BIO3153 Cell Biology Caroline Petit-Turcotte

Cytoskeleton (Filament Proteins) Lecture 4

Shivan Desai

The Cytoskeleton:
- Unique to eukaryotic cells
o Tubulin and actin homologs are found in bacteria (transient
structures)
o In eukaryotes they are not permanent and dynamic
o In prokaryotes they are fixed structures with a specific function
- Dynamic 3D structures that fill the cell
- Both muscle and skeleton for the cells
o Allows movement
o Gives shape
- Organization of intracellular
membranes
o In ER and Golgi (brings protein
to membrane, which are carried
along the cytoskeleton)
- Cells have elaborate arrays of protein
fibers which serve functions such as
establishing cell shape, structural
support, mechanical strength, locomotion, chromosome separation in
mitosis and meiosis, and intracellular transport of organelles
- 3 Types of Filaments:
o Actin filaments (AFs or microfilaments)
Found in the periphery of the cell (outside, occupies the
cortex of the cell)
The cell cortex is a specialized layer of cytoplasm on
the inner face of the plasma membrane which
functions as a mechanical support of the plasma
membrane
o In animal cells it is an actin-rich layer
responsible for movements of the cell surface
o Actin networks give a stiff cell cortex which
provides mechanical support for the plasma
membrane
Ex. Red blood cells springing back into
shape after squeezing through a capillary

They are also involved in cell migration (for pseudopodia,

which are stretched extensions of the cytoplasm)
They give shape to the cell

o Microtubules (MTs)
Maintains the cell structure, providing platforms of
transport where organelles can move
They contribute in the formation of the mitotic spindle and
in other cellular processes
In neurons they play the role of transport from cell bodies
to terminals
Two isoforms form a dimer
Dimers form a long chain to form a leaflet

o Intermediate filaments (IFs)

Reinforces cells to organize them into tissues
They provide mechanical support for the plasma
membrane when it comes into contact with other cells or
the extracellular environment

Ex. Keratin intermediate filaments are found in nails

and hair
Very fibrous proteins (not globular)
More flexible than MTs

Cytoskeletal Fibers:
- Monomers of proteins connected end to end to form polymers
- Diversity is in combination of fibers, proteins and proportions

Filament Construction:
- There are small subunits which form filaments
- Actin and tubulin are compact and globular (Ifs are fibers)
- Disassembly, diffusion, reassembly are the steps in formation
- Under given conditions, reverse assembly can occur where large
polymers can disassemble into a common pool of smaller subunits
which is always maintained
- Steps of construction:
o 1) Signals such as a nutrient source signals for the disassembly
of actin filaments

o 2) The subunits diffuse

o 3) The subunits reassemble at a new site into filaments
Weak noncovalent bonds are important for speed of formation
o Hydrophobic, ionic, dipole-dipole bonds
o Harder to break a microfilament than a protofilaments
o The ends are less stable since there are fewer bonds present
One subunit is only bonded with 3 other subunits
A subunit in the middle is bonded with 4
other subunits
This is why addition and removal
of subunits occur at the ends of
the filaments
If they build every filament one by one they are
unstable
o To become stable, addition must occur as a sheet and become
stacked
Makes the filament very stable
Protofilaments in sheets is the most stable configuration
Thermally stable
Sheet structure gives stability to the center
Leaves the ends able to interact (dynamic)
o MTs form in the manner in which protofilaments (long linear
strings of subunits joined end to end) will stack and align in a
circular fashion
o 13 protofilaments = microtubule

Nucleation:
- Limiting step

Nucleation is the term used for

polymerization, or assembly where the
first steps are energetically less
favourable than the continuation of
growth
Actin subunits are proteins which get
the formation of the filaments activated
The monomers will form a dimer, which
will then form a protofilaments, where
two protofilaments wrap around each other
The concentration and availability of the species is called nucleation
In the cytoplasm there is a concentration of ions, and salt, where salt
favours the formation of dimers making it more stable to assemble
o When conditions change, they become less favourable (for
assembly)
o Salt induces nucleation
o The lag phase is the time it takes to nucleate to form the initial
oligomer (where lagging occurs)
After, elongation occurs at a faster rate
The dimers required until equilibrium is where a stable protofilaments
is formed
o The equilibrium state is called treadmilling (at steady-state)
Treadmilling is the addition and deletion of subunits at the
same rate (hence at equilibrium)

Tubulin:
- Heterodimer = 1 subunit
- Added head to tail gives polarity
- Plus ends and minus ends of tubulin
o ends binds GTP on its top (+end = -tubulin, -end = -tubulin)
o ends also binds it to the top (-tubulin)
Hence, -tubulin does not have GTP which is accessible (it
is sequestered)
Means it cannot become hydrolyzed
- Hydrolysis of GTP promotes addition of subunits
o Binding and hydrolysis of GTP occurs at the positive end since
elongation of the polymer occurs
o When GTP is hydrolysed it forms GDP

13 protofilaments fit in 360

Tubulin is made of globular
proteins of two different
subunits
o Dimers attach to each
other end to end

Actin:
- There are 2 protofilaments in
a right-handed helix
- ATP is sequestered due to the
structure of the protein
o Barbed on +end and blunt on end confers polarity
o Polarity has nothing to do with charge, it is simply based on
presence or absence of MTs
o ATP is sequestered since it is inside the protein
Once hydrolysed, ADP is trapped inside
Treadmilling and Dynamic Instability:
- Growth and shrinking of filament proteins
- Actin and tubulin catalyze hydrolysis of ATP and GTP respectively
(catalysis is faster as filaments)
o Catalysed by enzymes
o Loss of phosphate reduces ATP/GTP to ADP/GDP
o ATP cap lost gives ADP, GTP to GDP
o ATP/GTP caps are built up ATP or GTP since hydrolysis is lagged
At some point, hydrolysis catches up
MTs have a GTP cap since they are present on the unit
Easier to grow when GTP is bound to -tubulin
-end is easier to dissociate since GTP is hydrolysed
and sequestered
- T or D form indicates if a triphosphate or diphosphate form exists
- Critical concentration (Cc) is where subunit addition = subunit loss (=
rate constant loss/gain ratio = 1 at treadmilling equal)
o Treadmilling occurs both with actin filaments and microtubules
Can say treadmilling occurs with actin filaments and
dynamic instability occurs with microtubules
- Treadmilling:
o Based on rate of hydrolysis and Cc
o Linear relationship of [subunit] vs. elongation rate
Both ends are dynamic hence, there are two separate lines
on a graph

o end grows less than the +end of actin filaments (difference is

reflected in Cc for addition of monomers at both ends of the
filament)
Actin bound to ATP associates with rapidly growing +ends,
where ATp bound to actin is then hydrolysed into ADP
Since ADP-actin dissociates from filaments more readily
than ATP-actin, the Cc of actin monomers is higher for
deletion at the end than the +end for actin filaments
Treadmilling is where the net dissociation of monomers
(ADP bound) on end = net association of monomers (ATP
bound) at the +end
o Same method goes for tubulin where GTP cap binds monomers
since they are accessible

Actin filaments in treadmilling:

o Perform an experiment to view formation
1) Filaments added to ATP-actin
2) ATP-actin addition occurs at both ends
3) ATP-actin drops, addition is greater at +end
(affinity)
4) Steady state (gain and loss)
5) Treadmilling (no change in length, gain =
loss)
Nucleation is not a factor since pre-formed
filaments were added to actin solution
Growth or deterioration is based on cytosolic
concentrations of available monomer subunits
in the given area
If concentration of subunits is high, addition>loss
If concentration drops until equilibrium, gain=loss

If concentration of ATP-actin is limiting loss>gain

o Loss more at end, but if concentration drops
critically, there is loss at both ends
Microtubules in treadmilling:
o Same systematics as in actin
o Tubulin can be labelled with
rhodamine
Length is constant but the
tubulin moves around
o Dynamic instability of
microtubules implies on how they
may grow steadily, but then shrink
rapidly by the loss of tubulin dimers
at the +end
Dynamic instability only on MTs
GTP cap on MTs are not always blunt
Based on the growth and shrinkage of microtubules
dependant on the concentration present within the
cytosol
The rapid disassembly is referred to as a catastrophe
The tendency to grow or shrink may be a function of
tubulin concentration
As microtubules grow, tubulin dimers are depleted
As tubulin concentration decreases the rate of hydrolysis
catches up, where rapid shrinkage at the +end is
attributed to the loss of the GTP
cap
Hydrolysis affects conformation
If GTP tubulin dimer is
hydrolyzed then it is less
stable since GTP has a more
stable shape than GDP
o Once the cap is
hydrolyzed there is
shrinkage since there
are less bonds at the
ends
o Occurs when the ends
are tapered since GTP is becoming chewed up
o The rigid and strong cap becomes curved and
loses its ability to become a rigid structure

o The dimers cannot stay attached and then a

catastrophe occurs
Recovery is possible
o Can reattach the GTP cap and elongate as
necessary

o Kinetics dependent on tubulin concentration

The actual shape changes the kinetics
Tapered end adds more dimers than
blunt end
o Treadmilling and dynamic instability
ultimately results in spatial and temporal
flexibility with a high turnover,
exploration for attachment sites and
remodelling, and is the fastest way to
grow filaments without nucleation
Necessary for cell growth and
division

Intermediate Filaments:
- Different from actin and tubulin since they are not
small globular proteins (they are fibrous)
- They are organized differently than actin and tubulin
o IFs are able to withstand a greater amount of
deforming force without breaking
o Allows the cell endure force, torque, crushing, etc.
- They have a coiled dimer where two will assemble
- Since they are antiparallel there is no polarity
- Rope-like appearance
- Formation is by spontaneous interaction (no energy required)
- Disassembly likely regulated by phosphorylation
- Formations:
o Dimer formation
o Tetramer formation
Two dimers

 Packed into an array of 16 dimers in cross-section

o Tetramer-tetramer association
Formation of 8 molecules

Epithelial IFs (keratins, etc.)

o More diverse family
o Type I and II keratin chains
o Strength in hair, nails, etc.
Axonal IFs (neurofilaments, etc.)
o Found in central and peripheral axons of vertebrate neurons
o Type L, M or H
o Growth by increase in axon diameter