The Cytoskeleton Overview of cytoskeletal components

- dynamic system of fibres in cell cytoplasm - helical polymers of actin

- controlled by many different signalling pathways - throughout cell
- concentrated under plasma
responsible for control of: membrane
- cell motility
- maintaining cell structure
- intracellular movement
- hollow cylinders of tubulin
consists of:
- one end attached to centrosome
a) dense and apparently random array of fibres

microfilaments (7-9nm diameter)

intermediate filaments (10nm diameter)
microtubules (24nm diameter)
- rope-like; various proteins
• polymerisation of monomers - in cytoplasm and nucleus
• ordered structures

b) motor proteins – intra- and intercellular movement

Actin F-actin structure

polymer of actin monomers - each 43kDa Actin has structural

polarity
highly conserved, 5-10% of total cellular protein
All monomers point in
monomer is globular actin (G-actin) same direction

polymer is filamentous actin (F-actin) One end is called the

minus (-) end (ATP
ATP and Mg2+ important for polymerization binding cleft exposed)

Other end is called the

(G-actin)n + Mg2+ F-actin + Mg2+ plus (+) end (ATP binding
cleft closed)

ATP ADP + Pi

F- actin assembly is reversible Beads on a string

Three stages of actin polymerisation

Regulation of actin filament length

ATP Proteins which interact with actin influence its ability to polymerise.
ADP

1. G-actin-binding proteins - profilin promotes F-actin assembly

thymosin 4 inhibits F-actin assembly

2. F-actin severing proteins - gelsolin

3. F-actin capping proteins - CapZ binds to barbed end

Tropomodulin binds to pointed end
1. G-monomers all have ATP attached (orange) and slowly form
stable complexes of actin (purple) If one end of F-actin is “capped” growth can only occur from the
2. When G-monomers are incorporated into F-actin, ATP is other end.
hydrolysed and become stable ADP-F-actin (white)
The alteration in actin filament length creates forces which create
Note the ATP-binding clefts (black triangles) all point the same
MOVEMENT
way in F-actin and are closed at the barbed end.

4BBB0108 Lecture 1 2010-11 1


Cross-
Cross -linking proteins of microfilaments
hold adjacent filaments together
a) short cross-linkers e.g. fascin
b) long flexible cross-linkers e.g. filamin
F-actin structures within the cell
STRENGTH Bundles
parallel arrays (stress fibres)
project out to exterior (filopodia, lamellipodia)
Networks
- criss-cross, right angles, loosely packed.
- project into cell interior
- fan out as a network in cytosol

a) associated with plasma membrane – web like (“cortex”)

b) within the cell -gel like

Bundles and networks determine a cell’s shape

Functions of F-
F-actin Images of F-
F-actin cyoskeleton in cells
1. movement - muscle
- intracellular motion
- polarised cell locomotion
Formation of:
filopodia - finger like projections
lamellipodia - broad-membrane projection

2. stress fibres -form at edge of cell which directs

movement (the leading edge)
Electron Micrograph of the edge of a
3. influence cell shape- by altering length of filaments cell showing actin network in ruffles
and fibres

Dynamics of F-
F-actin during cell migration

QuickTime™ and a
decompressor
p
are needed to see this picture.

Moving cell - plasma membrane projections pointing upwards Movie of a fibroblast cell - F-actin shown in white (GFP-actin)
are no longer attached to cell substratum are called ruffles Actual movie time = 2 minutes

4BBB0108 Lecture 1 2010-11 2

 Microtubules radiate out from the centre of the cell
Microtubules (MT)
(i) cytosolic

• transport of vesicles across cell

• chromosome movement- mitotic spindle is made from MT form poles of mitotic spindle
• axonal migration
microtubule organising centre
(ii) cell exterior

• cilia (m)
( )
• flagella (2mm)

used for:
cell movement (sperm, protozoa, green algae)
move and bring food particles
MT break down and
mammalian respiratory tracts
mitotic spindle forms
from them

Microtubules show polarity: + and - ends (like F-actin)

Centrosome (area surrounding centrioles) is major microtubule Microtubule structure

organising centre in animal cells
Made from two monomers -  and  tubulin, each 50kDa
- have nucleating sites, like F-actin to start MT polymerisation both bind GTP

form heterodimers ()

heterodimers lie in a longitudinal

array

polymerise to form a protofilament

then 13 protofilaments

align to form hollow shaped cylinder

How do microtubules polymerise?

GTP binds non-exchangeable to -tubulin

+ end = faster growing (nearest cell surface)
GTP-GDP hydrolysis cycle operates for -tubulin.
- end = slower growing (nearest cell centre)
GTP is hydrolysed soon after polymerisation

4BBB0108 Lecture 1 2010-11 3

 Microtubules show dynamic instability Each MT grows and shrinks independently of its neighbours.
Under favourable conditions, MT are also able to undergo treadmilling
Array of MT from centrosome is continually changing.
However:
New MT grow (red arrows) and old MT shrink (blue arrows)
Plus end of MT can grow for many minutes, then suddenly stop.

Undergoes a transition so loses subunits from its free end. +

It may partially shrink and then start growing again or disappear.
disappear
+
This occurs because -tubulin is a GTPase – hydrolysing GTP.

GTP-bound tubulin molecules are very stable, but once the

GTP is hydrolysed to GDP they become unstable and can leave the +
growing MT. + +

Thus: the rate of GTP hydrolysis controls the growth of MT

plus end
(red) Images of microtubule networks in cells

From time to time,

GTP cap is lost due
to hydrolysing to GDP,
faster than new GTP-
tubulin can bind

(dark green)

Fibroblast cells stained with antibodies to show

Actin (purple) and microtubule (yellow) cytoskeletons.
Nucleus is shown in blue

Dynamics of microtubule networks in cells Intermediate filaments

• monomer - very variable

- many types
- tissue dependent

I acidic keratins - epithelia cells (skin, hair,

II basic keratins - nails)
III desmin - muscle specific
vimentin - wide distribution
Movie of dynamic MT
IV nerves and eye
growth and shrinking from
MTOC in a living cell V nuclear lamins

• central -helical rod domain

• flanked by globular N and C-termini

4BBB0108 Lecture 1 2010-11 4

 Functions of intermediate filaments
Relationship between filament types in the cytoplasm
1. Structural role - mechanical support
Intermediate
Microfilament cross-linkers plectin filaments
a) bind to cell junctions at plasma membrane
radiate into cell

b) bind to organelles - keep in place

2 Organise cells into tissues

2.

Occur as:
Bundles - nucleus to plasma membrane

Networks- nuclear lamina (inside of nuclear membrane)

Intermediate filaments form a more STABLE structure -

than microfilaments or microtubules Immunoelectron micrograph microtubules

MYOSIN
Molecular motors 18 classes of myosin - large filamentous-type proteins

Common features - one or two heavy protein chains

Proteins which move along microfilaments and several light chains

MYOSIN -helical neck region Large globular head

binds light chains (actin binding, ATPase)

Proteins which move along microtubules

Light chains -
DYNEIN and KINESIN regulate myosin
function

All three use the energy from ATP hydrolysis Tail - binds to other myosin
to power their movement molecules

Myosin forms bipolar filaments

Common types of myosin

Myosin’s tail domains associate in anti-parallel array

electronmicrograph

4BBB0108 Lecture 1 2010-11 5

 Myosin binds to actin-
actin- actomyosin
2. Non-muscle cells
1. Muscle contraction - in cardiac and skeletal muscle (striated due
to sarcomere structure) seen in: stress fibres (cell migration, controlling cell shape)
Myosin (thick) filaments bind to actin (thin) filaments through it’s
globular head regions, and “walks” along the thin filaments using
the energy from ATP hydrolysis. Myosin bipolar filaments
interact with F-actin
in anti-parallel array

Occur in bundles

Attach to cell surface

long where the cell touches
the substratum

short

2. Non-muscle cells (contd) DYNEIN and KINESIN

also seen in: contractile ring (cell division - cytokinesis) Motor proteins which move along microtubules
Actin and myosin accumulate at equator of dividing cell,
midway between the poles of the spindle dynein is minus end directed (cell centre)

As cell divides, diameter of contractile ring decreases kinesin is plus end directed (cell surface)

myosin blue
Functions

actin red 1.Determines

organelle and
chromosome position

Functions (contd.)
2. Movement of vesicles, containing proteins which need to be moved
around the cell or exported from the cell - (A)
Vesicle on a
microtubule
3. Movement of vesicles along axons i.e. axonal transport - (B)
vesicle

microtubule

Like a car pulling

a caravan along a
motorway!

Electron micrograph

4BBB0108 Lecture 1 2010-11 6

 DYNEIN FAMILY Kinesin
Dimer of two heavy chains (140,000D each) and two light chains

Three domains - pair of large globular heads -long central stalk-

small globular tail
All VERY large multimeric proteins - final complex
weight exceeds 1,000,000 Daltons Can mediate transport on its own - needs no supporting proteins

y chains and a
Each consists of two or three heavy
number of intermediate and lighter chains which
have not been fully characterised

ATP hydrolysis on Binds to vesicle- often

binding microtubules referred to as CARGO

dynein and kinesin binding to microtubules Vesicles can be transported bi-

bi-directionally simultaneously

Vesicles
WALK-
not roll

kinesin cytoplasmic axoneme

dynein dynein

schematic
Freeze-etch electronmicrograph

4BBB0108 Lecture 1 2010-11 7