The Cytoskeleton

Reading: Alberts et al. 5th edition,

pages 965-988 & 1010-1025
Tutorial on Thursday
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Protein Sorting Animations for Review

Animations:
http://bcs.whfreeman.com/lodish5e/
Select Chapter 16
Moving Proteins into Membranes and Organelles
http://www.dnatube.com/video/544/Signal-RecognitionParticle-SRP
Protein sorting to the ER

The Cytoskeleton
Provides structural support
Positions organelles
Directs intracellular
transport
Involved in locomotion
Required for cell
division
Green = Tubulin, Red = Actin, Blue = DNA
Figure 16-1 Molecular Biology of the Cell ( Garland Science 2008)

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

Three types of filaments form

the cytoskeleton

mainly in
periphery of
cell

also called actin filaments

Microfiloaments:

Actin
Diameter: 5-9nm

2 stranded helix
polymer

Intermediate Filaments:
Intermediate
filament proteins
Diameter: 10nm

Microtubules:
Tubulin
Diameter: 25nm

nuclear lamina under

nuclear membrane

all radiate from one point near nucleus

Panel16-1 Molecular Biology of the Cell ( Garland Science 2008)

Fluorescent secondary
antibody

Immunofluorescence
A technique used to determine the
location of proteins within the cell.
Cells are fixed (not live imaging)

actin
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

A fluoresence microscope is used to

excite the fluorescent molecule and
visualise the light emitted.
Figure 16-1 Molecular Biology of the Cell ( Garland Science 2008)

Green = Tubulin,
Red = Actin, Blue = DNA

Limits of Light Microscopy

The light microscope has a resolution limit:
Due to diffraction
Based on the wavelength of light
~250 nm
(microtubules have a diameter of 25 nm)

Electron microscope:
Electrons, shorter wavelength
Resolution: <10 Angstroms

(>250x better resolution)

Note: nm=10-9, Angstrom = 10-10

An Electron Micrograph of the

Microtubules in a Flagellum

Figure 16-81 Molecular Biology of the Cell ( Garland Science 2008)

Microscopy Techniques
1. Light Microscope:
Advantages:

Detect multiple proteins: Immunofluorescence

Live cell imaging: FRAP, single molecule tracking

Disadvantage:

Resolution limit of ~250 nm

(10x the diameter of microtubules)

2. Electron Microscope:
Advantages:

Best resolution, <10 Angstroms, resolves filament structures

Disadvantages:

Static images only

Difficult to detect multiple proteins
Uses harsher fixatives and treatments to prepare the sample
9

Its a dynamic structure!

For cell motility the actin filaments must rapidly:
Disassemble and Reassemble
At the leading edge (red)
A neutrophil chasing a clump
of bacteria (white arrow)

Microtubules:
Most interphase microtubules radiate
from one microtubule organising centre
(MTOC)
Are reorganised to form the bipolar
mitotic spindles in dividing cells
Figure 16-4 Molecular Biology of the Cell ( Garland Science 2008)

10

Cytoskeletal filaments are composed of

small soluble subunits that form polymers
(or a pathogen)

Leading edge
This feature allows them to be dynamic;
- can be rapidly assembled
and disassembled

Figure 16-7 Molecular Biology of the Cell ( Garland Science 2008)

New
leading
edge
11

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:
both breakages
equally likely

Single chain polymers with :

Thermal stability

Dynamic filament?

Strong Bonds
High
No

Figure 16-8 Molecular Biology of the Cell ( Garland Science 2008)

Weak bonds
Low
Yes
12

Multiple subunits are assembled into

bundles of protofilaments:
Individual protofilaments associate with each other
laterally
Strength is due
to multiple bonds
Each bond does
not need to be as strong

Actin filaments, intermediate filaments and microtubules are all

constructed of more than one protofilament.
thermally stable as well as
+ end is faster growing end

Figure 16-8 Molecular Biology of the Cell ( Garland Science 2008)

dynamic
13

Microtubules (1)
Involved in:

Intracellular transport
Structural support
Cell organisation
hold Golgi stacks
Mitosis
together

Made of:

Tubulin
Long hollow tubes
Stiff
Inextensible (they dont extend)

Figure 16-11 Molecular Biology of the Cell ( Garland Science 2008)

14

Microtubules (2)
Made of individual
subunits of:

non-covalent interactions

-tubulin
-tubulin

Two closely related

globular proteins form:
Tubulin heterodimers

This regular arrangement

of & subunits give the
microtubule polarity

It has a plus end () and a

minus end ()

- end usually radiates

from centrosome

(The minus ends are organised to the centre of the cell attached to the centrosome)
15

Microtubules (3)
13 parallel protofilaments make up the
hollow tubule.
All the bonds between the individual
subunits are non-covalent
The bonds between protofilaments are
weaker than the bonds within each
protofilament

Growth and disassembly of

microtubules occurs at:
the ends

plus
end

a-b bonds stronger

than a-a or b-b

minus
end
16

Microtubule growth is faster at the plus end

Isolated microtubules
are incubated with
tubulin.
Note: this is a bundle of
microtubules isolated from a
cillium
A bundle of microtubules
An individual microtubule
occurs under certain conditions
Figure 16-13 Molecular Biology of the Cell ( Garland Science 2008)

Cilium
17

Tubulin Dimers

free dimers not good at cleaving GTP, only good when as filaments

Free dimers are bound to

GTP (Guanosine triphosphate)

alpha subunit always bound

to GTP, does not hydrolyse it

the T form

Tubulin subunits are

enzymes:
hydrolyse GTP

When this occurs in the

filament GDP is trapped
in the tubulin subunits.
the D form

GTP much higher than GDP in

cytosol
Figure 16-11 Molecular Biology of the Cell ( Garland Science 2008)

18

Incorporation of tubulin into

microtubule filaments
T form = GTP bound
D form = GDP bound

T forms more attracted to

each other

Microtubules have a GTP cap at the plus end when :

- Dimer addition at the plus end is faster than GTP hydrolysis
Figure 16-14a Molecular Biology of the Cell ( Garland Science 2008)

19

The microtubule GTP cap stabilises the plus end

The GTP cap is at the plus end.
This is the faster growing end.

plus
end

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
20

Dynamic Instability

(Polymerisation)

curl off and depolymerizes 100x faster

(Deploymerisation) without GTP cap

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

21

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

The centrosome nucleates the

formation of microtubules.
The minus end is stable
The plus end is dynamic
(grows and shrinks)

Figure 16-30b Molecular Biology of the Cell ( Garland Science 2008)

22

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)
23

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/OrganelleMovement-on-Microtubules
24

Organelles are associated with

microtubules

Red = tubulin, Green = peroxisomal protein-GFP

25

The Dynein Motor Protein Complex

A microtubule motor

helps move things

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

Figure 16-67 Molecular Biology of the Cell ( Garland Science 2008)

26

Axonal Vesicular Transport

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

(where centrosomes are)

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

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Microfilaments/Actin Filaments (1)

Involved in:

Cell Motility
Contractile activity

Made of:

Actin monomers
Flexible
Inextensible
Helical filaments

Motor protein:
Myosin

Figure 16-47 Molecular Biology of the Cell ( Garland Science 2008)

28

Microfilaments/Actin Filaments (2)

Composed of a single type of
globular protein:
hydrophilic

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)

Figure 16-12 Molecular Biology of the Cell ( Garland Science 2008)

29

Actin Monomers
Free monomers are bound ATP:

ATP is bound in the centre of the protein

Actin is an ATPase:

Hydrolyses ATP
ADP remains bound

ATP hydrolysis occurs more

rapidly after actin monomers
have been incorporated into
the filament

Growth of the filament is:

faster at the plus end

Actin filaments have an ATP cap

Figure 16-12 Molecular Biology of the Cell ( Garland Science 2008)

30

Actin polymerisation in a test tube

CC=
critical concentration

elongation as soon as 3 get together

raising temperature also works
Figure 16-10a Molecular Biology of the Cell ( Garland Science 2008)

31

Actin Filament Treadmilling

Although an actin filament looks stable

There is continual exchange of dimers at the ends

Polymerisation is faster:

at the plus end, has higher affinity

for actin monomers

A pulse of
labelled actin
monomers

At the treadmilling concentration:

net loss of monomers at the minus end

As this happens over time all the

monomers are eventually replaced
treadmilling

Panel 16-2 Molecular Biology of the Cell ( Garland Science 2008)

32

Cc(T) =
critical concentration for
T form

Cc(D) =
critical concentration for
D form
[monomer]>Cc
to + end of filament
and <Cc for - end, treadmilling occurs
concentration where there is
equilibrium between free and polymer forms
net addition above Cc, net loss below

Figure 16-14b Molecular Biology of the Cell ( Garland Science 2008)

33

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

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

34

Myosin is a family of related proteins

Skeletal muscle:
Myosin II
N-terminus

C-terminus

The motor
domains are
conserved
within the
myosin family
termini sections very diverse
Figure 16-57 Molecular Biology of the Cell ( Garland Science 2008)

most move toward

+ end, except VI
35

Myosin II forms large bipolar

thick filaments in muscle

Dark band: myosin thick filaments

The light band contains:
Actin filaments

The sliding of Myosin II along actin

filaments causes muscles to contract
Figure 16-55 Molecular Biology of the Cell ( Garland Science 2008)
Figure 16-74 Molecular Biology of the Cell ( Garland Science 2008)

36

1) Myosin attached to an actin filament

2) ATP binding releases myosin from actin
3) ATP hydrolysis occurs along with a
conformational change that displaces the
myosin head moves toward + end
4) Myosin binding to actin releases the Pi
triggering the force-generating shape change
5) At the end of the cycle myosin is in the
initial conformation but has moved to a new
position in the actin filament
Figure 16-61 Molecular Biology of the Cell ( Garland Science 2008)

37

Summary of motor proteins

Actin motor protein:

Myosin, plus end directed

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).
38

Intermediate Filaments (1)

Involved in:

Structural support

Made of:

Several different proteins

Tough, flexible
Extensible
(they can extend)

Not found in plants

Not in all animal cells
Figure 16-9 Molecular Biology of the Cell ( Garland Science 2008)

39

Intermediate Filaments (2)

Coiled-coil dimer forms a
staggered antiparallel tetramer:
No filament polarity
No known motor proteins

The soluble form is a

tetramer

Figure 16-19 Molecular Biology of the Cell ( Garland Science 2008)

40

Intermediate Filaments (3)

Pack together into ropelike filaments
Prominent in cells subjected to mechanical stress:
Epithelial cells (keratin)
Neurons (neurofilament proteins)
Muscle cells (desmin)

Figure 16-19 Molecular Biology of the Cell ( Garland Science 2008)

41

Keratin Filaments in Epithelial Cells

Filaments in each cell are anchored:
at sites of cell-cell contact
by desmosomes
mechanical strength

Green = Keratin,
Blue = cell boundary
Figure 16-20 Molecular Biology of the Cell ( Garland Science 2008)

42

Remember.

Figure 11-11 Molecular Biology of the Cell ( Garland Science 2008)

43

Cytoskeletal Components in
Epithelial Cell Polarity
Bundled actin
Actin bands
Intermediate filaments
Microtubules
-cell polarity

for directional transport

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

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45

End

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