Biophysics of Molecules

Concepts of cell adhesion and cellular

mechanostransduction – part I

Dr. Carsten Grashoff

MPI of Biochemistry

E-mail: cgrasho@biochem.mpg.de
 The three filament networks

intermediate
f-actin tubulin filaments

thin (7 nm) thick (25 nm) intermediate (10 nm)

ADP/ATP-binding GDP/GTP-binding no nucleotide binding

polarity polarity no polarity

decentralized centralized decentralized

2
 Mechanosensitivity

- the ability to sense

and respond to
mechanical forces
is called
mechanosensitivity

osteocytes myocytes endothelial cells

- all prokaryotic and

eukaryotic cells are
mechanosensitive

epithelial cells cardiomyocytes keratinocytes

3
Many disorders are ‘mechanical diseases’ !

- endothelial cells line the blood

vessels and are constantly
exposed to the shear flow of
the blood

- endothelial cells sense and

respond to the shear flow
DeBakey et al.
1985

- the first atherosclerotic

plaques form in areas of
disturbed blood flow
atherosclerosis

4
 Cancer cells are mechanosensitive

- primary tumors are always

more rigid than their
environment (tumor cells are
more soft though)

- increased tissue rigidity

promotes tumor growth
Levental et al., Cell, 2009

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Many biological processes are mechanosensitive

push - cells generate mechanical

forces to migrate

- but they also sense their

mechanical environment

pull - cells will usually move

towards stiffer substrates; this
process is called durotaxis

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 What we would like to understand

1. How does the cell recognize its

mechanical environment ?

2. How does the cell transduce mechanical

information into a biological response ?

3. How can we measure that ?

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What we would like to understand

1. How does the cell recognize its

mechanical environment ?

What is the mechanical environment ?

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What characterizes the mechanical environment ?

- the mechanical
properties of the
cellular environment
are characterized by
the extracellular
matrix (ECM)

- the ECM dictates

the biochemical
and biophysical
properties of the
environment

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The extracellular matrix (ECM)

The ECM consists of many

different macromolecules.

We distinguish:

- filamentous proteins

- glykosaminoglycanes
(GAGs)

- proteoglycanes

- adhesion proteins

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Collagen: the most abundant EMC protein

- collagen is the most

abundant protein in the
human body

- we know 28 different
collagen subtypes
consisting of 46 distinct
polypeptides

- collagens are
evolutionary conserved

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Collagen and the collagen triple helix

- collagen is characterized
by a typical amino acid
repeat:

(G-X-Y)n

G: glycine
X: proline
Y: hydroxy-proline

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Collagen and the collagen triple helix

- collagen is characterized
by a typical amino acid
repeat:

(G-X-Y)n

G: glycine
X: proline
Y: hydroxy-proline

- the collagen repeat leads to

the formation of a helix with
one repeat per turn

- three helices assemble to

form the collagen triple-helix

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 The role of hydroxyproline

(G-X-Y)n
prolin
X: often Prolin
Y: often Hydroxy-Prolin

- hydroxyprolin stabilizes
the triple-helix through
prolylhydroxylase
stereo-electronic effects
+ Vitamin C
- hydroxyproline is not a
natural amino acid and has
hydroxy- to be synthesized by the cell
prolin
- lack of hydroxyproline
leads to scurvy

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Collagen fibers are very long

- an average collagen triple

helix is 300 nm long and
displays a diameter of less
than 2 nm

- collagen fibers can get 1

cm long and reach a
thickness of 500 nm

- this is achieved through

collagen fibrillogenesis

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Collagen fibrillogenesis is a multi-step process

- collagen synthesis occurs within the cell

- collagen fibrillogenesis occurs extracellulary

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Lysyloxidase (LOX) catalyzes collagen crosslinking

- collagen fibers are

crosslinked during
collagen fibrillogenesis

- lysyloxidase (LOX)
catalyzes the oxidation of
lysine groups

- LOX-expression correlates
with malignancy of breast
cancer

- most primary tumors

are more rigid because of
increased collagen
crosslinking
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Collagens can form many distinct networks

- collagens can form

sheets, fibrils, anchors,
membranes, etc.

- these networks have

distinct biochemical and
mechanical properties

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Defects in the collagen protein cause diseases

Scurvy Osteogenesis
(general) Imperfecta (Col1A)

Morbus Ehlers-Danlos Stickler-Syndrom (Col2A,

(Col1A, Col3A, Col5A) Col2A, Col11A) 19
Mechanical properties of the ECM

The problem

Collagens mediate tensile strength.

But the ECM has to be elastic as well.

What mediates matrix elasticity ?

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 Our tissue needs to be elastic !

skin blood vessels

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Elastin gives tissues their elasticity

- many tissues need to be

elastic such as the skin, lung or
blood vessels
- elastin content increases by
500 % in the uterus during
pregnancy

- the typical elastin repeat is

very similar to the collagen
repeat:

(P-G-V-G-V-A)n

- but elastin is essentially

unstructured and behaves as
an entropic elastomer
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 Elastin fibers behave as entropic springs

- individual elastin fibers are

cross-linked through covalent
bonds

- each elastin molecule in the

- ΔS + ΔS
network can extend and
contract in a manner
resembling an entropic spring
so that the elastin fiber will
recoil after transient stretch

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 Elastomeres and the elastic module

- ideal elastomeres show a

linear-elastic
linear-elastic behaviour
non-linear elastic

elastic modulus:
non-elastic
break
E=σ/ε
stress σ [ N / m2 ]

[ N / m2 ] = Pa
[ N / mm2 ] = MPa
[ kN / mm2 ] = GPa

Erubber ~ 0.01-0.1 GPa

strain ε [ Δl / l ]
Ecollagen ~ 1.2 GPa
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 Examples of naturally occurring elastomers

elastin flagelliform resilin

(P-G-V-G-V-A)n (G-P-G-G-A)n (G-G-R-P)n
E ~ 0.0011 GPa E ~ 0.003 GPa E ~ 0.002 GPa

Erubber ~ 0.01-0.1 GPa Ecollagen ~ 1.2 GPa

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 Elastin concentration in the aging skin

- the elastin concentration gradually decreases in aging skin and

tissue elasticity is reduced
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 Mechanical properties of the ECM

The problem

Collagens mediate tensile strength and elastin confers elasticity.

How about compressibility ?

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What mediates compressibility ?

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GAGs mediate compressibility

- glycosaminoglykane
(GAGs) consist of
repeating sugar subunits

- depending on the sugar

side-chain we distinguish:

hyaluronan
chrondoitin sulfate
dermatan sulfate
keratan sulfate
heparan sulfate
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GAGs occupy a large volume

- polysaccharide chains are too stiff

to adopt a compacted structure and
are very hydrophilic

- even at low concentration GAGs

form gels, which attract cations
(such as Na+) causing water
incorporation and swelling

- GAGs fill most of the space in the

cartilage

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 GAG example - Hyaluronan

- the concentration of hyaluronan is reduced in osteoarthritic joints

- injection of hyaluronan into the joint (vascosupplementation) is

used to reduce symptoms
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Proteoglycanes

- proteoglycanes are
composed of GAGs covalently
linked to a core protein

- proteoglycanes are a very

heterogenous group of
molecules and can be complex

- they have many functions

and may serve as adhesion
proteins (syndecan), linker
between other matrix proteins
(decorin) or as a sponge
(aggrecan)

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Proteoglycane - Aggrecan

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 Adhesive glycoproteins

- glycoproteins are smaller and more flexible than proteoglycanes

- glycoproteins only bind small saccharide side chains

- they often connect to other matrix molecules (such as collagen)

- they often self-assemble (i.e. laminin, fibronectin)

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 The adhesive glycoprotein laminin

- laminin can self-assemble (trimer formation)

- laminin interacts with proteoglycanes (perlecan)

- laminin is recognized by cell surface receptors called integrins

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 Summary - ECM

- the ECM composition determines the biochemical and

biophysical properties of the cellular environment

- the ECM contains a large variety of molecules that can

be classified into proteins, glycosaminoglycanes (sugars),
proteoglycanes and glycoproteins

- collagens mediate tensile strength

- elastins mediate elasticity

- GAGs mediate compressibility

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 Summary - ECM

sugars

elastomers

triple helix

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What we would like to understand

1. How does the cell recognize its

mechanical environment ?

How does the cell sense the ECM ?

38
Direct and indirect sensing mechanisms

- ion-channels can rather

unspecifically sense
mechanical forces
(stretch-dependent ion-
channels)

- cells express surface

receptors which bind
distinct ECM molecules:

- we distinguish:

cell-cell adhesions

cell-ECM adhesions
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Overview of cell adhesion structures

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Integrins mediate cell-matrix adhesions

- integrins are heterodimeric

transmembrane proteins and
consists of an a and a b-subunit
β
α extracellular

- integrins bind to various ECM

proteins such as collagens,
laminins or fibronectin

intracellular - integrins are the main ligands for

the ECM

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Human cells express many integrins

a1 β extracellular
a2
a3 α
a4
a5
b1
a6
b2
a7
b3
a8
a9 b4
a10 b5
a11 b6
aV b7
aIIb b8
aD
aX
intracellular
aL
aM
aE
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Integrin receptor classes

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Integrins are very important

- without integrins
embryos die even
before implantation
into the ueterus !

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Integrins are important for platelet activation

- platelets need to be activated

for aggregation

- b1 and b3 integrins are

essential for platelet
activation

- defective integrins on platelets

leads to thrombastenia
inactive active (bleeding disorder)

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Integrins can be active or inactive

EZM

inactive
conformation active
conformation

Integrin activation is regulated !

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Intracellular activation by talin and kindlin

Kindlin

Talin no (αIIbβ3): no platelet aggregation

no talin-1: no platelet aggregation

no kindlin-3: no platelet aggregation

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Integrin-connection to the cytoskeleton

The problem

Integrins can not bind to the cytoskeleton directly.

How do integrins connect the ECM with the

cytoskeleton?

48
Focal adhesions form at integrin tails

- upon integrin activation and

ECM binding cells form
intracellular complexes called
focal adhesions (FAs)

- FAs consist of 100‘s of proteins,

which link integrins to the
cytoskeleton

- FAs are very dynamic and

>100 Proteine
undergo a constant turnover

- FAs are the cells’ feet !

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 FAs affect many cellular processes

- FAs contain many different

proteins (structural proteins,
enzymes, etc.)

adapter proteins
kinases, phosphatases,
proteases, etc. cell division

cell migration

mechanical integration 50
 Summary – cell adhesion and integrins

- cells adhere to each other using cell-cell contacts

- cells adhere to the ECM using a family of cell surface

receptors called integrins

- integrins have to be activated by intracellular proteins

- integrins connect to the cytoskeleton in subcellular

structures called focal adhesions

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What we would like to understand

2. How does the cell transduce

mechanical stimulation into a biological
information ?

How do integrins (FAs) sense

mechanical forces ?

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Integrin-dependent force transduction

- cells pull on the ECM at

FAs !

- the ECM pulls at cells in

FAs!

What happens on the

molecular level ?

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Talin can interact with integrins and actin

FERM domain
(integrin binding)
vinculin binding domain

actin binding domain

dimerization domain

vinculin binding
domains

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A model of force transduction in FAs

f-actin-talin
binding

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A model of force transduction in FAs

forces of the actin

cytoskeleton

56
A model of force transduction in FAs

vinculin
recruitment

forces of the actin

cytoskeleton

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A model of force transduction in FAs

vinculin
recruitment
to talin

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A model of force transduction in FAs

higher forces at the

integrins

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A model of force transduction in FAs

integrin clustering

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 Measuring force transduction

The problem

We need to know which molecules are exposed to mechanical

forces within the cells.

How can we measure mechanical forces in cells ?

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