The crux of the problem in most cases stems from
vimentin, talin, desmin, troponin, dystrophin, and spectrin, calpain “overactivity” due to aberrant calcium handling
but substrates also include receptors, ion channels, tran- and is often associated with pathologies of membrane in-
scription factors, signaling proteins, and enzymes (102). jury, such as muscular dystrophy (262, 289), cardiac isch-
emia-reperfusion injury (124), traumatic brain injury (243)
2. Expression and localization and stroke (17, 64), Alzheimer’s disease (273), multiple
sclerosis (259, 271), and cataract formation (31). More-
There are 14 genes encoding large catalytic calpain subunits over, calpains are becoming increasingly implicated in can-
in humans. Calpain-1 and -2 are ubiquitously expressed cer biology, with roles in pro-survival or apoptotic decision-
and are also referred to as micro (␮)- and milli (m)-calpain, making, and cytoskeletal remodeling and migration, highly
respectively, thus named according to their activating cal- relevant to tumorigenesis and metastasis (268).

1222 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

A polymorphism within intron 3 of calpain-10 has been Calpains have also been shown to be vital for membrane
implicated as a susceptibility factor for type 2 diabetes resealing of mammalian somatic cells. In the acute setting of
(114). However, a direct role for calpains in human disease membrane scrape-injury, treatment with calpain inhibitors
is best described by a monogenic form of autosomal reces- markedly impairs cell survival of immortalized mouse em-
sive limb girdle muscular dystrophy (LGMD2A) due to mu- bryonic fibroblasts (MEFs) and primary human skin fibro-
tations in the gene encoding calpain 3a (CAPN3) (233). The blasts and neonatal rat cardiomyocytes (182, 183). Using
mechanism underpinning LGMD2A remains relatively mouse embryonic fibroblasts with targeted knockout of the
poorly understood. Calpain-3, although abundant, is ex- CAPNS1 regulatory subunit of calpain-1 and -2 (11), Mell-
tremely labile and partitions to the myofibrillar compart- gren et al. (183) were able to show that calpain-1 and/or
ment of skeletal muscle (194). The current paradigm for the calpain-2 were required for acute membrane repair (183).
pathogenesis of LGMD2A centers upon a role for calpain-3 In contrast, calpain-3a did not contribute to survival after
as a “sarcomeric remodeler” (24, 283), although different scrape damage of skeletal myoblasts or to acute repair of a
studies have separately implicated roles in muscle matura- laser damaged membrane in skeletal myotubes derived
tion (264), myonuclear apoptosis (15), and maintenance of from CAPN3 knockout mice (182).
the costameric dystrophin-associated complex via targeted
cleavage of filamen-C and ␦- and ␥-sarcoglycan (107). B) THE PUZZLE: HOW DO CALPAINS UNDERPIN THE CALCIUM-DEPEN-
DENCE OF MEMBRANE REPAIR? Significantly, these studies sug-
4. Role in membrane repair gested that calpains were primary mediators of the calcium
dependence of membrane repair. CAPNS1-deficient MEFs
A) ACTIVATION OF CALPAIN IS VITAL FOR THE ACUTE MEMBRANE
did not show improved membrane repair outcomes in the
REPAIR RESPONSE. Interestingly, modulating calpain activity presence of calcium, in contrast to wild-type MEFs, or vi-
bestows both a blessing and a curse for cellular survival rally rescued CAPNS1 knockout MEFs. The calcium depen-
following membrane injury. Although treatment with cal- dence of membrane repair is a central dogma within the
pain inhibitors improves physiological outcomes and the membrane repair field, historically attributed to a require-
degree of cell and tissue death following pathologies of ment for calcium-dependent exocytosis (27, 267) and vesi-
membrane injury such as ischemia-reperfusion injury in the cle-vesicle fusion to facilitate membrane “patching” of
heart, stroke, and traumatic brain injury (317), activation plasma membrane disruptions (181, 285). Thus how do
of calpain is vital for the survival of an acute membrane calpains regulate calcium-dependent exocytic fusion under-
injury (183). pinning the membrane repair paradigm?

In 1991, Xie and Barrett (313) presented evidence showing A role for calpain-1 and -2 in membrane repair of adult
that calcium-activated proteases facilitated membrane re- cardiomyocytes was recently confirmed using a cardiac-spe-
sealing of transected mammalian neurites; calpain inhibi- cific knockout of CAPNS1 (281). Cardiac CAPNS1 null
tors strongly inhibited resealing of severed axons, suggest- mice showed normal heart histology and function at base-
ing a requirement of calpain activity for neurite sealing. A line, although developed symptoms of congestive heart fail-
role for calpains in neurite sealing was confirmed by Godell ure following a surgical protocol to constrict the aorta and
et al. (101), who demonstrated that application of exoge- induce pressure overload to the heart. Cardiac CAPNS1-
nous calpain restored the resealing capacity of crayfish me- null mice showed increased levels of fibrosis, poorer cardiac
dial giant axons (MGAs) in calcium-free media, and could contractility, and greater permeability to Evan’s Blue dye
also induce sealing in transected squid giant axons (GAs) uptake following the hemodynamic stress protocol. Iso-
that otherwise do not seal (with or without calcium). Godell lated cardiomyocytes subjected to the two-photon laser
et al. (101) showed that both crayfish MGAs and squid GAs damage assay (18) showed a significant defect in plasma
recruited vesicles to the transected end of the axon. In the membrane repair, although whether CAPNS1-null cardio-
presence of calcium, vesicles recruited to the end of tran- myocytes still possessed calcium-dependent membrane re-
sected crayfish MGAs fuse to form a dye-impermeable bar- pair was not examined (281).
rier, whereas vesicles in squid GAs do not. Treatment of
transected MGA neurites with calpain inhibitors did not Although calpain is known to cleave numerous substrates,
attenuate vesicle formation or recruitment, but prevented the functional specialization that calpain cleavage imparts
their fusion, in some cases inducing the formation of abnor- upon target substrates is largely unknown, with few excep-
mally large vesicles that failed to fuse with the cut axonal tions. Recent results from our own research illuminate dys-
end. The authors therefore proposed that calpains en- ferlin as a specific substrate of injury-induced calpain acti-
hanced the fusion of recruited vesicles to reseal transected vation, with a clear link to membrane repair. Since dysferlin
neurites and suggested squid GAs possess all of the machin- is proposed to be the “calcium-sensing vesicle fusion pro-
ery for effective resealing, although must lack sufficient ac- tein” for exocytic membrane repair (18, 181), could calpain
tive calpain to facilitate vesicle fusion. cleavage of dysferlin to release a synaptotagmin-like mod-

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1223


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

ule, mini-dysferlinC72, explain how calpains may regulate ␤-sheet sandwich of the C2 domain, the helix-loop-helix
the exocytic fusion of membrane repair? module of EF hands, and the superhelical annexin calcium
binding domain. Annexins bear a conserved domain struc-
5. Perspectives ture of a variable NH2-terminal domain and a COOH-
terminal core domain of four, tandem, calcium-binding re-
Calpain inhibition is being explored as a therapeutic strat- peats (eight in annexin VI) (98). Each repeat is ⬃70 amino
egy in numerous pathologies (see Ref. 44). The challenge acids in length, forming a superhelix of 5 inter-wound ␣-he-
with emerging calpain-inhibitor-based therapies is to spe- lices (158). The four superhelical domains of annexin pack
cifically target the appropriate calpain and to define the into a compact disc, slightly convex on one side and slightly
therapeutic window that permits the acute resealing re- concave on the other side. The calcium-coordinating resi-
sponse, but protects against the uncontrolled calpain activ- dues lie on the convex face of the disc and are highly evo-
ity that follows, leading to a negative spiral of deleterious lutionarily conserved (190). Thus the annexin core func-
calpain-mediated degradation of cellular proteins that re- tions as a four-domain module that binds negatively
sults in cytotoxicity. charged phospholipids via its calcium-bound convex sur-
face, with its concave surface facing the cytoplasm.
The superphysiological activating calcium concentration
for m-calpain has puzzled biochemists for the last two de- The annexin NH2-terminal domains vary in length and are
cades, and much effort has been devoted to deciphering thought to lie on the concave side of the core module where
molecular modifications that occur in vivo to lower its re- they confer specificity for different binding partners. In the
quired calcium concentration for activation. Membrane in- case of annexin A1, the NH2-terminal domain was shown
jury seems tailor-made for m-calpain; full activating cal- to be buried within the core of the molecule and released in
cium concentrations, discrete substrate specificity, and a a conformational shift with calcium binding (241). Thus
repertoire of protein substrates that one can easily fit into a calcium coordination by annexin A1 simultaneously medi-
model for membrane repair: 1) dysferlin and voltage-gated ates phospholipid binding of the convex face, and, effector
calcium channels for vesicle fusion; 2) matrix proteins and interaction by the NH2 terminus on the cytoplasmic con-
submembraneous cytoskeleton to allow delivery of exocytic cave face. For annexins A1 and A2, the unique NH2-termi-
vesicles for patch-repair as well as internalization of endo- nal domains also convey specificity for binding the EF-hand
cytic vesicles to remove oxidized lipids and damaged recep- accessory proteins S100A10 (annexin II) and S100A11 (an-
tors; 3) dystrophin, talin, and spectrin to severe adhesion nexin I) (156).
anchors and improve membrane flexibility to allow remod-
eling and resealing of large injuries; and 4) annexins to 2. Expression and localization
compartmentalize and order the resulting mish-mash of
lipid membranes delivered in the emergency response to
restore membrane integrity. Many annexins are ubiquitously expressed (A1, 2, 4, 5, 6, 7,
11), highly abundant proteins, although several annexins
On balance, several lines of evidence are converging to tri- show tissue-specific expression; for example, A3 in neutro-
angulate calpains as central regulators of membrane repair. phils, A8 in placenta and skin, A9 in the tongue, and A10
One begins to question whether dysferlin is indeed a master and A13 in the gastrointestinal tract (98). The term annexin
regulator of membrane repair, or one of several effectors is derived from annexare (Latin) and annexer (French)
activated by calpain during its boisterous response to the meaning to join, to connect, to attach; the primordial role of
unregulated influx of calcium that uniquely characterizes all annexins is thought to relate to their capacity to aggre-
the local environs of a membrane injury. gate and organize membranes in response to calcium.

Annexins generally show a cytoplasmic distribution, but


D. Annexins partition to discrete membrane compartments upon cal-
cium signaling. Studies of GFP-annexin fusion proteins
1. Evolution and structure have revealed that different annexins target different sub-
cellular membrane compartments with calcium signaling,
The annexins are an ancient family of calcium-sensitive and indeed, respond to different activating calcium concen-
phospholipid binding proteins (98). There are 12 annexins trations (78, 188). Annexins A1 and A2 shuffle between
in chordates (A1-A11, A13), with more than 500 members plasma membrane and endosomal compartments (170,
found across all major eukaryotic phyla (190). Like dysfer- 231), with emerging evidence for extracellular activities in
lin (152), annexins are also present in primitive unicellular anti-inflammatory and anticoagulation pathways (108,
protists and are absent from prokaryotes and yeast (190). 214). Annexin 5 localizes to several different compartments
of the biosynthetic pathway, including the endoplasmic re-
There are three major classes of structurally diverse calci- ticulum, Golgi, late endosomes, and nuclear envelope (21,
um-binding domains found in eukaryotic proteins: the 73, 225). Annexin A5 is also widely used as a marker of

1224 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

apoptotic cells via its specificity for calcium-dependent thought to be the basis for thrombosis and pregnancy loss in
binding to exposed phosphatidlyserine (139). human anti-phospholipid syndrome (226).

3. Role in membrane repair Annexin A5 displays rapid recruitment to injury sites in


damaged C2C12 myoblasts (40) and perivascular cells (37).
Annexins are becoming widely implicated in different as- Moreover, exogenous application of recombinant annexin
pects of membrane compartmentalization, stabilization, A5 improves membrane repair in wild-type perivascular
and remodeling associated with membrane repair (76). An- cells and rescues the membrane repair defect in annexin A5
nexin A1 interacts with dysferlin (154) and was the first of null perivascular cells (37). Through design of an annexin
the annexins to be implicated in membrane repair (176). A5 mutant unable to form two-dimensional arrays from
Annexin A1 translocates to the plasma membrane with in- assembled trimers, Bouter et al. (37) were able to demon-
jury (14, 176), and an anti-annexin A1 blocking antibody, a strate that formation of crystalline arrays is crucial for the
small peptide inhibitor, and a calcium-binding annexin A1 role of annexin A5 in membrane repair.
mutant were each shown to inhibit resealing in HeLa cells
subjected to mechanical scrape injury or laser irradiation C) ANNEXIN A6 PARTICIPATES IN SKELETAL MUSCLE MEMBRANE RE-
(176). Levels of annexin A1 are upregulated in patients with PAIR. Intravital imaging of annexins in the zebrafish model
muscular dystrophy (304), and annexin A1 labels vacuol- of sarcolemmal membrane repair also identified a role for
ized longitudinal membranes of the t-tubule network (a annexin A6 (238). Annexin A6 showed immediate (10 –20
response to stretch injury) in patients with muscular dystro- s), dysferlin-independent translocation to injury sites, tem-
phy (304) and statin myopathy (302). porally preceding recruitment of annexin 11a (40 s), an-
nexin 2a (60 – 80 s), and annexin 1a (200 –240 s). This is
A) ANNEXINS INCREMENTALLY RESPOND TO DIFFERENT DEGREES OF consistent with the temporal progression of annexin re-
CALCIUM INFLUX. Subsequent studies of SLO perforated cruitment to injury sites in SLO-treated HEK293 cells
HEK293 cells have revealed unique interplay between an- (219).
nexins 6 and 1 following membrane injury (14, 219). An-
nexin 6 shows higher sensitivity to elevations in intracellu- Morpholino knockdown of annexin A6 resulted in simi-
lar calcium, and rapidly and reversibly translocates to the lar, though more mild, symptoms to dysferlin morphants,
plasma membrane in cells suffering minor SLO perforation, with a curved trunk, myofibrillar misalignment, and a
inducing local hot spots of elevated calcium in the range of reduction in birefringence (238). Double knockdown of
5–10 ␮M. Annexin 6 specifically targets these injured re- both dysferlin and annexin A6 caused a severe pheno-
gions of the plasma membrane and is encapsulated together type, with all animals presenting with a curved trunk,
with SLO perforated membranes into microvesicles that are severe cardiac edema, myofibrillar disorganization, and
then shed by the cell. With more severe SLO perforation, almost absent birefringence (238). Knockdown of an-
cytosolic [Ca]i exceeds 10 ␮M and activates the less sensi- nexin A6, dysferlin, or both prevented injury-activated
tive annexin A1, which translocates to the nuclear envelope
accumulation of annexin A1, and slowed recruitment of
and plasma membrane and is associated both with mi-
annexin A2, with more profound effects observed with
crovesicle shedding of perforated membranes as well as in-
the double knockout. Collectively, results suggest that
ternalization of ceramide platforms (14). The careful work
dysferlin and annexin A6 are independently recruited to
of this research group has revealed cooperative roles of
injury sites, and work cooperatively to facilitate the sub-
annexins A1 and A6 that can incrementally respond to dif-
sequent accumulation of slower responding annexins A1
ferent degrees of membrane damage according to local ele-
vations in [Ca]i, and either package small lesions into shed and A2. These data are supported by studies in human
microvesicles, or alternately internalize larger areas of myotubes, where dysferlin rapidly recruits to injury sites
membrane damage demarked by ceramide plaques (76). and specifically labels the edges of the lesion, whereas
annexins A1 and A2 do not show enrichment until 30 s
B) ANNEXIN A5: A SHIELD FOR INJURY SITES? A recent study has postinjury, and do not specifically label injury sites but
shown that calcium-dependent binding of annexin A5 to instead show more generalized enrichment in a large zone
exposed phosphatidylserine plays a biological role in the around the ballistics lesion (150).
membrane repair response of damaged perivascular cells
(37). Annexin A5 is known to self-assemble into trimers Recently, annexin A6 has been identified as a potential dis-
that interconnect to form two-dimensional structural ar- ease modifier of muscular dystrophy, which is characterized
rays once bound to negatively charged phospholipids (206). by a high degree of pathological variability in human pa-
This property plays an important role in its anti-thrombo- tients. A truncated version of A6 was found in a genome-
lytic and anticoagulant activity (237, 278, 286). Maternal wide scan of mice, and this abnormal A6 was shown to
anti-annexin A5 autoantibodies that disrupt this crystalline interfere with membrane repair by disrupting the recruit-
annexin A5 anticoagulant shield that covers the placenta is ment of normal A6 to disruption sites (277).

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1225


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

4. Perspectives hetero- and homo-oligomerization (130, 232). TRIM pro-


teins play diverse roles in development, differentiation, on-
The ancient phylogeny of the annexins, their high abun- cogenesis, and immunity (175, 197, 208). Phylogenetic
dance, and their ability to stabilize and order phospholipid studies of TRIM-family specific B-box sequences reveal the
membranes makes them good candidates as primordial me- presence of this domain in protozoa and unicellular eu-
diators of a membrane repair response. It makes sense that karyotes, but the tripartite motif that characterizes the
cells will activate and employ different coping mechanisms, TRIM family E3 ligases is exclusive to multicellular meta-
depending on the location and extent of the membrane zoans (249).
injury, whether that be rapid shedding of microvesicles or
internalization of larger sections of the bilayer with more The NH2-terminal tripartite motif is generally thought to
substantial damage. Ceramide accumulation at annexin collectively mediate E3 ligase activity of TRIM proteins,
A1-labeled injury sites, or crystalline annexin A5 arrays whereas the COOH-terminal motif is though to confer sub-
may function threefold: to provide a mechanism for the cell strate specificity and varies throughout the TRIM protein
to recognize a damage site and target endocytic machinery, family. MG53 (TRIM72) is a group 2 TRIM protein bear-
to provide a scaffold to aide in the recruitment and assem- ing a COOH-terminal PRY-SPRY domain (249). This class
bly of other repair and remodeling factors, and, more in- of TRIM proteins are evolutionary newcomers, present
triguingly, perhaps to provide a temporary shield to delay only in chordates and showing rapid gene expansion in
an immune or apoptotic response to allow time for repair. humans. The PRY-SPRY domain of MG53 has been crys-
tallized, revealing a compact globular structure character-
Although calcium signaling may provide the ultimate ized by a convex surface on one side and a concave surface
“membrane injury” signal to activate a tailored response by on the other that includes a large binding pocket (212).
different cytoplasmic annexins in many cells and tissues
throughout the body, it is difficult to reconcile how such a 2. Role in human disease
mechanism works effectively in the muscle myoplasm. The
cytoplasm of contractile muscle cells is routinely flooded
A) MG53. MG53 has not been implicated in human muscle
with extraordinarily high levels of calcium during contrac-
tion; ⬃1–3 ␮M cytoplasmic calcium with ⬃100 ␮M locally disease but shows upregulation in patients with muscular
at the triad junction (23, 148). These cytoplasmic calcium dystrophy (304). Recently, MG53 has been implicated in
concentrations would rarely be encountered in other cell the metabolic syndrome that results in type II diabetes and
types, except in the local environs of specific calcium signal- insulin resistance (261). Levels of MG53 were elevated in
ing or a small membrane rupture. Perhaps it is this specific animal models with insulin resistance and metabolic syn-
property of muscle that confounds an ancestral membrane drome, as well as in obese humans. Upregulation of MG53
repair response, which perhaps requires further refinement in skeletal muscle via transgenic overexpression caused
by muscle-specific effectors such as MG53. whole body insulin resistance and metabolic syndrome,
whereas mice deficient in MG53 were protected from the
metabolic effects of high fat feeding. Biochemical studies
E. TRIM72/MG53 showed that MG53 overexpression induced ubiquitination
and degradation of the insulin receptor and the insulin re-
1. Evolution, structure, and localization ceptor substrate IRS1, therefore functioning as a muscle-
specific negative regulator of insulin signaling (261).
Mitsigumin-53 (MG53) was first identified as part of an
immunoproteomic study to derive monoclonal antibodies B) OTHER TRIM FAMILY PROTEINS. Mutations in several other
against skeletal muscle triad proteins, mitsigumin being the TRIM-family ubiquitin ligases cause different forms of in-
Japanese word for triad. MG53 is highly expressed in skel- herited disorders in humans. MID1 (midline-1) causes
etal and cardiac muscle and immunolocalizes to the t-tu- Opitz Syndrome (223), and TRIM37 causes Mulibrey
bule/sarcoplasmic reticulum triad junction in murine mus- (muscle-liver-brain-eye) nanism (12); both are developmen-
cle (308), with more variable cytosolic, sarcolemmal, and tal disorders affecting several organs and tissues. Interest-
t-tubule labeling in human skeletal muscle fibers (304). ingly, another TRIM protein is implicated in the pathogen-
MG53 is a member of the TRIM family of E3 ubiquitin esis of muscular dystrophy in humans. Mutations in
ligases and is also known as TRIM72. TRIM32 cause an autosomal recessive muscular dystrophy
(LGMD2H) (92), although the disease mechanism is un-
There are more than 70 TRIM family proteins in mammals, known. TRIM32 belongs to a more ancient subgroup of
thus named due to their characteristic NH2-terminal tripar- TRIM proteins present in invertebrates, but shows only
tite motif, a RING finger domain conferring ubiquitination weak amino acid identity to MG53 and does not contain
activity, one or two zinc-binding B-box domains; TRIM- the COOH-terminal PRY-SPRY domain. It remains to be
family specific sequences thought to mediate protein-pro- determined whether TRIM32 mutations impact skeletal
tein interactions, and a coiled-coiled domain that mediates muscle membrane repair in affected patients.

1226 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

3. Role in membrane repair unregulated calcium influx, many plasma membrane pro-
teins and receptors will be damaged by oxidation and cal-
In 2009, a MG53 knockout mouse was derived and found pain cleavage. Therefore, it seems likely that the trigger for
to show exercise intolerance with downhill running and MG53 recruitment relates to its role as a ubiquitin ligase,
increased muscle damage detected by Evans Blue dye up- utilizing existing trafficking machinery that ubiquitin li-
take (40). Closer examination revealed histopathological gases transit to rapidly label target substrate(s) for endocy-
signs of a mild, progressive muscular dystrophy. Membrane tosis and degradation.
damage assays using laser irradiation and microinjection
revealed that MG53 was rapidly recruited to sites of mem- The recent discovery of the insulin receptor as a specific
brane injury in a calcium-independent manner and that substrate of MG53 ubiquitin ligase activity (261) is not
MG53⫺/⫺ null myofibers and myotubes possessed a pri- easily reconciled with the role of MG53 in membrane repair
mary defect in membrane resealing. MG53 was shown to (40). It is possible that MG53 has several ubiquitination
oligomerize via cysteine 242, with mutation of C242 to targets and that one of these additional targets plays an
alanine ablating MG53 injury recruitment and membrane important role in the response to, and the signaling of, a
repair activity (40). MG53 was thus proposed to be an membrane injury. Given the ancient eukaryotic origins of
oxidative sensor of membrane injury, forming an oligomer- other proteins implicated in membrane repair (SNAREs,
ized scaffold for assembly of the membrane repair complex ferlins, annexins, and calpains), one might suppose that the
(for review, see Ref. 177). ubiquitination target of MG53 belongs to an ancient recep-
tor or channel family, which has perhaps evolved a chor-
MG53 was shown to coimmunoprecipitate dysferlin and date-specific or tissue-specific isoform that MG53 has re-
the muscle-specific caveolin-3 (42). Caveolins are the major cently evolved to specifically target.
protein constituents of membrane caveolae, and mutations
in caveolin-3 cause a form of autosomal dominant muscular
dystrophy (LGMD1C) (186). A Golgi-retained muscular F. Late Endosomal Pathway And Membrane
dystrophy mutant of caveolin-3 (P104L) caused coaccumu- Repair
lation of dysferlin and MG53 in the Golgi apparatus of
skeletal myofibers, resulting in defective membrane repair Recent research is expanding the body of evidence implicat-
(42). Overexpression of MG53 in C2C12 mouse myoblasts ing machinery of the late endosomal pathway in membrane
was shown to activate membrane exocytosis, inducing repair, including components of the endosomal sorting
masses of filopodic extensions (41), a process reversible by complex ESCRT-III (129, 250) and an endolysosomal tran-
coexpression of caveolin-3. Collectively, these findings im- sient receptor potential (TRP) cation channel, mucolipin-1
plicated functional interplay between MG53, caveolin-3, (TRPML-1) (49).
and dysferlin in membrane trafficking events required for
membrane repair. MG53 recruitment and anchorage at 1. ESCRT
sites of membrane injury have since been to shown to re-
quire the cytoskeletal motor nonmuscle myosin IIA (160) A) EVOLUTION, STRUCTURE, AND LOCALIZATION. ESCRT (endo-
and caveolae regulatory protein PTRF (polymerase I and somal sorting complexes required for transport) plays a key
transcript release factor, also known as cavin-1) (319). role in the endocytic trafficking and lysosomal targeting of
ubiquitinylated transmembrane receptors and proteins (for
Application of recombinant MG53 can improve resealing recent reviews, see Refs. 110, 224). ESCRT proteins were
outcomes when applied to the extracellular media bathing first identified in yeast, as class E vacuolar protein sorting
dysferlin-deficient myofibers and can improve dystrophic (Vps) genes, required for sorting of transmembrane pro-
pathologies when systemically delivered to the mdx mouse, teins into intraluminal endosomal vesicles (228, 298). The
a model of Duchenne muscular dystrophy (309). MG53 has ESCRT complex is comprised of five subcomplexes: ES-
also been implicated in the response to membrane injury in CRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and the VPS4 AT-
the heart (43, 306). Pase, and it works in concert with a wide range of adaptor
proteins. Components of ESCRT complexes-I, -II, -III, and
4. Perspectives the VPS4 ATPase are conserved throughout eukaryotic evo-
lution, present in protozoan parasites through to metazoa
MG53 is expressed only in chordates, with no ortholog (155). Furthermore, there is evidence for ancestral ESCRT-
identified in chicken or zebrafish (249). As membrane repair III proteins and the VPS4 ATPase in archae-bacteria (89,
is a feature of both vertebrate and invertebrate cells, one 203). However, ESCRT-0 is found only in Opisthokonts
must consider that MG53 is not an ancestral membrane (animal and fungi kingdoms) and is not present in primitive
repair component, although it may have assumed the func- eukaryotes (155).
tion of an evolutionarily more ancient predecessor or per-
haps lent a tissue-specific specialization to the membrane The ESCRT complexes are best known for their role in
repair response. In the setting of a membrane injury with endosomal sorting, whereby ubiquitinylated receptors are

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1227


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

endocytosed into clathrin-coated endosomes, clustered A subsequent study used a proteomics approach to identify
within the endosomal membrane, then partitioned into proteins exocytosed to the plasma membrane after an acute
membrane compartments that invaginate and bud-off into elevation in submembraneous Ca2⫹ (using the Ca2⫹ iono-
the endosomal lumen to form multivesicular bodies (MVBs) phore ionomycin), and highlighted several components of
(204). MVBs then fuse with lysosomes, resulting in the the ESCRT-III machinery (250). Importantly, Scheffer et al.
eventual degradation of the ubiquitinylated cargo (117). (250) established that the ALIX-binding protein ALG-2
ESCRT-0 bears a ubiquitin-interacting domain and a FYVE (apoptosis-linked gene-2), a penta-EF hand protein, pro-
domain that specifically recognizes phosphatidylinositol vides the Ca2⫹-dependent component for sequential re-
3-phosphate lipids highly enriched within the endosomal cruitment and assembly of the ESCRT-III complex (250).
membrane (95, 144). ESCRT-0 is thought to recognize the Using siRNA knockdown of ALG-2, ALIX, and Vsp4
ubiquitinylated cargo within the endosomal membrane and ATPase, they demonstrated each of these components was
facilitate recruitment of ESCRT-I, which in turn mediates required for repair, closure, and exosomal shedding of
interaction with ESCRT-II. The serial assembly of ES- plasma membrane injuries. By studying the capacity of each
CRT-0, -I and -II complexes provides the platform for as- knockdown cell line to recruit the different components of
sembly of ESCRT-III, the core workhorse of the ESCRT the ESCRT-III machinery, a model emerged whereby
machinery that forms filamentous circular arrays to drive ALG-2 responds to the Ca2⫹ influx and facilitates recruit-
deformation of the endosomal membrane and formation of ment of its binding partner ALIX. In turn, ALIX mediates
intraluminal endosomal vesicles (109, 224). The VPS4 the sequential assembly of CHMP proteins, then the Vsp4
ATPase disassembles the ESCRT-III machinery and facili- ATPase (250). The ESCRT-III machinery deforms the
tates recycling of its protein constituents (244). plasma membrane into outwardly forming buds that are
pinched off to close and/or remodel the wound, with abscis-
In addition to a role in endosomal sorting, components of sion steps involving the ESCRT Vsp4-ATPase (129, 250).
ESCRT play key roles in viral budding (269, 303) and the
C) PERSPECTIVES. Interestingly, although data presented by
abscission step of cytokinesis (189, 246, 265) (for recent
Jimenez et al. (129) suggest ESCRT-III machinery does not
reviews, see Refs. 171, 174, 245).
influence the closure of wounds larger than 100 nM, Schef-
fer et al. (250) found cells with ⬎90% knockdown of
B) ROLE IN MEMBRANE REPAIR. Recently, membrane fission
ALG-2, ALIX, and Vsp4 ATPase were more vulnerable to
roles of the ESCRT complex have been implicated in the
larger laser injuries (⬃1 ␮M) than control cells. Thus
closure of small wounds to the plasma membrane (129)
whether ESCRT-III plays a greater role in repair of smaller
(FIGURE 4). Using several modes of membrane injury (mi-
versus larger injuries requires further clarification. Impor-
croinjection, laser ablation injury, pore formation), Jimenez
tantly, Scheffer et al. (250) showed ESCRT-III plays a role
et al. (129) show recruitment to injury sites of several mem-
in membrane repair in C2C12 myoblasts and primary myo-
bers of the ESCRT-III complex (CHMP4, 3, 2A, and 2B),
fibers, and thus is a ubiquitous response, and not a pecu-
the Vsp4 ATPase, and the adaptor protein ALIX known to liarity of the immortal HeLa cell line. Notably, both studies
facilitate assembly of ESCRT-III in the absence of ESCRT-I carefully demonstrated that recruitment of ESCRT-III ma-
and II complexes. Interestingly, recruitment of ESCRT pro- chinery was not accompanied by lysosomal exocytosis at
teins was calcium-dependent, but not accompanied by ve- the site of injury, although lysosomal exocytosis was ob-
sicular recruitment of late endosomes/lysosomes, and was served to occur distally in response to the membrane injury
not blocked by microtubule deploymerization. These data (129, 250).
suggest ESCRT proteins are not delivered via vesicular
transport but are recruited as soluble, cytoplasmic proteins. Although one begins to sniff a tantalizing potential link
between the E3 ubiquitin-ligase MG53 (TRIM72) and ES-
Using quantitative analyses of live imaging data combined CRT machinery in membrane repair, immunolabeling for
with scanning electron microscopy, the authors revealed the polyubiquitination of plasma membrane proteins following
ESCRT proteins play a role in repair of small lesions ⬍100 injury suggested assembly of ESCRT-III preceded polyubiq-
nM, including small lesions made by bacterial pore-forming uitination events (129). Again, Ca2⫹ provides a key trigger
toxins. Repair and removal of the bacterial pores or small for ESCRT-III exosomal shedding for membrane repair, via
microinjuries are related to outward budding of the deco- ALG-2. On this basis, it will be important to determine
rated wounds and pinching-off via exocytic shedding (129) whether other settings of ESCRT-mediated fission are also
(see FIGURE 4). The capacity to survive the membrane injury Ca2⫹-dependent and, furthermore, whether the concentra-
and shed ESCRT-positive exosomes were both ATP-depen- tion of calcium required to activate recruitment of ESCRT-
dent, suggesting the Vsp4 ATPase may be required to dis- III machinery to injury sites is consistent with concentra-
assemble the ESCRT-III complexes so the protein constitu- tions required to activate calcium-dependent membrane re-
ents can be recycled, and/or play a direct role in the pinching pair in mammalian cells [⬃150 ␮M in human muscle cells
off the exocytic buds (129). (230) and ⬃300 ␮M in 3T3 fibroblasts (267)].

1228 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

Yet to be determined and vital for this field are the connec- ⬎5 mo of age) (49). Skeletal muscle fibers isolated from
tions and interplay between the rapid Ca2⫹-dependent ex- mucolipin-1⫺/⫺ mice show a major defect in Ca2⫹-depen-
ocytosis triggered in the immediate surrounds of a mem- dent membrane repair, characterized by defective lysosomal
brane injury (⬍10 s; Ref. 150), the rapid compensatory exocytosis. Similarly, when mucolipin-1 channels are
endocytosis (57, 123, 173), the slower phase of lysosomal blocked in C2C12 myoblasts or MEFs using synthetic in-
exocytosis at regions distal to the injury site (126, 229), and hibitors, membrane repair is inhibited and lysosomal exo-
the vesicle-independent accumulation of ESCRT-III ma- cytosis is reduced. Thus lysosomal Ca2⫹ flux via mucoli-
chinery for exosomal shedding of the injured membrane pin-1 is an important event for both membrane repair and
(49, 129). for lysosomal exocytosis. The importance of lysosomal
Ca2⫹ stores was supported through treatment with glycyl-
2. Mucolipin-1 L-phenylalanine 2-naphthylamide (a substrate hydrolysed
by cathepsin C leading to lysosomal osmotic injury and
A) EVOLUTION, STRUCTURE, AND LOCALIZATION. Mucolipin-1 depletion of Ca2⫹), and BAPTA-AM (a cell-permeable,
(also known as TRPML-1) is one of three members of the rapid, and highly effective Ca2⫹ chelator). Both treatments
vertebrate mucolipin-subfamily of the TRP (transient recep- inhibited membrane repair, suggesting intracellular Ca2⫹,
tor potential) cation channels, a superfamily of nonselective and lysosomal Ca2⫹ in particular, is important for mem-
cation channels bearing six transmembrane domains (S1– brane repair. Lastly, electrophysiological analyses of wild-
S6) (299). Functional TRP channels are homo- or hetero- type cells reveals an increase in whole cell mucolipin-1 cur-
oligomers of four subunits, with a pore domain located rents following membrane perforation with SLO, implying
between transmembrane domains S5 and S6 consisting of
mucolipin-1 channels become incorporated into the plasma
negatively charged glutamate and aspartate residues to con-
membrane following lysosomal exocytosis in injured cells.
fer cation selectivity (157). TRP channels are best known
for their roles as sensory channels for photoreception, pro-
C) PERSPECTIVES.
This study provides additional evidence that
prioception, mechanosensation, nociception, and ther-
lysosomal exocytosis plays an important role in membrane
mosensation (299). Members of the TRP superfamily are
repair. Mucolipin-1 mediates lysosomal Ca2⫹ release in re-
present in yeast through to metazoa, and although TRP-like
sponse to injury, and this Ca2⫹ release is required for nor-
genes are identified in unicellular phytoplankton, TRP-like
mal lysosomal exocytosis and membrane repair. In the con-
genes are absent in plants (201). The mucolipin subfamily
text of membrane repair, it is not yet clear whether lyso-
of TRP channels emerged as a single gene in invertebrates,
somal Ca2⫹ release mediated by mucolipin-1 is required for
with gene duplication events producing three mucolipin
hetero- or homotypic fusion of endolysosomal compart-
genes in vertebrates (201). The mucolipin-1 channel local-
izes to Rab-7-positive late endosomal and lysosomal com- ments (215, 222) that precede lysosomal exocytosis, or
partments and is permeable to Ca2⫹ and Fe2⫹ (75). whether it provides a specific Ca2⫹ flux important for dock-
ing and fusion of lysosomes with the plasma membrane.
B) ROLE IN DISEASE. Mutations in the mucolipin-1 gene Notably, mucolipin-1 is a binding partner of ALG-2 (300),
(MCOLN-1) cause an autosomal recessive lysosomal stor- potentially providing a key link to the ESCRT-III mem-
age disease, mucolipidosis type IV (20). Mucolipidosis type brane repair pathway.
IV is a devastating neurodegenerative disorder character-
ized by progressive intellectual disability and motor dys- In addition to the endoplasmic reticulum, mitochondria
function, muscle weakness, retinal degeneration and cloud- and lysosomes are major reservoirs of Ca2⫹, with intralu-
ing of the cornea, and anemia due to deficient acid secretion minal lysosomal Ca2⫹ around 0.4 – 0.6 ␮M (52). One pro-
in the gut (acid is required for iron absorption), likely con- vocative possibility is whether the dystrophic pathology of
founded at the cellular level by defects in iron-conducting mucolipin-1⫺/⫺ mice may relate to defects in Ca2⫹ seques-
properties of the mucolipin-1 channel itself. Abnormally tration, or more intriguingly, Ca2⫹ extrusion. Persistent
swollen and enlarged lysosomal compartments filled with elevation of intracellular Ca2⫹ in skeletal muscle, induced
membrane and protein aggregates are a feature of patients by transgenic overexpression of the stretch-activated
with mucolipidosis type IV, suggesting abnormal matura- TRPC3 channel, induces a severe muscular dystrophy phe-
tion and fusion of endolysosomal organelles (48, 147). notype (185). This study shows that development of a mus-
cular dystrophy phenotype is strongly related to unregu-
C) ROLE IN MEMBRANE REPAIR. Somewhat unexpectedly, the lated Ca2⫹ influx. If, like mitochondria, lysosomes play a
major presentation of a mouse knockout model of mucoli- role in Ca2⫹ sequestration following membrane injury, then
pin-1 is an early-onset progressive muscular dystrophy, levels of lysosomal Ca2⫹ will increase in the seconds and
characterized by abundant internal nuclei, fibrotic and in- minutes following membrane injury. Despite the vast Ca2⫹-
flammatory infiltrate, and elevated serum creatine kinase handling capabilities of the sarcoplasmic reticulum, it is
(49). Dystrophic pathology is apparent as early as 1 mo of conceivable that lysosomal exocytosis triggered by mem-
age, preceding overt histopathological symptoms of lyso- brane injury may also play some role in Ca2⫹ extrusion, to
somal storage disease or neuronal degeneration (apparent help purge the cell of unwanted Ca2⫹, protect mitochondria

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1229


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

from Ca2⫹ overload, and protect a cell from deleterious are present in virtually all eukaryotes, as are microtubule-
calpain overactivity. dependent motors, the kinesins. Indeed, structural similar-
ity between that catalytic core of myosin and kinesin sug-
gests a common evolutionary ancestor of both ATPase mo-
G. Cytoskeletal Networks tor domains (141).

1. Structure and evolution 2. Role in human disease

Eukaryotic cells are differentiated from their prokaryotic Mutations in tubulin, actin, and myosin underpin a broad
and archaeal ancestors not only by their endomembrane spectrum of inherited disorders in humans. Tubulin muta-
system, but also by their complex cytoskeleton. Prokaryotic tions have recently been implicated in inherited neurode-
cells do not possess direct homologs of the major cytoskel- generative disorders, consistent with the vital role of the
etal proteins or tubulin or actin, although they express di- microtubule cytoskeleton for neuronal function (134, 290).
vergent filamentous proteins with remarkable structural Mutations in members of the actin and myosin gene families
similarity to eukaryotic tubulin (bacterial FtsZ; Refs. 69, cause a wide range of clinical defects, typically affecting
227) and actin (bacterial MreB; Ref. 36). A dynamic cyto- tissues and organs where they are dominantly expressed
skeleton underpins eukaryotic cell division, cell shape, mo- (63). For example, mutations in skeletal actin (ACTA1) and
tility, as well as the capacity to phagocytose nutrients for myosin (MYH7 and MYH2) cause a skeletal myopathy,
growth and secrete signals for cellular communication. mutations in cardiac actin (ACTC1) and myosin (MYH7)
cause cardiomyopathy, and mutations in smooth muscle
Isoforms of tubulin and actin are present in every phyla of actin (ACTA2) and myosin (MYH11) cause thoracic aortic
the eukaryotic kingdom, together with their molecular mo- aneurysms and dissections. Mutations within nonmuscle
tors kinesin/dynein and myosin (for reviews, see Refs. 82, members of the actin and myosin gene families can induce
311). Tubulin microfilaments possess an inherent instabil- deafness, blindness, and other neurological and immune
ity intrinsic to their dynamic function. Mature microtubules cell dysfunction, depending on the isoform involved. Cur-
typically consist of 13 protofilaments that interact to form a rently, no pathology caused by mutations in cytoskeletal
hollow cylinder. Each protofilament results from the po- proteins has been linked directly to defects in membrane
lymerization of GTP-bound ␣- and ␤-tubulin heterodimers; repair.
free GTP at the growing (plus) end of the filament promotes
further polymerization, whereas hydrolysis of GTP to GDP- 3. Role in membrane repair
tubulin exerts a structural and energetic change that renders
the microtubule prone to deploymerization (83). Dynamic A role for cytoskeletal motors in membrane repair was pro-
microtubules form mitotic spindles for cell division and posed by Xie and Barrett (313) in the early 1990s, who
cytoskeletal highways for vesicular transport via their mo- revealed that microtubule destabilizing agents (colchicine)
lecular motors dynein and kinesin. promoted resealing of transected neurons, whereas micro-
tubule stabilizing agents (taxol) inhibited resealing. Dis-
Actin is one of the most highly conserved eukaryotic pro- creet roles for microtubule/kinesin and actin/myosin mo-
teins, showing ⬃85% identity between human, yeast, and tors for membrane repair was elaborated upon in greater
plant actin isoforms. Actin filaments consist of two strands detail by Bi et al. (28), who presented evidence for three
of polymerized actin, interwound into a tight right-handed phases of vesicle fusion associated with membrane repair of
helix (74). Actin filaments are not as intrinsically unstable embryonic sea urchin cells: immediate, fast, and slow
as microtubules, but nevertheless are capable of rapidly phases. Immediate exocytosis (⬍5 s) that occurred in re-
polymerizing and depolymerizing through an active tread- sponse to membrane wounding was insensitive to both ki-
milling process where GTP-bound actin monomers are nesin and myosin inhibitors, and presumed to involve pre-
added to the plus end of the growing filament and removed docked vesicles that did not require kinesin or myosin mo-
from the minus end of the filament, regulated by conserved tors for delivery or fusion. The next “fast” phase (5–15 s) of
families of monomer-binding factors, capping proteins, fil- vesicle fusion was sensitive to myosin inhibitors, but not
ament-stabilizing proteins, and actin-severing proteins. Ac- kinesin inhibitors, suggesting a local pool of vesicles that
tin-myosin contractile cycling is best characterized in skel- can quickly respond to the calcium flux of membrane in-
etal and cardiac muscle, with nonmuscle isoforms of actin jury. The third “slow” phase (20 – 60 s post injury) of ves-
and myosin playing vital roles in cytokinesis, cell motility, icle fusion was sensitive to kinesin inhibition, and also to
and endomembrane transport (34). myosin inhibition, suggestive of longer distance recruitment
of vesicles trafficked along microtubules toward the site of
Myosins are also an evolutionary ancient gene family, char- membrane injury.
acterized by the myosin head domain that encompasses a
powerful ATPase that utilizes ATP hydrolysis to power Inhibition of kinesin motor activity was similarly shown to
movement along an actin filament. Myosin head proteins impair membrane resealing of mammalian Swiss 3T3 fibro-

1230 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

blasts (267). Treatment with the actin depolymerizing agent tile actomyosin ring mediating wound closure. Interest-
DNAse I improved resealing of gastric epithelial cells, ingly, this study revealed evidence for periodic connections
whereas actin filament stabilizing agents phalloidin and jas- between the actomyosin ring and the circumference of the
plakinolide strongly inhibited resealing (187). There is wound sites through interactions mediated by DE-cadherin,
some discrepancy regarding the effects of cytochalasin D, suggesting that final stages of wound closure and remodel-
an actin severing agent, shown to exert inhibitory (urchin ing following injury occurs via tethering of the contractile
embryos, Ref. 28), facilitative (3T3 fibroblasts, Refs. 123, machinery to stable adhesive connections with the plasma
292), or no effect (gastric epithelia, Ref. 187) on membrane membrane.
resealing from a single wound. However, collectively, evi-
dence using different actin stabilizing and destabilizing 4. Perspectives
agents in different models suggests local deploymerization
of subcortical actin facilitates the vesicle fusion of mem- The importance of both the actin cytoskeleton (172) and
brane repair. microtubule/kinesin motors (173) has been confirmed for
membrane resealing of murine skeletal myofibers, and it is
Further research later refined a specific role for nonmuscle very likely that acto-myosin contractile rings and microtu-
myosin IIB in wound-activated exocytosis and membrane bule transport networks underpin plasma membrane repair
repair of injured 3T3 fibroblasts (293). Knockdown of non- and remodeling in all of eukaryota. Whereas endocytosis
muscle myosin IIA did not affect exocytosis or membrane and exocytic shedding provide an effective means for re-
repair, but rather inhibited facilitated resealing, a more moval and repair of small injuries (14, 129, 164), larger
rapid resealing response to a second injury at the same site injuries present a more complicated problem for resealing.
due to vesicle formation and recruitment from endomem- As one reflects upon the central themes of membrane re-
brane organelles such as the trans-Golgi network (TGN). pair– calcium, vesicle fusion, calpains, cytoskeletal remod-
eling, contractile rings–there is remarkable complementar-
ity and consistency among major findings by different re-
A) AN ACTO-MYOSIN RING FOR MEMBRANE REPAIR. A series of
search groups, despite huge variance in experimental
elegant imaging experiments in Xenopus oocytes revealed
models and approaches. Acknowledging the oversimplifica-
evidence for complex interplay between acto-myosin and
tion of what is a complex and incompletely understood
microtubule cytoskeletal motors for wound repair (25, 168,
pathway, as well as the caveats of comparison between
169). Xenopus oocyte injury sites were shown to be encir-
different model systems, the timelines separately proposed
cled by a ring of nonmuscle myosin II positioned at the
by individual research groups fit surprisingly well into a
inside edge of a wider band of actin. This acto-myosin ring
collective model for membrane repair and makes for inter-
was surrounded by a radial array of microtubules that were
esting contemplation.
integrally associated with actin filaments and indeed ap-
peared to be pulled into the active zone of actin polymer-
ization by the acto-myosin motors themselves (168). In IV. LITERATURE OVERVIEW: A
turn, the microtubule networks controlled the breadth of COLLECTIVE MODEL FOR REPAIR OF
the zone of actin assembly, facilitating formation of the LARGE MEMBRANE INJURIES
actin-myosin II contractile ring that gradually contracts to
seal the wound. Inhibition of either acto-myosin or micro- A. 0 –10 s
tubule motor systems impaired wound healing and cell sur-
vival. Importantly, wounds made in calcium-free medium Calcium floods down a steep concentration to create a mi-
failed to reseal, with injected fluorophores leaking out and croenvironment of high local intracellular calcium, activat-
failing to successfully label cytoskeletal compartments. This ing calcium-activated signaling molecules and calpains,
suggests that formation of the acto-myosin contractile ring which cleave dysferlin and other cytoskeletal and plasma
occurs after formation of a membrane barrier that is depen- membrane substrates. In skeletal muscle cells, MG53 re-
dent on extracellular calcium. sponds to an unknown signal and is rapidly recruited to
injury sites. The immediate and fast phases of vesicle fusion
These studies were mirrored in wounded early Drosophila occur, utilizing predocked and local vesicle pools, including
embryos that do not undergo cytokinesis for the first few nearby lysosomes. Dysferlin is endocytosed from regions
nuclear divisions and exist as large syncytial cells. Both distal to the injury site, and dysferlin is delivered to the
actin and myosin showed rapid accumulation at injury sites plasma membrane in cytoplasmic vesicles, perhaps trigger-
(from ⬃30 s after injury), aligning in concentric circles that ing their fusion, and upon integration into the plasma mem-
gradually contract to reseal the injury (2). Resealing was brane intensely labels the circumference of injury sites.
shown to occur via discrete stages of expansion, contraction There is reduced labeling for filamentous cortical actin sur-
and closure. Immediately after wounding, the lesions ex- rounding the membrane injury, due to proteolytic cleavage
pand until actin is observed to accumulate at the periphery. of membrane tethers by calpain and actin deploymeriza-
Myosin II accumulated at the wound edge forms a contrac- tion. Lesions expand.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1231


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

B. 10 –30 s needed to treat the huge spectrum of human disorders char-


acterized by membrane injury.
The slow phase of vesicle fusion begins, requiring cytoskel-
etal transport of vesicles upon microtubule and actin-myo- ACKNOWLEDGMENTS
sin motors. ALG-2 and annexins A6 and A11 are delivered
to injury sites. Calcium influx continues to trigger exocytic Address for reprint requests and other correspondence:
fusion of lysosomes in zones surrounding the lesion site. S. T. Cooper, Discipline of Paediatrics and Child Health,
Calpains cleave cytoskeletal networks and adhesion pro- Univ. of Sydney, Institute for Neuroscience and Muscle
teins. Lesions continue to expand. Research, The Children’s Hospital at Westmead, Locked
Bag 4001, Sydney, NSW 2145, Australia (e-mail: Sandra.
cooper@sydney.edu.au).
C. 30 – 60 s
GRANTS
Actin accumulates at the wound periphery, forming a dy-
namic zone of rapidly polymerizing and depolymerizing S. T. Cooper is supported by an Australian National Health
actin. Actin filaments interconnect with local polymerizing and Medical Research Council (NHMRC) Career Develop-
microtubule filaments, pulling them toward the wound site ment Fellowship APP1048816, NHMRC Project Grant
and anchoring these microtubule highways in position for APP1048814, and funding from the Jain Foundation. P. L.
rapid transport of vesicles, signaling proteins and cytoskel- McNeil was supported by National Institutes of Health
etal remodeling proteins. Microtubules in turn stabilize the Grants AR-060565 and DK-090703.
active zone of actin polymerization to facilitate formation
of an acto-myosin contractile ring. ALIX, CHMP, and Vsp4 DISCLOSURES
ATPase ESCRT-III machinery sequentially accumulate at
injury sites. Acid sphingomyelinase released by lysosomal No conflicts of interest, financial or otherwise, are declared
exocytosis hydrolyzes sphingomyelin to form ceramide-rich by the authors.
platforms to activate endocytosis. Damaged plasma mem-
brane is removed by endocytosis and exosomal shedding.
Lesions contract. REFERENCES

1. Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG. A pore-forming toxin
D. 60 –240 s interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic
reticulum. J Cell Biol 140: 525–540, 1998.

The acto-myosin ring gradually contracts and closes, rees- 2. Abreu-Blanco MT, Verboon JM, Parkhurst SM. Cell wound repair in Drosophila occurs
through three distinct phases of membrane and cytoskeletal remodeling. J Cell Biol
tablishing the network of cytoskeletal connections beneath 193: 455– 464, 2011.
the wounded plasma membrane. Annexins A1 and A2 ac-
3. Achanzar WE, Ward S. A nematode gene required for sperm vesicle fusion. J Cell Sci
cumulate at injury sites. Phases of exocytosis, endocytosis 110: 1073–1081, 1997.
and exosomal shedding remodel the hastily repaired plasma
membrane barrier, replacing damaged proteins and recep- 4. Adibhatla RM, Hatcher JF. Lipid oxidation and peroxidation in CNS health and disease:
from molecular mechanisms to therapeutic opportunities. Antioxidants Redox Signal
tors, and reconstituting the normal repertoire of plasma 12: 125–169, 2010.
membrane lipids and microdomains.
5. Allen DG. Eccentric muscle damage: mechanisms of early reduction of force. Acta
Physiol Scand 171: 311–319, 2001.

V. CONCLUDING REMARKS 6. Allen DG, Gervasio OL, Yeung EW, Whitehead NP. Calcium and the damage path-
ways in muscular dystrophy. Can J Physiol Pharmacol 88: 83–91, 2010.

It seems no accident that membrane repair uses ancient and 7. Ampong BN, Imamura M, Matsumiya T, Yoshida M, Takeda S. Intracellular localiza-
robust molecular machinery, such as contractile rings and tion of dysferlin and its association with the dihydropyridine receptor. Acta Myol 24:
134 –144, 2005.
vesicle fusion; these are fundamental biological processes
vital for early eukaryotic life. Along these lines, one suspects 8. Anderluh G, Macek P. Cytolytic peptide and protein toxins from sea anemones (An-
the vesicle fusion of membrane repair may be more rudi- thozoa: Actiniaria). Toxicon 40: 111–124, 2002.

mentary than that of synaptic neurotransmission, evolving 9. Anderson LV, Davison K, Moss JA, Young C, Cullen MJ, Walsh J, Johnson MA, Bashir
500 million years later. Importantly for the membrane re- R, Britton S, Keers S, Argov Z, Mahjneh I, Fougerousse F, Beckmann JS, Bushby KM.
Dysferlin is a plasma membrane protein and is expressed early in human develop-
pair field, the biology of vesicle fusion and cytokinesis are ment. Hum Mol Genet 8: 855– 861, 1999.
well defined, each with established pharmacological modi-
10. Arroyo CM, Kramer JH, Dickens BF, Weglicki WB. Identification of free radicals in
fiers. The key now is to connect each research field impli- myocardial ischemia/reperfusion by spin trapping with nitrone DMPO. FEBS Lett 221:
cated in membrane repair and better elucidate the molecu- 101–104, 1987.
lar signaling governing the temporal progression of each
11. Arthur JS, Elce JS, Hegadorn C, Williams K, Greer PA. Disruption of the murine calpain
stage in the membrane repair process. It is only then we will small subunit gene, Capn4: calpain is essential for embryonic development but not for
be able to strategize novel interventions so desperately cell growth and division. Mol Cell Biol 20: 4474 – 4481, 2000.

1232 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

12. Avela K, Lipsanen-Nyman M, Idanheimo N, Seemanova E, Rosengren S, Makela TP, 33. Blanchard H, Grochulski P, Li Y, Arthur JS, Davies PL, Elce JS, Cygler M. Structure of
Perheentupa J, Chapelle AD, Lehesjoki AE. Gene encoding a new RING-B-box- a calpain Ca2⫹-binding domain reveals a novel EF-hand and Ca2⫹-induced conforma-
Coiled-coil protein is mutated in mulibrey nanism. Nature Genet 25: 298 –301, 2000. tional changes. Nature Struct Biol 4: 532–538, 1997.

13. Azam M, Andrabi SS, Sahr KE, Kamath L, Kuliopulos A, Chishti AH. Disruption of the 34. Blanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J. Actin dynamics, architecture,
mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol 21: and mechanics in cell motility. Physiol Rev 94: 235–263, 2014.
2213–2220, 2001.
35. Bommert K, Charlton MP, DeBello WM, Chin GJ, Betz H, Augustine GJ. Inhibition of
14. Babiychuk EB, Monastyrskaya K, Potez S, Draeger A. Intracellular Ca2⫹ operates a neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocy-
switch between repair and lysis of streptolysin O-perforated cells. Cell Death Differ- tosis. Nature 363: 163–165, 1993.
entiation 16: 1126 –1134, 2009.
36. Bork P, Sander C, Valencia A. An ATPase domain common to prokaryotic cell cycle
15. Baghdiguian S, Martin M, Richard I, Pons F, Astier C, Bourg N, Hay RT, Chemaly R,
proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci USA
Halaby G, Loiselet J, Anderson LV, Lopez de Munain A, Fardeau M, Mangeat P,
89: 7290 –7294, 1992.
Beckmann JS, Lefranc G. Calpain 3 deficiency is associated with myonuclear apoptosis
and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limb-girdle 37. Bouter A, Gounou C, Berat R, Tan S, Gallois B, Granier T, d’Estaintot BL, Poschl E,
muscular dystrophy type 2A. Nature Med 5: 503–511, 1999.
Brachvogel B, Brisson AR. Annexin-A5 assembled into two-dimensional arrays pro-
16. Baines CP. The mitochondrial permeability transition pore and ischemia-reperfusion motes cell membrane repair. Nature Commun 2: 270, 2011.
injury. Basic Res Cardiol 104: 181–188, 2009.
38. Brose N, Petrenko AG, Sudhof TC, Jahn R. Synaptotagmin: a calcium sensor on the
17. Bano D, Nicotera P. Ca2⫹ signals and neuronal death in brain ischemia. Stroke 38: synaptic vesicle surface. Science 256: 1021–1025, 1992.
674 – 676, 2007.
39. Buki A, Povlishock JT. All roads lead to disconnection? Traumatic axonal injury revis-
18. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL, Campbell ited. Acta Neurochir 148: 181–193, 2006.
KP. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature
423: 168 –172, 2003. 40. Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, Ko JK, Lin P,
Thornton A, Zhao X, Pan Z, Komazaki S, Brotto M, Takeshima H, Ma J. MG53
19. Baran K, Dunstone M, Chia J, Ciccone A, Browne KA, Clarke CJ, Lukoyanova N, Saibil nucleates assembly of cell membrane repair machinery. Nat Cell Biol 11: 56 – 64, 2009.
H, Whisstock JC, Voskoboinik I, Trapani JA. The molecular basis for perforin oli-
gomerization and transmembrane pore assembly. Immunity 30: 684 – 695, 2009. 41. Cai C, Masumiya H, Weisleder N, Pan Z, Nishi M, Komazaki S, Takeshima H, Ma J.
MG53 regulates membrane budding and exocytosis in muscle cells. J Biol Chem 284:
20. Bargal R, Avidan N, Ben-Asher E, Olender Z, Zeigler M, Frumkin A, Raas-Rothschild 3314 –3322, 2009.
A, Glusman G, Lancet D, Bach G. Identification of the gene causing mucolipidosis type
IV. Nature Genet 26: 118 –123, 2000. 42. Cai C, Weisleder N, Ko JK, Komazaki S, Sunada Y, Nishi M, Takeshima H, Ma J.
Membrane repair defects in muscular dystrophy are linked to altered interaction
21. Barwise JL, Walker JH. Subcellular localization of annexin V in human foreskin fibro- between MG53, caveolin-3, and dysferlin. J Biol Chem 284: 15894 –15902, 2009.
blasts: nuclear localization depends on growth state. FEBS Lett 394: 213–216, 1996.
43. Cao CM, Zhang Y, Weisleder N, Ferrante C, Wang X, Lv F, Zhang Y, Song R, Hwang
22. Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, Richard I, Marchand S,
M, Jin L, Guo J, Peng W, Li G, Nishi M, Takeshima H, Ma J, Xiao RP. MG53 constitutes
Bourg N, Argov Z, Sadeh M, Mahjneh I, Marconi G, Passos-Bueno MR, Moreira Ede S,
a primary determinant of cardiac ischemic preconditioning. Circulation 121: 2565–
Zatz M, Beckmann JS, Bushby K. A gene related to Caenorhabditis elegans spermato-
2574, 2010.
genesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nature
Genet 20: 37– 42, 1998.
44. Carragher NO. Calpain inhibition: a therapeutic strategy targeting multiple disease
23. Baylor SM, Hollingworth S. Intracellular calcium movements during excitation-con- states. Curr Pharmaceutical Design 12: 615– 638, 2006.
traction coupling in mammalian slow-twitch and fast-twitch muscle fibers. J Gen
45. Casademont J, Carpenter S, Karpati G. Vacuolation of muscle fibers near sarcolemmal
Physiol 139: 261–272, 2012.
breaks represents T-tubule dilatation secondary to enhanced sodium pump activity. J
24. Beckmann JS, Spencer M. Calpain 3, the “gatekeeper” of proper sarcomere assembly, Neuropathol Exp Neurol 47: 618 – 628, 1988.
turnover and maintenance. Neuromuscular Disorders 18: 913–921, 2008.
46. Cassidy SK, O’Riordan MX. More than a pore: the cellular response to cholesterol-
25. Bement WM, Mandato CA, Kirsch MN. Wound-induced assembly and closure of an dependent cytolysins. Toxins 5: 618 – 636, 2013.
actomyosin purse string in Xenopus oocytes. Curr Biol 9: 579 –587, 1999.
47. Chakrabarti S, Kobayashi KS, Flavell RA, Marks CB, Miyake K, Liston DR, Fowler KT,
26. Bennett MK, Calakos N, Scheller RH. Syntaxin: a synaptic protein implicated in dock- Gorelick FS, Andrews NW. Impaired membrane resealing and autoimmune myositis
ing of synaptic vesicles at presynaptic active zones. Science 257: 255–259, 1992. in synaptotagmin VII-deficient mice. J Cell Biol 162: 543–549, 2003.

27. Bi GQ, Alderton JM, Steinhardt RA. Calcium-regulated exocytosis is required for cell 48. Chen CS, Bach G, Pagano RE. Abnormal transport along the lysosomal pathway in
membrane resealing. J Cell Biol 131: 1747–1758, 1995. mucolipidosis, type IV disease. Proc Natl Acad Sci USA 95: 6373– 6378, 1998.

28. Bi GQ, Morris RL, Liao G, Alderton JM, Scholey JM, Steinhardt RA. Kinesin- and 49. Cheng X, Zhang X, Gao Q, Ali Samie M, Azar M, Tsang WL, Dong L, Sahoo N, Li X,
myosin-driven steps of vesicle recruitment for Ca2⫹-regulated exocytosis. J Cell Biol Zhuo Y, Garrity AG, Wang X, Ferrer M, Dowling J, Xu L, Han R, Xu H. The intracel-
138: 999 –1008, 1997. lular Ca2⫹ channel MCOLN1 is required for sarcolemma repair to prevent muscular
dystrophy. Nature Med 20: 1187–1192, 2014.
29. Binz T, Blasi J, Yamasaki S, Baumeister A, Link E, Sudhof TC, Jahn R, Niemann H.
Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J Biol Chem 269:
50. Cho SJ, Kelly M, Rognlien KT, Cho JA, Horber JK, Jena BP. SNAREs in opposing
1617–1620, 1994.
bilayers interact in a circular array to form conducting pores. Biophys J 83: 2522–2527,
30. Bischofberger M, Gonzalez MR, van der Goot FG. Membrane injury by pore-forming 2002.
proteins. Curr Opin Cell Biol 21: 589 –595, 2009.
51. Cho W, Stahelin RV. Membrane binding and subcellular targeting of C2 domains.
31. Biswas S, Harris F, Dennison S, Singh J, Phoenix DA. Calpains: targets of cataract Biochim Biophys Acta 1761: 838 – 849, 2006.
prevention? Trends Mol Med 10: 78 – 84, 2004.
52. Christensen KA, Myers JT, Swanson JA. pH-dependent regulation of lysosomal cal-
32. Bittner RE, Anderson LV, Burkhardt E, Bashir R, Vafiadaki E, Ivanova S, Raffelsberger cium in macrophages. J Cell Sci 115: 599 – 607, 2002.
T, Maerk I, Hoger H, Jung M, Karbasiyan M, Storch M, Lassmann H, Moss JA, Davison
K, Harrison R, Bushby KM, Reis A. Dysferlin deletion in SJL mice (SJL-Dysf) defines a 53. Claflin DR, Brooks SV. Direct observation of failing fibers in muscles of dystrophic
natural model for limb girdle muscular dystrophy 2B. Nature Genet 23: 141–142, mice provides mechanistic insight into muscular dystrophy. Am J Physiol Cell Physiol
1999. 294: C651–C658, 2008.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1233


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

54. Clarke MS, Khakee R, McNeil PL. Loss of cytoplasmic basic fibroblast growth factor 77. Draeger A, Sanchez-Freire V, Monastyrskaya K, Hoppeler H, Mueller M, Breil F,
from physiologically wounded myofibers of normal and dystrophic muscle. J Cell Sci Mohaupt MG, Babiychuk EB. Statin therapy and the expression of genes that regulate
106: 121–133, 1993. calcium homeostasis and membrane repair in skeletal muscle. Am J Pathol 177: 291–
299, 2010.
55. Cohn RD, Campbell KP. Molecular basis of muscular dystrophies. Muscle Nerve 23:
1456 –1471, 2000. 78. Draeger A, Wray S, Babiychuk EB. Domain architecture of the smooth-muscle plasma
membrane: regulation by annexins. Biochem J 387: 309 –314, 2005.
56. Cooper ST, Head SI. Membrane injury and repair in the muscular dystrophies. The
Neuroscientist. In press. 79. Dutt P, Croall DE, Arthur JS, Veyra TD, Williams K, Elce JS, Greer PA. m-Calpain is
required for preimplantation embryonic development in mice. BMC Dev Biol 6: 3,
57. Corrotte M, Almeida PE, Tam C, Castro-Gomes T, Fernandes MC, Millis BA, Cortez 2006.
M, Miller H, Song W, Maugel TK, Andrews NW. Caveolae internalization repairs
wounded cells and muscle fibers. eLife 2: e00926, 2013. 80. Edwards JN, Launikonis BS. The accessibility and interconnectivity of the tubular
system network in toad skeletal muscle. J Physiol 586: 5077–5089, 2008.
58. Corrotte M, Fernandes MC, Tam C, Andrews NW. Toxin pores endocytosed during
plasma membrane repair traffic into the lumen of MVBs for degradation. Traffic 13: 81. Elferink LA, Peterson MR, Scheller RH. A role for synaptotagmin (p65) in regulated
483– 494, 2012. exocytosis. Cell 72: 153–159, 1993.

59. Covian-Nares JF, Koushik SV, Puhl HL 3rd, Vogel SS. Membrane wounding triggers 82. Erickson HP. Evolution of the cytoskeleton. BioEssays 29: 668 – 677, 2007.
ATP release and dysferlin-mediated intercellular calcium signaling. J Cell Sci 123:
1884 –1893, 2010. 83. Erickson HP, O’Brien ET. Microtubule dynamic instability and GTP hydrolysis. Annu
Rev Biophys Biomol Struct 21: 145–166, 1992.
60. Cowell S, Aschauer W, Gruber HJ, Nelson KL, Buckley JT. The erythrocyte receptor
for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-an- 84. Ersfeld K, Barraclough H, Gull K. Evolutionary relationships and protein domain ar-
chored protein. Mol Microbiol 25: 343–350, 1997. chitecture in an expanded calpain superfamily in kinetoplastid parasites. J Mol Evol 61:
742–757, 2005.
61. Craxton M. Synaptotagmin gene content of the sequenced genomes. BMC Genomics
5: 43, 2004. 85. Ervasti JM. Costameres: the Achilles’ heel of Herculean muscle. J Biol Chem 278:
13591–13594, 2003.
62. Croall DE, Ersfeld K. The calpains: modular designs and functional diversity. Genome
Biol 8: 218, 2007. 86. Ervasti JM, Campbell KP. Membrane organization of the dystrophin-glycoprotein
complex. Cell 66: 1121–1131, 1991.
63. Cruz M, Kaemd L. Actin-Binding Proteins and Disease. Berlin: Springer, 2008, p. 348.
87. Essen LO, Perisic O, Cheung R, Katan M, Williams RL. Crystal structure of a mam-
64. Czogalla A, Sikorski AF. Spectrin and calpain: a “target” and a “sniper” in the pathology malian phosphoinositide-specific phospholipase C delta. Nature 380: 595– 602, 1996.
of neuronal cells. Cell Mol Life Sci 62: 1913–1924, 2005.
88. Evesson FJ, Peat RA, Lek A, Brilot F, Lo HP, Dale RC, Parton RG, North KN, Cooper
65. Dacks JB, Doolittle WF. Novel syntaxin gene sequences from Giardia, Trypanosoma ST. Reduced plasma membrane expression of dysferlin mutants is attributed to ac-
and algae: implications for the ancient evolution of the eukaryotic endomembrane celerated endocytosis via a syntaxin-4-associated pathway. J Biol Chem 285: 28529 –
system. J Cell Sci 115: 1635–1642, 2002. 28539, 2010.

66. Dacks JB, Field MC. Evolution of the eukaryotic membrane-trafficking system: origin, 89. Field MC, Dacks JB. First and last ancestors: reconstructing evolution of the endo-
tempo and mode. J Cell Sci 120: 2977–2985, 2007. membrane system with ESCRTs, vesicle coat proteins, and nuclear pore complexes.
Curr Opin Cell Biol 21: 4 –13, 2009.
67. Dacks JB, Peden AA, Field MC. Evolution of specificity in the eukaryotic endomem-
brane system. Int J Biochem Cell Biol 41: 330 –340, 2009. 90. Franzini-Armstrong C, Landmesser L, Pilar G. Size and shape of transverse tubule
openings in frog twitch muscle fibers. J Cell Biol 64: 493– 497, 1975.
68. Damle SS, Moore EE, Babu AN, Meng X, Fullerton DA, Banerjee A. Hemoglobin-
based oxygen carrier induces heme oxygenase-1 in the heart and lung but not brain. J 91. Friden J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric
Am Coll Surg 208: 592–598, 2009. exercise in man. Int J Sports Med 4: 170 –176, 1983.

69. De Boer P, Crossley R, Rothfield L. The essential bacterial cell-division protein FtsZ is 92. Frosk P, Weiler T, Nylen E, Sudha T, Greenberg CR, Morgan K, Fujiwara TM, Wro-
a GTPase. Nature 359: 254 –256, 1992. gemann K. Limb-girdle muscular dystrophy type 2H associated with mutation in
TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet 70: 663– 672, 2002.
70. De Sousa MV, Morhy L. Enterolobin, a hemolytic protein from Enterolobium contor-
tisiliquum seeds (Leguminosae–Mimosoideae). Purification and characterization. 93. Galitsky N, Cody V, Wojtczak A, Ghosh D, Luft JR, Pangborn W, English L. Structure
Anais da Academia Brasileira de Ciencias 61: 405– 412, 1989. of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis. Acta
Crystallographica Sect D Biol Crystallogr 57: 1101–1109, 2001.
71. Demonbreun AR, Fahrenbach JP, Deveaux K, Earley JU, Pytel P, McNally EM. Im-
paired muscle growth and response to insulin like growth factor 1 in dysferlin medi- 94. Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the
ated muscular dystrophy. Hum Mol Genet 20: 779 –789, 2011. reperfused rat heart using electron spin resonance spectroscopy. Circ Res 61: 757–
760, 1987.
72. Detrait E, Eddleman CS, Yoo S, Fukuda M, Nguyen MP, Bittner GD, Fishman HM.
Axolemmal repair requires proteins that mediate synaptic vesicle fusion. J Neurobiol 95. Gaullier JM, Simonsen A, D’Arrigo A, Bremnes B, Stenmark H, Aasland R. FYVE
44: 382–391, 2000. fingers bind PtdIns(3)P. Nature 394: 432– 433, 1998.

73. Diakonova M, Gerke V, Ernst J, Liautard JP, van der Vusse G, Griffiths G. Localization 96. Geoffroy C, Gaillard JL, Alouf JE, Berche P. Purification, characterization, and toxicity
of five annexins in J774 macrophages and on isolated phagosomes. J Cell Sci 110: of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. In-
1199 –1213, 1997. fection Immunity 55: 1641–1646, 1987.

74. Dominguez R, Holmes KC. Actin structure and function. Annu Rev Biophys 40: 169 – 97. Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Sudhof TC. Synap-
186, 2011. totagmin I: a major Ca2⫹ sensor for transmitter release at a central synapse. Cell 79:
717–727, 1994.
75. Dong XP, Cheng X, Mills E, Delling M, Wang F, Kurz T, Xu H. The type IV mucolipi-
dosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 98. Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev 82: 331–371,
455: 992–996, 2008. 2002.

76. Draeger A, Monastyrskaya K, Babiychuk EB. Plasma membrane repair and cellular 99. Gil-Parrado S, Popp O, Knoch TA, Zahler S, Bestvater F, Felgentrager M, Holloschi A,
damage control: the annexin survival kit. Biochem Pharmacol 81: 703–712, 2011. Fernandez-Montalvan A, Auerswald EA, Fritz H, Fuentes-Prior P, Machleidt W, Spiess

1234 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

E. Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain. J 122. Idone V, Tam C, Andrews NW. Two-way traffic on the road to plasma membrane
Biol Chem 278: 16336 –16346, 2003. repair. Trends Cell Biol 18: 552–559, 2008.

100. Glaser T, Schwarz-Benmeir N, Barnoy S, Barak S, Eshhar Z, Kosower NS. Calpain 123. Idone V, Tam C, Goss JW, Toomre D, Pypaert M, Andrews NW. Repair of injured
(Ca2⫹-dependent thiol protease) in erythrocytes of young and old individuals. Proc plasma membrane by rapid Ca2⫹-dependent endocytosis. J Cell Biol 180: 905–914,
Natl Acad Sci USA 91: 7879 –7883, 1994. 2008.

101. Godell CM, Smyers ME, Eddleman CS, Ballinger ML, Fishman HM, Bittner GD. Cal- 124. Inserte J, Hernando V, Garcia-Dorado D. Contribution of calpains to myocardial
pain activity promotes the sealing of severed giant axons. Proc Natl Acad Sci USA 94: ischaemia/reperfusion injury. Cardiovasc Res 96: 23–31, 2012.
4751– 4756, 1997.
125. Ishiharajima S, Aida T, Nakagawa R, Kameyama K, Sugano K, Oguro T, Asano G. Early
102. Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev 83: membrane damage during ischemia in rat heart. Exp Mol Pathol 44: 1– 6, 1986.
731– 801, 2003.
126. Jaiswal JK, Chakrabarti S, Andrews NW, Simon SM. Synaptotagmin VII restricts fusion
103. Golstein P, Kroemer G. Cell death by necrosis: towards a molecular definition. Trends pore expansion during lysosomal exocytosis. PLoS Biol 2: E233, 2004.
Biochem Sci 32: 37– 43, 2007.
127. Jena BP. Role of SNAREs in membrane fusion. Adv Exp Med Biol 713: 13–32, 2011.
104. Golstein P, Kroemer G. A multiplicity of cell death pathways. Symposium on apoptotic
and non-apoptotic cell death pathways. EMBO Reports 8: 829 – 833, 2007. 128. Jeremic A, Kelly M, Cho JA, Cho SJ, Horber JK, Jena BP. Calcium drives fusion of
SNARE-apposed bilayers. Cell Biol Int 28: 19 –31, 2004.
105. Gonzalez MR, Bischofberger M, Pernot L, van der Goot FG, Freche B. Bacterial
pore-forming toxins: the (w)hole story? Cell Mol Life Sci 65: 493–507, 2008. 129. Jimenez AJ, Maiuri P, Lafaurie-Janvore J, Divoux S, Piel M, Perez F. ESCRT machinery
is required for plasma membrane repair. Science 343: 1247136, 2014.
106. Gozen I, Dommersnes P. Pore dynamics in lipid membranes. Eur Phys J Spec Top 223:
1813–1829, 2014. 130. Joazeiro CA, Weissman AM. RING finger proteins: mediators of ubiquitin ligase ac-
tivity. Cell 102: 549 –552, 2000.
107. Guyon JR, Kudryashova E, Potts A, Dalkilic I, Brosius MA, Thompson TG, Beckmann
JS, Kunkel LM, Spencer MJ. Calpain 3 cleaves filamin C and regulates its ability to 131. Jones DA, Newham DJ, Round JM, Tolfree SE. Experimental human muscle damage:
interact with gamma- and delta-sarcoglycans. Muscle Nerve 28: 472– 483, 2003. morphological changes in relation to other indices of damage. J Physiol 375: 435– 448,
1986.
108. Hajjar KA, Krishnan S. Annexin II: a mediator of the plasmin/plasminogen activator
system. Trends Cardiovasc Med 9: 128 –138, 1999. 132. Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell biology of ischemia/reperfusion
injury. Int Rev Cell Mol Biol 298: 229 –317, 2012.
109. Hanson PI, Roth R, Lin Y, Heuser JE. Plasma membrane deformation by circular arrays
of ESCRT-III protein filaments. J Cell Biol 180: 389 – 402, 2008. 133. Kasai H, Takahashi N, Tokumaru H. Distinct initial SNARE configurations underlying
110. Henne WM, Buchkovich NJ, Emr SD. The ESCRT pathway. Dev Cell 21: 77–91, 2011. the diversity of exocytosis. Physiol Rev 92: 1915–1964, 2012.

111. Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Kang C. Crystal structure of the 134. Keays DA, Tian G, Poirier K, Huang GJ, Siebold C, Cleak J, Oliver PL, Fray M, Harvey
cytotoxic bacterial protein colicin B at 2.5 A resolution. Mol Microbiol 51: 711–720, RJ, Molnar Z, Pinon MC, Dear N, Valdar W, Brown SD, Davies KE, Rawlins JN, Cowan
2004. NJ, Nolan P, Chelly J, Flint J. Mutations in alpha-tubulin cause abnormal neuronal
migration in mice and lissencephaly in humans. Cell 128: 45–57, 2007.
112. Ho M, Post CM, Donahue LR, Lidov HG, Bronson RT, Goolsby H, Watkins SC, Cox
GA, Brown RH Jr. Disruption of muscle membrane and phenotype divergence in two 135. Kerr JP, Ziman AP, Mueller AL, Muriel JM, Kleinhans-Welte E, Gumerson JD, Vogel
novel mouse models of dysferlin deficiency. Hum Mol Genet 13: 1999 –2010, 2004. SS, Ward CW, Roche JA, Bloch RJ. Dysferlin stabilizes stress-induced Ca2⫹ signaling in
the transverse tubule membrane. Proc Natl Acad Sci USA 110: 20831–20836, 2013.
113. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duch-
enne muscular dystrophy locus. Cell 51: 919 –928, 1987. 136. Keyel PA, Loultcheva L, Roth R, Salter RD, Watkins SC, Yokoyama WM, Heuser JE.
Streptolysin O clearance through sequestration into blebs that bud passively from the
114. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner plasma membrane. J Cell Sci 124: 2414 –2423, 2011.
TH, Mashima H, Schwarz PE, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I,
Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus 137. Kitagaki H, Tomioka S, Yoshizawa T, Sorimachi H, Saido TC, Ishiura S, Suzuki K.
C, Groop L, Boerwinkle E, Hanis CL, Bell GI. Genetic variation in the gene encoding Autolysis of calpain large subunit inducing irreversible dissociation of stoichiometric
calpain-10 is associated with type 2 diabetes mellitus. Nature Genet 26: 163–175, heterodimer of calpain. Biosci Biotechnol Biochem 64: 689 – 695, 2000.
2000.
138. Klinge L, Laval S, Keers S, Haldane F, Straub V, Barresi R, Bushby K. From T-tubule to
115. Hosfield CM, Elce JS, Davies PL, Jia Z. Crystal structure of calpain reveals the struc- sarcolemma: damage-induced dysferlin translocation in early myogenesis. FASEB J 21:
tural basis for Ca2⫹-dependent protease activity and a novel mode of enzyme activa- 1768 –1776, 2007.
tion. EMBO J 18: 6880 – 6889, 1999.
139. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH.
116. Howard AC, McNeil AK, Xiong F, Xiong WC, McNeil PL. A novel cellular defect in Annexin V for flow cytometric detection of phosphatidylserine expression on B cells
diabetes: membrane repair failure. Diabetes 60: 3034 –3043, 2011. undergoing apoptosis. Blood 84: 1415–1420, 1994.
117. Hurley JH. ESCRT complexes and the biogenesis of multivesicular bodies. Curr Opin 140. Krahn M, Wein N, Bartoli M, Lostal W, Courrier S, Bourg-Alibert N, Nguyen K, Vial C,
Cell Biol 20: 4 –11, 2008. Streichenberger N, Labelle V, DePetris D, Pecheux C, Leturcq F, Cau P, Richard I,
118. Husmann M, Beckmann E, Boller K, Kloft N, Tenzer S, Bobkiewicz W, Neukirch C, Levy N. A naturally occurring human minidysferlin protein repairs sarcolemmal le-
Bayley H, Bhakdi S. Elimination of a bacterial pore-forming toxin by sequential endo- sions in a mouse model of dysferlinopathy. Science Transl Med 2: 50ra69, 2010.
cytosis and exocytosis. FEBS Lett 583: 337–344, 2009.
141. Kull FJ, Sablin EP, Lau R, Fletterick RJ, Vale RD. Crystal structure of the kinesin motor
119. Huynh C, Roth D, Ward DM, Kaplan J, Andrews NW. Defective lysosomal exocytosis domain reveals a structural similarity to myosin. Nature 380: 550 –555, 1996.
and plasma membrane repair in Chediak-Higashi/beige cells. Proc Natl Acad Sci USA
142. Kumamoto T, Kleese WC, Cong JY, Goll DE, Pierce PR, Allen RE. Localization of the
101: 16795–16800, 2004.
Ca2⫹-dependent proteinases and their inhibitor in normal, fasted, and denervated rat
120. Iacovache I, van der Goot FG, Pernot L. Pore formation: an ancient yet complex form skeletal muscle. Anat Rec 232: 60 –77, 1992.
of attack. Biochim Biophys Acta 1778: 1611–1623, 2008.
143. Kuru S, Yasuma F, Wakayama T, Kimura S, Konagaya M, Aoki M, Tanabe M,
121. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Camp- Takahashi T. [A patient with limb girdle muscular dystrophy type 2B (LGMD2B)
bell KP. Primary structure of dystrophin-associated glycoproteins linking dystrophin manifesting cardiomyopathy]. Rinsho Shinkeigaku Clin Neurol 44: 375–378,
to the extracellular matrix. Nature 355: 696 –702, 1992. 2004.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1235


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

144. Kutateladze TG, Ogburn KD, Watson WT, de Beer T, Emr SD, Burd CG, Overduin 164. Los FC, Kao CY, Smitham J, McDonald KL, Ha C, Peixoto CA, Aroian RV. RAB-5- and
M. Phosphatidylinositol 3-phosphate recognition by the FYVE domain. Mol Cell 3: RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane
805– 811, 1999. repair after attack by bacterial pore-forming toxin. Cell Host Microbe 9: 147–157,
2011.
145. Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, Green DR,
Newmeyer DD. Bid, Bax, and lipids cooperate to form supramolecular openings in 165. Los FC, Randis TM, Aroian RV, Ratner AJ. Role of pore-forming toxins in bacterial
the outer mitochondrial membrane. Cell 111: 331–342, 2002. infectious diseases. Microbiol Mol Biol Rev 77: 173–207, 2013.

146. Lai Y, Thomas GD, Yue Y, Yang HT, Li D, Long C, Judge L, Bostick B, Chamberlain JS, 166. Lostal W, Bartoli M, Roudaut C, Bourg N, Krahn M, Pryadkina M, Borel P, Suel L,
Terjung RL, Duan D. Dystrophins carrying spectrin-like repeats 16 and 17 anchor Roche JA, Stockholm D, Bloch RJ, Levy N, Bashir R, Richard I. Lack of correlation
nNOS to the sarcolemma and enhance exercise performance in a mouse model of between outcomes of membrane repair assay and correction of dystrophic changes in
muscular dystrophy. J Clin Invest 119: 624 – 635, 2009. experimental therapeutic strategy in dysferlinopathy. PloS One 7: e38036, 2012.

147. LaPlante JM, Ye CP, Quinn SJ, Goldin E, Brown EM, Slaugenhaupt SA, Vassilev PM. 167. Lovelace LL, Cooper CL, Sodetz JM, Lebioda L. Structure of human C8 protein
Functional links between mucolipin-1 and Ca2⫹-dependent membrane trafficking in provides mechanistic insight into membrane pore formation by complement. J Biol
mucolipidosis IV. Biochem Biophys Res Commun 322: 1384 –1391, 2004. Chem 286: 17585–17592, 2011.

148. Launikonis BS, Stephenson DG, Friedrich O. Rapid Ca2⫹ flux through the transverse 168. Mandato CA, Bement WM. Actomyosin transports microtubules and microtubules
tubular membrane, activated by individual action potentials in mammalian skeletal control actomyosin recruitment during Xenopus oocyte wound healing. Curr Biol 13:
muscle. J Physiol 587: 2299 –2312, 2009. 1096 –1105, 2003.

149. Law RH, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, Dunstone MA, 169. Mandato CA, Bement WM. Contraction and polymerization cooperate to assemble
D’Angelo ME, Orlova EV, Coulibaly F, Verschoor S, Browne KA, Ciccone A, Kuiper and close actomyosin rings around Xenopus oocyte wounds. J Cell Biol 154: 785–797,
MJ, Bird PI, Trapani JA, Saibil HR, Whisstock JC. The structural basis for membrane 2001.
binding and pore formation by lymphocyte perforin. Nature 468: 447– 451, 2010.
170. Mayran N, Parton RG, Gruenberg J. Annexin II regulates multivesicular endosome
150. Lek A, Evesson FJ, Lemckert FA, Redpath GM, Lueders AK, Turnbull L, Whitchurch biogenesis in the degradation pathway of animal cells. EMBO J 22: 3242–3253, 2003.
CB, North KN, Cooper ST. Calpains, cleaved mini-dysferlinC72, and L-type channels
underpin calcium-dependent muscle membrane repair. J Neurosci 33: 5085–5094, 171. McCullough J, Colf LA, Sundquist WI. Membrane fission reactions of the mammalian
2013. ESCRT pathway. Annu Rev Biochem 82: 663– 692, 2013.

151. Lek A, Evesson FJ, Sutton RB, North KN, Cooper ST. Ferlins: regulators of vesicle 172. McDade JR, Archambeau A, Michele DE. Rapid actin-cytoskeleton-dependent re-
fusion for auditory neurotransmission, receptor trafficking and membrane repair. cruitment of plasma membrane-derived dysferlin at wounds is critical for muscle
Traffic 13: 185–194, 2012. membrane repair. FASEB J 28: 3660 –3670, 2014.

152. Lek A, Lek M, North KN, Cooper ST. Phylogenetic analysis of ferlin genes reveals 173. McDade JR, Michele DE. Membrane damage-induced vesicle-vesicle fusion of dysfer-
ancient eukaryotic origins. BMC Evol Biol 10: 231, 2010. lin-containing vesicles in muscle cells requires microtubules and kinesin. Hum Mol
Genet 23: 1677–1686, 2014.
153. Leloup L, Shao H, Bae YH, Deasy B, Stolz D, Roy P, Wells A. m-Calpain activation is
regulated by its membrane localization and by its binding to phosphatidylinositol 174. McDonald B, Martin-Serrano J. No strings attached: the ESCRT machinery in viral
4,5-bisphosphate. J Biol Chem 285: 33549 –33566, 2010. budding and cytokinesis. J Cell Sci 122: 2167–2177, 2009.

154. Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, Brown RH Jr. Dysferlin 175. McNab FW, Rajsbaum R, Stoye JP, O’Garra A. Tripartite-motif proteins and innate
interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing. J Biol immune regulation. Curr Opin Immunol 23: 46 –56, 2011.
Chem 278: 50466 –50473, 2003.
176. McNeil AK, Rescher U, Gerke V, McNeil PL. Requirement for annexin A1 in plasma
155. Leung KF, Dacks JB, Field MC. Evolution of the multivesicular body ESCRT machin- membrane repair. J Biol Chem 281: 35202–35207, 2006.
ery: retention across the eukaryotic lineage. Traffic 9: 1698 –1716, 2008.
177. McNeil P. Membrane repair redux: redox of MG53. Nat Cell Biol 11: 7–9, 2009.
156. Lewit-Bentley A, Rety S, Sopkova-de Oliveira Santos J, Gerke V. S100-annexin com-
plexes: some insights from structural studies. Cell Biol Int 24: 799 – 802, 2000. 178. McNeil PL. Cellular and molecular adaptations to injurious mechanical stress. Trends
Cell Biol 3: 302–307, 1993.
157. Liao M, Cao E, Julius D, Cheng Y. Structure of the TRPV1 ion channel determined by
electron cryo-microscopy. Nature 504: 107–112, 2013. 179. McNeil PL, Baker MM. Cell surface events during resealing visualized by scanning-
electron microscopy. Cell Tissue Res 304: 141–146, 2001.
158. Liemann S, Huber R. Three-dimensional structure of annexins. Cell Mol Life Sci 53:
516 –521, 1997. 180. McNeil PL, Khakee R. Disruptions of muscle fiber plasma membranes. Role in exer-
cise-induced damage. Am J Pathol 140: 1097–1109, 1992.
159. Lin GD, Chattopadhyay D, Maki M, Wang KK, Carson M, Jin L, Yuen PW, Takano E,
Hatanaka M, DeLucas LJ, Narayana SV. Crystal structure of calcium bound domain VI 181. McNeil PL, Kirchhausen T. An emergency response team for membrane repair.
of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor Nature Rev Mol Cell Biol 6: 499 –505, 2005.
binding. Nature Struct Biol 4: 539 –547, 1997.
182. Mellgren RL, Miyake K, Kramerova I, Spencer MJ, Bourg N, Bartoli M, Richard I, Greer
160. Lin P, Zhu H, Cai C, Wang X, Cao C, Xiao R, Pan Z, Weisleder N, Takeshima H, Ma PA, McNeil PL. Calcium-dependent plasma membrane repair requires m- or mu-
J. Nonmuscle myosin IIA facilitates vesicle trafficking for MG53-mediated cell mem- calpain, but not calpain-3, the proteasome, or caspases. Biochim Biophys Acta 1793:
brane repair. FASEB J 26: 1875–1883, 2012. 1886 –1893, 2009.

161. Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, Serrano C, Urtizberea JA, Hentati F, 183. Mellgren RL, Zhang W, Miyake K, McNeil PL. Calpain is required for the rapid,
Hamida MB, Bohlega S, Culper EJ, Amato AA, Bossie K, Oeltjen J, Bejaoui K, McK- calcium-dependent repair of wounded plasma membrane. J Biol Chem 282: 2567–
enna-Yasek D, Hosler BA, Schurr E, Arahata K, de Jong PJ, Brown RH Jr. Dysferlin, a 2575, 2007.
novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular
dystrophy. Nature Genet 20: 31–36, 1998. 184. Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic trans-
mission with pH-sensitive green fluorescent proteins. Nature 394: 192–195, 1998.
162. Liu Z, Cao J, Gao X, Ma Q, Ren J, Xue Y. GPS-CCD: a novel computational program
for the prediction of calpain cleavage sites. PloS One 6: e19001, 2011. 185. Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, Molkentin JD. Cal-
cium influx is sufficient to induce muscular dystrophy through a TRPC-dependent
163. Lo HP, Cooper ST, Evesson FJ, Seto JT, Chiotis M, Tay V, Compton AG, Cairns AG, mechanism. Proc Natl Acad Sci USA 106: 19023–19028, 2009.
Corbett A, MacArthur DG, Yang N, Reardon K, North KN. Limb-girdle muscular
dystrophy: diagnostic evaluation, frequency and clues to pathogenesis. Neuromuscular 186. Minetti C, Sotgia F, Bruno C, Scartezzini P, Broda P, Bado M, Masetti E, Mazzocco M,
Disorders 18: 34 – 44, 2008. Egeo A, Donati MA, Volonte D, Galbiati F, Cordone G, Bricarelli FD, Lisanti MP, Zara

1236 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

F. Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular 210. Pang ZP, Sudhof TC. Cell biology of Ca2⫹-triggered exocytosis. Curr Opin Cell Biol 22:
dystrophy. Nature Genet 18: 365–368, 1998. 496 –505, 2010.

187. Miyake K, McNeil PL, Suzuki K, Tsunoda R, Sugai N. An actin barrier to resealing. J Cell 211. Pangrsic T, Lasarow L, Reuter K, Takago H, Schwander M, Riedel D, Frank T, Taran-
Sci 114: 3487–3494, 2001. tino LM, Bailey JS, Strenzke N, Brose N, Muller U, Reisinger E, Moser T. Hearing
requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells.
188. Monastyrskaya K, Babiychuk EB, Hostettler A, Rescher U, Draeger A. Annexins as Nat Neurosci 13: 869 – 876, 2010.
intracellular calcium sensors. Cell Calcium 41: 207–219, 2007.
212. Park EY, Kwon OB, Jeong BC, Yi JS, Lee CS, Ko YG, Song HK. Crystal structure of
189. Morita E, Sandrin V, Chung HY, Morham SG, Gygi SP, Rodesch CK, Sundquist WI.
PRY-SPRY domain of human TRIM72. Proteins 78: 790 –795, 2010.
Human ESCRT and ALIX proteins interact with proteins of the midbody and function
in cytokinesis. EMBO J 26: 4215– 4227, 2007. 213. Parker MW, Feil SC. Pore-forming protein toxins: from structure to function. Prog
Biophys Mol Biol 88: 91–142, 2005.
190. Moss SE, Morgan RO. The annexins. Genome Biol 5: 219, 2004.
214. Perretti M, Gavins FN. Annexin 1: an endogenous anti-inflammatory protein. News
191. Murphy E, Cross H, Steenbergen C. Sodium regulation during ischemia versus rep-
Physiol Sci 18: 60 – 64, 2003.
erfusion and its role in injury. Circ Res 84: 1469 –1470, 1999.
215. Peters C, Mayer A. Ca2⫹/calmodulin signals the completion of docking and triggers a
192. Murphy E, Cross HR, Steenbergen C. Is Na/Ca exchange during ischemia and reper-
late step of vacuole fusion. Nature 396: 575–580, 1998.
fusion beneficial or detrimental? Ann NY Acad Sci 976: 421– 430, 2002.
216. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL. Dystrophin protects the
193. Murphy E, Steenbergen C. Mechanisms underlying acute protection from cardiac
sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci
ischemia-reperfusion injury. Physiol Rev 88: 581– 609, 2008.
USA 90: 3710 –3714, 1993.
194. Murphy RM. Calpains, skeletal muscle function and exercise. Clin Exp Pharmacol
217. Petros AM, Medek A, Nettesheim DG, Kim DH, Yoon HS, Swift K, Matayoshi ED,
Physiol 37: 385–391, 2010.
Oltersdorf T, Fesik SW. Solution structure of the antiapoptotic protein bcl-2. Proc
195. Murphy RM, Verburg E, Lamb GD. Ca2⫹ activation of diffusible and bound pools of Natl Acad Sci USA 98: 3012–3017, 2001.
mu-calpain in rat skeletal muscle. J Physiol 576: 595– 612, 2006.
218. Piccolo F, Moore SA, Ford GC, Campbell KP. Intracellular accumulation and reduced
196. Nalefski EA, Falke JJ. Location of the membrane-docking face on the Ca2⫹-activated sarcolemmal expression of dysferlin in limb– girdle muscular dystrophies. Ann Neurol
C2 domain of cytosolic phospholipase A2. Biochemistry 37: 17642–17650, 1998. 48: 902–912, 2000.

197. Napolitano LM, Meroni G. TRIM family: pleiotropy and diversification through homo- 219. Potez S, Luginbuhl M, Monastyrskaya K, Hostettler A, Draeger A, Babiychuk EB.
multimer and heteromultimer formation. IUBMB Life 64: 64 –71, 2012. Tailored protection against plasmalemmal injury by annexins with different Ca2⫹
sensitivities. J Biol Chem 286: 17982–17991, 2011.
198. Nelson KL, Raja SM, Buckley JT. The glycosylphosphatidylinositol-anchored surface
glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin. J Biol Chem 220. Pramono ZA, Tan CL, Seah IA, See JS, Kam SY, Lai PS, Yee WC. Identification and
272: 12170 –12174, 1997. characterisation of human dysferlin transcript variants: implications for dysferlin mu-
tational screening and isoforms. Hum Genet 125: 413– 420, 2009.
199. Newham DJ, Jones DA, Edwards RH. Plasma creatine kinase changes after eccentric
and concentric contractions. Muscle Nerve 9: 59 – 63, 1986. 221. Proske U, Morgan DL. Muscle damage from eccentric exercise: mechanism, mechan-
ical signs, adaptation and clinical applications. J Physiol 537: 333–345, 2001.
200. Nguyen K, Bassez G, Krahn M, Bernard R, Laforet P, Labelle V, Urtizberea JA, Fig-
arella-Branger D, Romero N, Attarian S, Leturcq F, Pouget J, Levy N, Eymard B. 222. Pryor PR, Mullock BM, Bright NA, Gray SR, Luzio JP. The role of intraorganellar Ca2⫹
Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes
atypical phenotypes. Arch Neurol 64: 1176 –1182, 2007. from hybrid organelles. J Cell Biol 149: 1053–1062, 2000.

201. Nilius B, Owsianik G. The transient receptor potential family of ion channels. Genome 223. Quaderi NA, Schweiger S, Gaudenz K, Franco B, Rugarli EI, Berger W, Feldman GJ,
Biol 12: 218, 2011. Volta M, Andolfi G, Gilgenkrantz S, Marion RW, Hennekam RC, Opitz JM, Muenke M,
Ropers HH, Ballabio A. Opitz G/BBB syndrome, a defect of midline development, is
202. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for
due to mutations in a new RING finger gene on Xp22. Nature Genet 17: 285–291,
cellular regulation. Nature 334: 661– 665, 1988.
1997.
203. Obita T, Saksena S, Ghazi-Tabatabai S, Gill DJ, Perisic O, Emr SD, Williams RL.
224. Raiborg C, Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated
Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4.
membrane proteins. Nature 458: 445– 452, 2009.
Nature 449: 735–739, 2007.

204. Odorizzi G, Babst M, Emr SD. Fab1p PtdIns(3)P 5-kinase function essential for protein 225. Rambotti MG, Spreca A, Donato R. Immunocytochemical localization of annexins V
sorting in the multivesicular body. Cell 95: 847– 858, 1998. and VI in human placentae of different gestational ages. Cell Mol Biol Res 39: 579 –588,
1993.
205. Ohsako T, Hirai K, Yamamoto MT. The Drosophila misfire gene has an essen-
tial role in sperm activation during fertilization. Genes Genet Syst 78: 253–266, 226. Rand JH, Wu XX, Lapinski R, van Heerde WL, Reutelingsperger CP, Chen PP,
2003. Ortel TL. Detection of antibody-mediated reduction of annexin A5 anticoagulant
activity in plasmas of patients with the antiphospholipid syndrome. Blood 104:
206. Oling F, Bergsma-Schutter W, Brisson A. Trimers, dimers of trimers, and trimers of 2783–2790, 2004.
trimers are common building blocks of annexin a5 two-dimensional crystals. J Struct
Biol 133: 55– 63, 2001. 227. RayChaudhuri D, Park JT. Escherichia coli cell-division gene ftsZ encodes a novel
GTP-binding protein. Nature 359: 251–254, 1992.
207. Oyler GA, Higgins GA, Hart RA, Battenberg E, Billingsley M, Bloom FE, Wilson MC.
The identification of a novel synaptosomal-associated protein, SNAP-25, differentially 228. Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classifica-
expressed by neuronal subpopulations. J Cell Biol 109: 3039 –3052, 1989. tion of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar com-
partment in class E vps mutants. Mol Biol Cell 3: 1389 –1402, 1992.
208. Ozato K, Shin DM, Chang TH, Morse HC 3rd. TRIM family proteins and their emerg-
ing roles in innate immunity. Nature Rev Immunol 8: 849 – 860, 2008. 229. Reddy A, Caler EV, Andrews NW. Plasma membrane repair is mediated by Ca2⫹-
regulated exocytosis of lysosomes. Cell 106: 157–169, 2001.
209. Pang ZP, Melicoff E, Padgett D, Liu Y, Teich AF, Dickey BF, Lin W, Adachi R, Sudhof
TC. Synaptotagmin-2 is essential for survival and contributes to Ca2⫹ triggering of 230. Redpath G, Woolger N, Piper A, Lemckert F, Lek A, Greer P, North K, Cooper S.
neurotransmitter release in central and neuromuscular synapses. J Neurosci 26: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for
13493–13504, 2006. membrane repair. Mol Biol Cell 25: 3037–3048, 2014.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1237


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

231. Rescher U, Zobiack N, Gerke V. Intact Ca2⫹-binding sites are required for targeting 253. Schiavo G, Stenbeck G, Rothman JE, Sollner TH. Binding of the synaptic vesicle v-SNARE,
of annexin 1 to endosomal membranes in living HeLa cells. J Cell Sci 113: 3931–3938, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at
2000. neurotoxin-treated synapses. Proc Natl Acad Sci USA 94: 997–1001, 1997.

232. Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, Riganelli D, Zanaria E, 254. Schwane JA, Johnson SR, Vandenakker CB, Armstrong RB. Delayed-onset muscular
Messali S, Cainarca S, Guffanti A, Minucci S, Pelicci PG, Ballabio A. The tripartite motif soreness and plasma CPK and LDH activities after downhill running. Med Sci Sports
family identifies cell compartments. EMBO J 20: 2140 –2151, 2001. Exercise 15: 51–56, 1983.

233. Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Bren- 255. Shao H, Chou J, Baty CJ, Burke NA, Watkins SC, Stolz DB, Wells A. Spatial localization
guier L, Devaud C, Pasturaud P, Roudaut C. Mutations in the proteolytic enzyme of m-calpain to the plasma membrane by phosphoinositide biphosphate binding dur-
calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27– 40, 1995. ing epidermal growth factor receptor-mediated activation. Mol Cell Biol 26: 5481–
5496, 2006.
234. Rizo J, Rosenmund C. Synaptic vesicle fusion. Nature Struct Mol Biol 15: 665– 674,
2008. 256. Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, Shaheen F, Sheibani N,
Bernatchez P. A new role for the muscle repair protein dysferlin in endothelial cell
235. Roche JA, Lovering RM, Bloch RJ. Impaired recovery of dysferlin-null skeletal muscle
adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 30: 2196 –2204, 2010.
after contraction-induced injury in vivo. Neuroreport 19: 1579 –1584, 2008.
257. Shatursky O, Heuck AP, Shepard LA, Rossjohn J, Parker MW, Johnson AE, Tweten
236. Roche JA, Lovering RM, Roche R, Ru LW, Reed PW, Bloch RJ. Extensive mononuclear
RK. The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a
infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle
novel paradigm for pore-forming toxins. Cell 99: 293–299, 1999.
from contraction-induced injuries. Am J Physiol Cell Physiol 298: C298 –C312, 2010.
258. Shen SS, Tucker WC, Chapman ER, Steinhardt RA. Molecular regulation of membrane
237. Romisch J, Seiffge D, Reiner G, Paques EP, Heimburger N. In-vivo antithrombotic
resealing in 3T3 fibroblasts. J Biol Chem 280: 1652–1660, 2005.
potency of placenta protein 4 (annexin V). Thromb Res 61: 93–104, 1991.
259. Shields DC, Schaecher KE, Saido TC, Banik NL. A putative mechanism of demyelina-
238. Roostalu U, Strahle U. In Vivo imaging of molecular interactions at damaged sarco-
tion in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA 96:
lemma. Dev Cell 22: 515–529, 2012.
11486 –11491, 1999.
239. Rosado CJ, Buckle AM, Law RH, Butcher RE, Kan WT, Bird CH, Ung K, Browne KA,
Baran K, Bashtannyk-Puhalovich TA, Faux NG, Wong W, Porter CJ, Pike RN, Ellisdon 260. Shogomori H, Kobayashi T. Lysenin: a sphingomyelin specific pore-forming toxin.
AM, Pearce MC, Bottomley SP, Emsley J, Smith AI, Rossjohn J, Hartland EL, Vosko- Biochim Biophys Acta 1780: 612– 618, 2008.
boinik I, Trapani JA, Bird PI, Dunstone MA, Whisstock JC. A common fold mediates
261. Song R, Peng W, Zhang Y, Lv F, Wu HK, Guo J, Cao Y, Pi Y, Zhang X, Jin L, Zhang M,
vertebrate defense and bacterial attack. Science 317: 1548 –1551, 2007.
Jiang P, Liu F, Meng S, Zhang X, Jiang P, Cao CM, Xiao RP. Central role of E3 ubiquitin
240. Rosado CJ, Kondos S, Bull TE, Kuiper MJ, Law RH, Buckle AM, Voskoboinik I, Bird PI, ligase MG53 in insulin resistance and metabolic disorders. Nature 494: 375–379, 2013.
Trapani JA, Whisstock JC, Dunstone MA. The MACPF/CDC family of pore-forming
262. Sorimachi H, Ono Y. Regulation and physiological roles of the calpain system in
toxins. Cell Microbiol 10: 1765–1774, 2008.
muscular disorders. Cardiovasc Res 96: 11–22, 2012.
241. Rosengarth A, Luecke H. A calcium-driven conformational switch of the N-terminal
263. Sorimachi H, Suzuki K. The structure of calpain. J Biochem 129: 653– 664, 2001.
and core domains of annexin A1. J Mol Biol 326: 1317–1325, 2003.
264. Spencer MJ, Guyon JR, Sorimachi H, Potts A, Richard I, Herasse M, Chamberlain J,
242. Roux I, Safieddine S, Nouvian R, Grati M, Simmler MC, Bahloul A, Perfettini I, Le Gall
Dalkilic I, Kunkel LM, Beckmann JS. Stable expression of calpain 3 from a muscle
M, Rostaing P, Hamard G, Triller A, Avan P, Moser T, Petit C. Otoferlin, defective in
transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in
a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell
muscle maturation. Proc Natl Acad Sci USA 99: 8874 – 8879, 2002.
127: 277–289, 2006.
265. Spitzer C, Schellmann S, Sabovljevic A, Shahriari M, Keshavaiah C, Bechtold N, Her-
243. Saatman KE, Creed J, Raghupathi R. Calpain as a therapeutic target in traumatic brain
zog M, Muller S, Hanisch FG, Hulskamp M. The Arabidopsis elch mutant reveals
injury. Neurotherapeutics 7: 31– 42, 2010.
functions of an ESCRT component in cytokinesis. Development 133: 4679 – 4689,
244. Saksena S, Sun J, Chu T, Emr SD. ESCRTing proteins in the endocytic pathway. Trends 2006.
Biochem Sci 32: 561–573, 2007.
266. Stein A, Weber G, Wahl MC, Jahn R. Helical extension of the neuronal SNARE
245. Samson RY, Bell SD. Ancient ESCRTs and the evolution of binary fission. Trends complex into the membrane. Nature 460: 525–528, 2009.
Microbiol 17: 507–513, 2009.
267. Steinhardt RA, Bi G, Alderton JM. Cell membrane resealing by a vesicular mechanism
246. Samson RY, Obita T, Freund SM, Williams RL, Bell SD. A role for the ESCRT system similar to neurotransmitter release. Science 263: 390 –393, 1994.
in cell division in archaea. Science 322: 1710 –1713, 2008.
268. Storr SJ, Carragher NO, Frame MC, Parr T, Martin SG. The calpain system and
247. Sanada S, Komuro I, Kitakaze M. Pathophysiology of myocardial reperfusion injury: cancer. Nature Rev Cancer 11: 364 –374, 2011.
preconditioning, postconditioning, and translational aspects of protective measures.
Am J Physiol Heart Circ Physiol 301: H1723–H1741, 2011. 269. Strack B, Calistri A, Craig S, Popova E, Gottlinger HG. AIP1/ALIX is a binding partner
for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114: 689 – 699, 2003.
248. Sanderfoot A. Increases in the number of SNARE genes parallels the rise of multicel-
lularity among the green plants. Plant Physiol 144: 6 –17, 2007. 270. Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A,
Sorimachi H, Bourenkow G, Bartunik H, Suzuki K, Bode W. The crystal structure of
249. Sardiello M, Cairo S, Fontanella B, Ballabio A, Meroni G. Genomic analysis of the TRIM calcium-free human m-calpain suggests an electrostatic switch mechanism for activa-
family reveals two groups of genes with distinct evolutionary properties. BMC Evol Biol tion by calcium. Proc Natl Acad Sci USA 97: 588 –592, 2000.
8: 225, 2008.
271. Stys PK. General mechanisms of axonal damage and its prevention. J Neurol Sci 233:
250. Scheffer LL, Sreetama SC, Sharma N, Medikayala S, Brown KJ, Defour A, Jaiswal JK. 3–13, 2005.
Mechanism of Ca2⫹-triggered ESCRT assembly and regulation of cell membrane
repair. Nature Commun 5: 5646, 2014. 272. Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K. Crystal structure of human
serum albumin at 2.5 A resolution. Protein Engineering 12: 439 – 446, 1999.
251. Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed JC. Channel formation
by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA 94: 5113–5118, 1997. 273. Supnet C, Bezprozvanny I. Neuronal calcium signaling, mitochondrial dysfunction, and
Alzheimer’s disease. J Alzheimer’s Dis 20 Suppl 2: S487– 498, 2010.
252. Schiavo G, Santucci A, Dasgupta BR, Mehta PP, Jontes J, Benfenati F, Wilson MC,
Montecucco C. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct 274. Sutton RB, Davletov BA, Berghuis AM, Sudhof TC, Sprang SR. Structure of the first C2 domain
COOH-terminal peptide bonds. FEBS Lett 335: 99 –103, 1993. of synaptotagmin I: a novel Ca2⫹/phospholipid-binding fold. Cell 80: 929–938, 1995.

1238 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
MEMBRANE REPAIR: MECHANISMS AND PATHOPHYSIOLOGY

275. Sutton RB, Fasshauer D, Jahn R, Brunger AT. Crystal structure of a SNARE complex 296. Tweten RK, Parker MW, Johnson AE. The cholesterol-dependent cytolysins. Curr Top
involved in synaptic exocytosis at 2.4 A resolution. Nature 395: 347–353, 1998. Microbiol Immunol 257: 15–33, 2001.

276. Suzuki K, Hata S, Kawabata Y, Sorimachi H. Structure, activation, and biology of 297. Vandre DD, Ackerman WEt Kniss DA, Tewari AK, Mori M, Takizawa T, Robinson JM.
calpain. Diabetes 53 Suppl 1: S12–18, 2004. Dysferlin is expressed in human placenta but does not associate with caveolin. Biol
Reprod 77: 533–542, 2007.
277. Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Fahrenbach JP, Holley-
Cuthrell J, Eskin A, Chen Z, Squire K, Heydemann A, Palmer AA, Nelson SF, McNally 298. Vater CA, Raymond CK, Ekena K, Howald-Stevenson I, Stevens TH. The VPS1 pro-
EM. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proc tein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces
Natl Acad Sci USA 111: 6004 – 6009, 2014. cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol 119:
773–786, 1992.
278. Tait JF, Cerqueira MD, Dewhurst TA, Fujikawa K, Ritchie JL, Stratton JR. Evaluation of
annexin V as a platelet-directed thrombus targeting agent. Thrombosis Res 75: 491– 299. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 76: 387– 417, 2007.
501, 1994.
300. Vergarajauregui S, Martina JA, Puertollano R. Identification of the penta-EF-hand
279. Talukder MA, Yang F, Nishijima Y, Chen CA, Kalyanasundaram A, Periasamy M, protein ALG-2 as a Ca2⫹-dependent interactor of mucolipin-1. J Biol Chem 284:
Zweier JL. Reduced SERCA2a converts sub-lethal myocardial injury to infarction and 36357–36366, 2009.
affects postischemic functional recovery. J Mol Cell Cardiol 46: 285–287, 2009.
301. Vetter IR, Parker MW, Tucker AD, Lakey JH, Pattus F, Tsernoglou D. Crystal struc-
280. Tam C, Idone V, Devlin C, Fernandes MC, Flannery A, He X, Schuchman E, Tabas I,
ture of a colicin N fragment suggests a model for toxicity. Structure 6: 863– 874, 1998.
Andrews NW. Exocytosis of acid sphingomyelinase by wounded cells promotes en-
docytosis and plasma membrane repair. J Cell Biol 189: 1027–1038, 2010. 302. Voigt T, Sebald HJ, Schoenauer R, Levano S, Girard T, Hoppeler HH, Babiychuk EB,
Draeger A. Annexin A1 is a biomarker of T-tubular repair in skeletal muscle of
281. Taneike M, Mizote I, Morita T, Watanabe T, Hikoso S, Yamaguchi O, Takeda T, Oka
nonmyopathic patients undergoing statin therapy. FASEB J 27: 2156 –2164, 2013.
T, Tamai T, Oyabu J, Murakawa T, Nakayama H, Nishida K, Takeda J, Mochizuki N,
Komuro I, Otsu K. Calpain protects the heart from hemodynamic stress. J Biol Chem 303. Von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE,
286: 32170 –32177, 2011. Davis T, He GP, Cimbora DM, Scott A, Krausslich HG, Kaplan J, Morham SG,
282. Tang J, Maximov A, Shin OH, Dai H, Rizo J, Sudhof TC. A complexin/synaptotagmin 1 Sundquist WI. The protein network of HIV budding. Cell 114: 701–713, 2003.
switch controls fast synaptic vesicle exocytosis. Cell 126: 1175–1187, 2006.
304. Waddell LB, Lemckert FA, Zheng XF, Tran J, Evesson FJ, Hawkes JM, Lek A, Street
283. Taveau M, Bourg N, Sillon G, Roudaut C, Bartoli M, Richard I. Calpain 3 is activated NE, Lin P, Clarke NF, Landstrom AP, Ackerman MJ, Weisleder N, Ma J, North KN,
through autolysis within the active site and lyses sarcomeric and sarcolemmal com- Cooper ST. Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular
ponents. Mol Cell Biol 23: 9127–9135, 2003. dystrophy and localize to longitudinal tubules of the T-system with stretch. J Neuro-
pathol Exp Neurol 70: 302–313, 2011.
284. Teng FY, Wang Y, Tang BL. The syntaxins. Genome Biol 2: 3012, 2001.
305. Walev I, Martin E, Jonas D, Mohamadzadeh M, Muller-Klieser W, Kunz L, Bhakdi S.
285. Terasaki M, Miyake K, McNeil PL. Large plasma membrane disruptions are rapidly resealed by Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma
Ca2⫹-dependent vesicle-vesicle fusion events. J Cell Biol 139: 63–74, 1997. membrane for monovalent ions. Infect Immun 61: 4972– 4979, 1993.

286. Thiagarajan P, Benedict CR. Inhibition of arterial thrombosis by recombinant annexin 306. Wang X, Xie W, Zhang Y, Lin P, Han L, Han P, Wang Y, Chen Z, Ji G, Zheng M,
V in a rabbit carotid artery injury model. Circulation 96: 2339 –2347, 1997. Weisleder N, Xiao RP, Takeshima H, Ma J, Cheng H. Cardioprotection of ischemia/
reperfusion injury by cholesterol-dependent MG53-mediated membrane repair. Cir-
287. Thiery J, Keefe D, Boulant S, Boucrot E, Walch M, Martinvalet D, Goping IS, Bleackley
culation Research 107: 76 – 83, 2010.
RC, Kirchhausen T, Lieberman J. Perforin pores in the endosomal membrane trigger
the release of endocytosed granzyme B into the cytosol of target cells. Nature Immu- 307. Washington NL, Ward S. FER-1 regulates Ca2⫹-mediated membrane fusion during C.
nol 12: 770 –777, 2011. elegans. J Cell Sci 119: 2552–2562, 2006.
288. Thiery J, Keefe D, Saffarian S, Martinvalet D, Walch M, Boucrot E, Kirchhausen T, 308. Weisleder N, Takeshima H, Ma J. Immuno-proteomic approach to excitation-con-
Lieberman J. Perforin activates clathrin- and dynamin-dependent endocytosis, which traction coupling in skeletal and cardiac muscle: molecular insights revealed by the
is required for plasma membrane repair and delivery of granzyme B for granzyme- mitsugumins. Cell Calcium 43: 1– 8, 2008.
mediated apoptosis. Blood 115: 1582–1593, 2010.
309. Weisleder N, Takizawa N, Lin P, Wang X, Cao C, Zhang Y, Tan T, Ferrante C, Zhu H,
289. Tidball JG, Spencer MJ. Calpains and muscular dystrophies. Int J Biochem Cell Biol 32:
Chen PJ, Yan R, Sterling M, Zhao X, Hwang M, Takeshima M, Cai C, Cheng H,
1–5, 2000.
Takeshima H, Xiao RP, Ma J. Recombinant MG53 protein modulates therapeutic cell
290. Tischfield MA, Baris HN, Wu C, Rudolph G, Van Maldergem L, He W, Chan WM, Andrews C, membrane repair in treatment of muscular dystrophy. Science Translational Med 4:
Demer JL, Robertson RL, Mackey DA, Ruddle JB, Bird TD, Gottlob I, Pieh C, Traboulsi EI, 139ra185, 2012.
Pomeroy SL, Hunter DG, Soul JS, Newlin A, Sabol LJ, Doherty EJ, de Uzcategui CE, de
310. Wenzel K, Geier C, Qadri F, Hubner N, Schulz H, Erdmann B, Gross V, Bauer D,
Uzcategui N, Collins ML, Sener EC, Wabbels B, Hellebrand H, Meitinger T, de Berardinis T,
Dechend R, Dietz R, Osterziel KJ, Spuler S, Ozcelik C. Dysfunction of dysferlin-
Magli A, Schiavi C, Pastore-Trossello M, Koc F, Wong AM, Levin AV, Geraghty MT, Descartes
deficient hearts. J Mol Med 85: 1203–1214, 2007.
M, Flaherty M, Jamieson RV, Moller HU, Meuthen I, Callen DF, Kerwin J, Lindsay S, Meindl A,
Gupta ML Jr, Pellman D, Engle EC. Human TUBB3 mutations perturb microtubule dynamics,
311. Wickstead B, Gull K. The evolution of the cytoskeleton. J Cell Biol 194: 513–525, 2011.
kinesin interactions, and axon guidance. Cell 140: 74–87, 2010.
312. Wiener M, Freymann D, Ghosh P, Stroud RM. Crystal structure of colicin Ia. Nature
291. Togo T, Alderton JM, Bi GQ, Steinhardt RA. The mechanism of facilitated cell mem-
385: 461– 464, 1997.
brane resealing. J Cell Sci 112: 719 –731, 1999.
313. Xie XY, Barrett JN. Membrane resealing in cultured rat septal neurons after neurite
292. Togo T, Krasieva TB, Steinhardt RA. A decrease in membrane tension precedes
transection: evidence for enhancement by Ca2⫹-triggered protease activity and cy-
successful cell-membrane repair. Mol Biol Cell 11: 4339 – 4346, 2000.
toskeletal disassembly. J Neurosci 11: 3257–3267, 1991.
293. Togo T, Steinhardt RA. Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-
314. Yamasaki S, Baumeister A, Binz T, Blasi J, Link E, Cornille F, Roques B, Fykse EM,
dependent process of cell membrane repair. Mol Biol Cell 15: 688–695, 2004.
Sudhof TC, Jahn R. Cleavage of members of the synaptobrevin/VAMP family by types
294. Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, D and F botulinal neurotoxins and tetanus toxin. J Biol Chem 269: 12764 –12772, 1994.
Friedrich P. On the sequential determinants of calpain cleavage. J Biol Chem 279:
20775–20785, 2004. 315. Yasunaga S, Grati M, Cohen-Salmon M, El-Amraoui A, Mustapha M, Salem N,
El-Zir E, Loiselet J, Petit C. A mutation in OTOF, encoding otoferlin, a FER-1-like
295. Trimble WS, Cowan DM, Scheller RH. VAMP-1: a synaptic vesicle-associated integral protein, causes DFNB9, a nonsyndromic form of deafness. Nature Genet 21:
membrane protein. Proc Natl Acad Sci USA 85: 4538 – 4542, 1988. 363–369, 1999.

Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org 1239


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.
SANDRA T. COOPER AND PAUL L. McNEIL

316. Yeung EW, Whitehead NP, Suchyna TM, Gottlieb PA, Sachs F, Allen DG. Effects of 320. Zitzer A, Palmer M, Weller U, Wassenaar T, Biermann C, Tranum-Jensen J,
stretch-activated channel blockers on [Ca2⫹]i and muscle damage in the mdx mouse. Bhakdi S. Mode of primary binding to target membranes and pore formation
J Physiol 562: 367–380, 2005. induced by Vibrio cholerae cytolysin (hemolysin). Eur J Biochem 247: 209 –216,
1997.
317. Zatz M, Starling A. Calpains and disease. N Engl J Med 352: 2413–2423, 2005.
321. Zitzer A, Wassenaar TM, Walev I, Bhakdi S. Potent membrane-permeabilizing and
318. Zhang M, Fishman Y, Sher D, Zlotkin E. Hydralysin, a novel animal group-selective
cytocidal action of Vibrio cholerae cytolysin on human intestinal cells. Infect Immun 65:
paralytic and cytolytic protein from a noncnidocystic origin in hydra. Biochemistry 42:
8939 – 8944, 2003. 1293–1298, 1997.

319. Zhu H, Lin P, De G, Choi KH, Takeshima H, Weisleder N, Ma J. Polymerase tran- 322. Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation
scriptase release factor (PTRF) anchors MG53 protein to cell injury site for initiation of following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84: 1404 –1407,
membrane repair. J Biol Chem 286: 12820 –12824, 2011. 1987.

1240 Physiol Rev • VOL 95 • OCTOBER 2015 • www.prv.org


Downloaded from www.physiology.org/journal/physrev (213.196.113.090) on July 23, 2019.