The cytoskeleton(December 6, 2006)

1. What is the cytoskeleton?

2. Filament types, and polymerization

3. Motor proteins

Cytoskeleton A dynamic framework

Three types:A. IntermediateB. MicrotubulesC. Microfilaments

Cellular distribution of intermediate filaments and microtubules is similar

PolimerizationThree phases: 1. Lag phase: nucleation 2. Elongation 3. Equilibrium

Equilibrium

1. Dynamic equilibrium

2. Dynamic unstability: slow elongation followed by rapid (catastrophic) depolymerisation

3. ‘Tread-milling’

-Intrinsic flexibility-Thermal (entropy) flexibility (persistence length)

A = persistence length

F

Z = end-to-end distance

Lc = contour length

Polymer mechanics

Bending stiffness:

F

Longitudinal stiffness:F

Torsion:F

Mechanism:

The direction of force:

Microfilaments (actin)

Actin was discovered and named by a Hungarian scientist, Straub F. Brúnó

Globular (G-) actin MW: 43 kDa, 375 aa, 1 bound ATP or ADPSubdomains (4)

Actin monomer

1

4

3

2

nucleotide

Actin filament (F-actin)

37 nm

~7 nm thick, length in vitro is more than 10 µm, in vivo 1-2 µm

Double helix

Semi-flexible polymer chain (persistence length: ~10 µm)

"barbed end“ and "pointed end" (“barbed” =+ rapid polymerization, “pointed” =- slow polymerization)

Movement

• Subcellular, cellular levels

• Requires ATP (energy)

• Cytoskeleton-mediated– Assembly and disassembly of cytoskeletal fibers

(microfilaments and microtubules)– Motor proteins use cytoskeletal fibers (microfilaments

and microtubules) as tracks

Migrating melanocyte expressing GFP-tagged actin.(Vic. SMALL).

Cell Crawling

Growth of Filopodia

Motility with actin polymerizationIntracellular pathogens

Biophysical methods to study the cytoskeleton

-Fluorescence spectroscopy

-Fluorescence microscopy

-Atomic force microscopy

-EPR spectroscopy

-Calorimetry

-In vitro motility assays

…etc

Microtubules

Subunit: tubulinMW: ~50 kD, - és -tubulin -> heterodimer1 bound GTP or GDP;

Microtubuls

Microtubules

~25nm thick, tube shape13 protofilaments Right hand, short helixLeft hand, long helixStiff polymer chain (persistence length: a few mm!)Structural polarization:

+ end: rapid polymerization, - end: slow polymerization

GTP-cap

Intermediate filaments

The monomer is not globular, a fiber!

Tissue specific IF types

 Nuclear lamins A, B, C lamins

(65-75kDa)

Vimentin type Vimentin (54kDa)

Desmin (53kDa)

Peripherin (66kDa)

Keratins Type I (acidic) (40-70kDa)

Type II (neutral/basic) (40-70kDa)

Neuronal IF neurofilament proteins (60-130kDa)

The subunit of filaments: „coiled-coil” dimerVimentin dimer

Polymerisation of IF

protofilamentum

filamentum

Polymerised in celllack of dynamic equilibrium

Central rods (-helix) hydrofob-hydrofob interactions -> colied-coil dimer

2 dimer -> tetramer (antiparallel structure)

Tetramers connected longitudinally -> protofilaments

8 protofilaments -> filament

Cytoskeleton associated proteins

Many families of proteins which can bind specifically to actin

A. According to filaments1. Actin-associated (e.g. myosin)2. MT- associated (e.g. Tau protein)3. IF- associated

B. According to the binding site1. End binding proteins („capping”, pl. gelsolin)2. Side binding proteins (pl. tropomyosin)

C. According to function 1. Cross-linkers

a. Gel formation (pl. filamin, spectrin)b. Bundling (pl. alpha-aktinin, fimbrin, villin)

2. Polymerization effectsa. Induce depolymerization („severing”, pl. gelsolin)b. Stabilizing (pl. profilin, tropomiozin)

3. Motor proteins

Motor proteins

1. They can bind to specific filament types

2. They can travel along filaments

3. They hydrolyze ATP

Motor proteins

1. Actin-based: myosinsConventional (miozin II) and nonconventional

myosinsMyosin families: myosin I-XVIII

2. Microtubule based motorsa. Dynein

Flagellar and cytoplasmic dyneins. MW~500kDaThey move towards the minus end of MT

b. Kinesin Cytoskeletal kinesins Neurons, cargo transport along the axons Kinesin family: conventional kinesins + isoforms. MW~110 kDa They move towards the minus end of MT

3. Nucleic acid basedDNA and RNA polymerasesThey move along a DNA and produce force

Types of motor proteins

Motor proteins

• “Walk” or slide along cytoskeletal fibers– Myosin on microfilaments– Kinesin and dynein on microtubules

• Use energy from ATP hydrolysis

• Cytoskeletal fibers:– Serve as tracks to carry organelles or vesicles– Slide past each other

1. StructureN-terminal globular head:

motor domain, nucleotide binding and hydrolysis specific binding sites for the corresponding filaments

C-terminal: structural and functional role (e.g. myosins)

2. Mechanical properties, functionIn principle: cyclic function and workMotor -> binding to a filament -> force -> dissociation -> relaxation1 cycle requires 1 ATP hydrolysis

They can either move (isotonic conditions) or produce force (isometric conditions)

Common properties

N

C

r on

onoff

on total

The working cycle of motor proteins

von

total1

V

on v

Duty ratio:In vitro sliding

velocity:Cycle time:Attached time:

attachedon

detachedoff

ATP cyclepower stroke

back stroke

attachment detachment

= working distance

=working distance (or step size); V=ATPase activity; v=In vitro sliding velocity

r Vv

Duty ratio

Processive motor: r->1pl. kinesin, DNA-, RNA-polimerasethe motor is attached to the track in most of the working cycle

Nonprocessive motor: r->0pl. conventional myosin

A motor protein can produce force in the pN range.

=working distance or step sizeV=ATPase activityv=in vitro motility velocity

Kinesin scheme

How to follow polymerisation?

Pyrene fluorescence

Monomer

filament

fluo

resc

ence

0 5 10 15 200.0

0.5

1.0

0 10 20 30

0.0

0.2

0.4

0.6

0.8

1.0

elo

ngation r

ate

mDia1 or mDia3 (M)

no

rma

lise

d p

yre

ne

fluo

resce

nce

time (min)

Dia3

Dia1

Elo

ngác

iós

sebe

sség

The effect of Formin FH2

FH2 decreased the rate of polymerisation.

F-actin

myosin

mikroscopcover slit

In vitro motility assay

Laser tweezer Laser tweezer

Micro bead

Laser tweezer

Polystyrene beads of different diameters (0.5, 1, 3µm) have been functionalized with N-WASP and placed in a reconstitued motility medium containing actin, Arp2/3 complex, ADF/Cofilin, gelsolin (or any capping protein) and profilin..

Movement of  a migrating Keratocyte (Vic. Small).

A glass rod (Diam. 1µm, lenght 30 µm) has been functionalized with N-WASP and placed in the reconstitued motility medium.

Evidence for treadmilling is provided by light phase contrast recording of the movement of the rod (with A. Verkovsky): the size of the actin array remains stationnary, polymerization at rod surface being balanced by depolymerization in the actin meshwork.

Aktin:

1. cytochalasinok (a filamentum növekvô végéhez köt, polimerizációt gátol)

2. phalloidin (Amanita phalloides, polimert stabilizál)

Mikrotubulusok:

1. Colchicin (sáfrány, őszi kikerics, antimitoticum, köszvényben ôsi idôk óta használt, MT polimerizációt gátol)

2. Vinca alkaloidok (vinblastin, vincristin, antimitoticumok, MT polimerizációt gátolnak)

3. Taxol (tiszafából, MT stabilizáló, antimitoticum)

A polimerizáció kémiailag befolyásolható

Az ATP hidrolízis ciklusa

Other cell functions for actin and myosin

Other cell functions for actin and myosin

The head group of the myosin walks toward the plus end of the actin filament it contacts.

Functions of Actin Filaments

Actin filaments are concentrated beneath the plasma membrane (cell cortex) and give the cell mechanical strength.

Assembly of actin filaments can determine cell shape and cause cell movement.

Association of actin filaments with myosin can form contractile structures.