The Cytoskeleton

Reading: Alberts et al. 5th edition,

pages 965-988 & 1010-1022
Tutorial on Thursday
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 The Cytoskeleton:
• Provides structural support
• Positions organelles
• Directs vesicular transport
• Involved in locomotion

• Required for cell division

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 The cytoskeleton provides structural support

• A red blood cell poked with laser tweezers

returns to its original shape due to the
structural support provided by the
cytoskeleton

• http://www.dnatube.com/video/4159/Membr
ane-Effects-in-a-Red-Blood-Cell

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 Three types of filaments form
the cytoskeleton
• Microfiloaments:
– Actin
– Diameter: 5-9nm
around cell periphery, cell cortex

• Intermediate Filaments:
– Intermediate
filament proteins
– Diameter: 10nm made of keratin

• Microtubules:

– Tubulin
– Diameter: 25nm hollow tube, and begin at centrosome,
near nucleus

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Panel16-1 Molecular Biology of the Cell (© Garland Science 2008)
 Fluorescent secondary
Immunofluorescence antibody

• A technique used to determine the

location of proteins within the cell. actin
• Cells are fixed (not live imaging)
Primary antibody
• An antibody is used which binds
specifically to the protein of interest.
– The primary antibody

• A second antibody binds to the first

antibody and is covalently tagged with
a fluorescent molecule
– The secondary antibody

has a fluorescent molecule on it

• A fluorescence microscope is used to
excite the fluorescent molecule and Green = Tubulin,
visualise the light emitted. Red = Actin, Blue = DNA
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Figure 16-1 Molecular Biology of the Cell (© Garland Science 2008) actin at periphery, tubulin extending from near nucleus
 Limits of Light Microscopy
• The light microscope has a resolution limit:
– Due to diffraction - bending of light waves
– Based on the wavelength of light
– ~250 nm
(microtubules have a diameter of 25 nm)

• Electron microscope: skeletal muscles - work-out muscles

– Electrons, shorter wavelength

– Resolution: <10 Angstroms
(>250x better resolution)
Note: nm=10-9, Angstrom = 10-10 6
 The Microtubules in a Flagellum
Can't even see this with a light microscope; must have an electron microscope**

Tubulin localization in the

biflagellate green alga
www.haverford.edu/biology/Johnson/
Figure 16-81 Molecular Biology of the Cell (© Garland Science 2008)
Chlamydomonas 7
 It’s a dynamic structure!
• For cell motility/crawling the actin filaments must rapidly:
– Disassemble and reassemble flagellum motion - microtubules;
"amoeba-like" motion - actin filaments/
– At the leading edge (red) microfilaments

A neutrophil chasing a clump

of bacteria (white arrow)
neutrophils use ACTIN FILAMENTS,
OR MICROFILAMENTS.

• Microtubules:
– Most interphase microtubules radiate
from one microtubule organising centre

– Are reorganised to form the bipolar

mitotic spindles in dividing cells
- moves item within the cell (i.e. chromosomes in
metaphase) 8
Figure 16-4 Molecular Biology of the Cell (© Garland Science 2008)
 Cytoskeletal filaments are composed of
small soluble subunits that form polymers
(or a pathogen)

leading edge

-where cell is moving towards

-actin will (de/) polymerise here

This feature allows them to be dynamic; new leading edge

- can be rapidly assembled responds to signal
and disassembled - stretches, grabs,
moves forward

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Figure 16-7 Molecular Biology of the Cell (© Garland Science 2008)
 How are these polymers constructed?
• They need to be: strong, flexible and easy to disassemble and reassemble

• Are cytoskeletal filaments constructed as single chains of soluble subunits

or multiple chains?

• Properties of single chain polymers:

red: intermolecular
bonds

–Single chain polymers with : Strong Bonds Weak bonds

–
Thermal stability High
High Low
Low
–
Dynamic filament? No
No Yes
Yes
harder to disassemble with stronger bonds; either not stable or not dynamic
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Figure 16-8 Molecular Biology of the Cell (© Garland Science 2008)
 Multiple subunits are assembled into
bundles of protofilaments:
– Individual protofilaments associate with each other
laterally

 Strength is due
to many weak bonds that associate
laterally made of more than one
protofilament
 Each bond does
not need to be as strong
intermolecular bonds in four directions***

 Actin filaments, intermediate filaments and microtubules are all

constructed of more than one protofilament.

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Figure 16-8 Molecular Biology of the Cell (© Garland Science 2008)
 Actin polymerisation in a test tube

here some
critical concentration addition and
some
subtraction, in
a steady state

starts going really fast

start to polymerise

^ all in monomer form here

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Figure 16-10a Molecular Biology of the Cell (© Garland Science 2008)
 Microtubules (1)
• Involved in:
– Intracellular transport - within the cell
– Structural support
– Cell organisation
– Mitosis - metaphase chromosomal movement
– Cell motility (flagella and cillia)

• Made of:
– Tubulin
– Long hollow tubes
– Stiff
– Inextensible (they don’t extend)
- not much elasticity, can't strech; however,
you can ADD or SUBTRACT to change length
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Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008)
 Microtubules (2)
• Made of individual beta end is plus end
subunits of:
– alpha-tubulin
– beta-tubulin

• Two closely related intermolecular interactions

globular proteins form: between alpha and beta
– Tubulin heterodimers Also occurs between heterodimers

• This regular arrangement

of α & β subunits give the
microtubule polarity
– It has a plus end (β) and a
minus end (α) end with an alpha is
minus end
(The minus ends are organised to the centre of the cell attached to the centrosome)
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 Microtubules (3)
• 13 parallel protofilaments make up the
hollow tubule.

– All the bonds between the individual plus

end
subunits are noncovalent
non-covalent
– The bonds between protofilaments are
weaker than the bonds within each
protofilament
the sideways bonds are weaker than the alpha-beta in
the heterodimers
• Growth and disassembly of
microtubules occurs at:
minus
 the ends, add and subtract new ones end

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 In vitro microtubule growth is faster at the plus end

• Isolated microtubules
are incubated with a
high concentration of
tubulin and GTP.

Note: this is a bundle of

microtubules isolated from a
cillium
bundle of microtubules (darker)

individual microtubule (lighter)

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Figure 16-13 Molecular Biology of the Cell (© Garland Science 2008)
 Tubulin Dimers
• Free dimers are bound to
GTP (Guanosine triphosphate)
 the "T" form

• Tubulin subunits are

enzymes:
– can hydrolyse GTP
cleaves into GDP + P

• When this occurs in the

filament GDP is trapped
in the tubulin subunits.
 the "D" form
"Guanosine diphosphate"
when assembled into protofilament, can
convert into GDP much better - the beta is much better at this, with alpha usually as "T" form
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Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008)
 Incorporation of tubulin into
microtubule filaments
(one heterodimer- alpha & beta)

D form has alpha stuck to GTP and beta

cleaved GTP to GDP

T form = GTP bound

D form = GDP bound

Adds T-form, but T-form is added quickly

much slower
GTP cap

cap later becomes GDP, new cap

always forming!
Microtubules have a GTP cap at the plus end when :
- Dimer addition at the plus end is faster than
faster than GTP hydrolysis
GTP hydrolysis
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Figure 16-14a Molecular Biology of the Cell (© Garland Science 2008)
The microtubule GTP cap stabilises the plus end

• The GTP cap is at the plus end.

plus
• This is the faster growing end. end
heterodimers like to stick with others in the T-form

• The GTP cap stabilises the plus end

– favours tubule growth

– Dimers in the T form bind more strongly

to other dimers in the tubule.
– Hydrolysis of bound GTP reduces the
binding affinity of the subunit.
minus
end

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 Dynamic Instability
if the cap is lost, the protofilaments curl apart at 100x the rate of polymerisation...
-because lateral bonds are very weak - minus end is usually anchored to something
preventing depolymerisation

(Polymerisation) (Deploymerisation)
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Figure 16-16c Molecular Biology of the Cell (© Garland Science 2008)
 In cells Microtubules are Nucleated at the MTOC

• MTOC: Microtubule Organising Centre:

– Microtubules radiate from MTOC
– Joined at their minus ends
– Plus ends radiate out towards
the plasma membrane
-minus end is anchored near the nucleus, very stable

• The centrosome is a MTOC:

– nucleates the formation of microtubules.
– minus end is very stable, anchored
– plus end is dynamic, always changing
the GTP cap, grows and shrinks.

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Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008)
Nerve cells in your spinal cord extend
to your finger tips!
• These neurons can be a
meter long!

Problem:
How does neurotransmitter
synthesized in the ER get to
the synapse?

(The ER and Golgi are located

in the cell body)

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 How do transport vesicles get
to their destination?

• Directed movement of transport vesicles, pulled by

motor proteins along microtubules.

• http://www.dnatube.com/video/4168/Organelle-
Movement-on-Microtubules

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 Organelles are associated with
microtubules

Red = tubulin, Green = peroxisomal protein-GFP

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 The Dynein Motor Protein Complex
• A microtubule motor
move along MICROTUBULES ONLY, NOT ACTINS.

• Minus-end directed:
– Moves towards the minus end

• Dynein is a large protein complex

and is associated with other
protein complexes that together
transport cargo along
microtubules.

• Dynein uses ATP:

– provides the energy to move along the
microtubule

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Figure 16-67 Molecular Biology of the Cell (© Garland Science 2008)
 Axonal Vesicular Transport
minus end at NTOC, near nucleus

plus end
axon

cell body

Dynein movement:
 towards the cell body
(the microtubule minus end)

Kinesin movement:
 towards the axon terminus
(the microtubule plus end)

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 Microfilaments/Actin Filaments (1)
• Involved in:
– Cell Motility (crawling)
– Contractile activity
– Cytokinesis

• Made of:
– Actin monomers
– Flexible
– Inextensible
– Helical filaments

• Motor protein: can contract

movement
structure & stability
– myosin

moves along actin filaments, looks nothing like walky guys

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Figure 16-47 Molecular Biology of the Cell (© Garland Science 2008)
 Microfilaments/Actin Filaments (2)
• Composed of a single type of
globular protein:
– Actin monomers
– Two protofilments twisted in a
right-handed helix

• Are actin filaments polar?

– Yes
– Due to the regular orientation of
actin monomers in each
protofilament.
(Head to tail arrangement of actin monomers)

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Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008)
 Actin Monomers
• Free monomers are bound ATP:
– ATP is bound in the centre of the protein N and C-terminus, alpha-helix

• Actin is an ATPase:
– Hydrolyses ATP
– ADP remains bound

• ATP hydrolysis occurs more

rapidly after actin monomers
have been incorporated into
the filament

none of the extra stuff

• Growth of the filament is:
– faster at the plus end
– Actin filaments have an ATP cap, like
in microtubules with GTP
Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008)
has T and D form, but uses ATP instead of
GTP, it is an ATPase, and can cleave ATP
ATP hydrolysis decreases bond strength between
monomers in the filament, like tubules 29
 Actin Filament Treadmilling
• Although an actin filament looks “stable”
– continual exchange of monomers at the ends
because neither end is anchored, unlike microtubules A pulse of labelled
actin monomers
• Polymerisation is faster:
– at the plus end

• At the treadmilling concentration: gain

– net addition at the plus end loss

– net loss at the minus end

• As this happens over time all the

monomers are eventually replaced
– “treadmilling”
D-form with ADP T-form with
ATP30
Panel 16-2 Molecular Biology of the Cell (© Garland Science 2008)
 Actin treadmilling
• http://www.youtube.com/watch?v=EvqM2JO8R0w

– Also shows other proteins involved in filament

assembly:
• Profilin – speeds elongation
• Cofilin – accelerates disassembly

– In cells several proteins act to regulate filament

dynamics.
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The plus end has a red is new
higher affinity for actin actin

monomers than the monomers

minus end
addition at both ends, lots
at plus, bit at minus

add and subtract is

equal at minus end

Treadmilling
concentration of
monomers reached

THINK OF A TANK.
 Cc(T) =
critical concentration for
the T form

Cc(D) =
critical concentration for
the D form

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Figure 16-14b Molecular Biology of the Cell (© Garland Science 2008)
 Actin Motor Proteins
• Myosins:
– Tails of the two heavy chains organised in:
• a coiled-coil
– Heads of the heavy chains:
• Associated with four light chains (2 each at each head)
– ATP hydrolysed by the myosin head Cleaves ATP at
the head

Figure 16-54 Molecular Biology of the Cell (© Garland Science 2008)

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 Myosin is a family of related proteins

• Skeletal muscle:
– Myosin II

• The motor
domains are
conserved
within the
myosin family

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Figure 16-57 Molecular Biology of the Cell (© Garland Science 2008)
 Muscle cell contractile apparatus
don't memorise structures

• The sliding of Myosin II along

actin filaments causes muscles
to contract
Figure 16-76 Molecular Biology of the Cell (© Garland Science 2008)
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Figure 16-74 Molecular Biology of the Cell (© Garland Science 2008)
 Memorise THESE

1) Myosin attached to an actin filament

default state

2) ATP binding releases myosin from actin

atp stuck to myosin head

3) ATP hydrolysis occurs along with a

conformational change that displaces the
myosin head
myosin moves up a bit

4) Myosin binding to actin releases the Pi

triggering the force-generating shape change
myosin stuck again to actin

5) At the end of the cycle myosin is in the

initial conformation but has moved to a new
position in the actin filament
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Figure 16-61 Molecular Biology of the Cell (© Garland Science 2008)
 Summary of motor proteins
• Actin motor protein:
– Myosin II is plus end directed (goes to axon terminus

• Microtubule motor proteins:

– Dynein, minus end directed

– Kinesin, plus end directed

• Common mechanism:
– They all couple ATP hydrolysis with conformational
changes to generate force
– All move in a specific direction along filaments that have
polarity (plus/minus end).
Both are (de/)polymerised based on the needs of the CELL ******

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 Intermediate Filaments (1)
• Involved in:
– Structural support

• Made of:
– Several different proteins
– Tough, flexible rpoe-like

– Extensible
(they can extend)

• not found in plant cells

• not in all animal cells

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Figure 16-9 Molecular Biology of the Cell (© Garland Science 2008)
 Intermediate Filaments (2)
• Coiled-coil dimer forms a
staggered antiparallel tetramer:
– no filament polarity as with the others

– no known motor proteins (dynein, kinesin, nope.)

• The soluble form is a

tetramer

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Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008)
 Intermediate Filaments (3)
• Pack together into ropelike filaments
• Prominent in cells subjected to mechanical stress:
– Epithelial cells (keratin) keratin sub-units
– Neurons (neurofilament proteins)
– Muscle cells (desmin)

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Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008)
 Keratin Filaments in Epithelial Cells
line surfaces or cavities

• Filaments in each cell are anchored:

– at sites of cell-cell contact
– by desmosomes
– mechanical strength

Green = Keratin,
Blue = cell boundary
desmosomes
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Figure 16-20 Molecular Biology of the Cell (© Garland Science 2008)
 Remember….
apical side - where food is digested :P

basal-
lateral
side

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Figure 11-11 Molecular Biology of the Cell (© Garland Science 2008)
 Cytoskeletal Components in
Epithelial Cell Polarity
Bundled actin

Actin bands

Intermediate filaments
intermediates
anchored here

Microtubules
-cell polarity
microtubules have opposite
polarity! epithelial cells are
special

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Figure 16-5 Molecular Biology of the Cell (© Garland Science 2008)