The cytoskeleton Miklós Nyitrai Department of Biophysics, University of Pécs, Pécs, Hungary. EMBO Ph.D. course Heidelberg, Germany September, 2005.

The cytoskeleton

Miklós NyitraiDepartment of Biophysics, University of

Pécs, Pécs, Hungary.EMBO Ph.D. course

Heidelberg, Germany

September, 2005

1. What is the cytoskeleton?

2. Filament types and the process of polymerization

3. Motor proteins

So, what is the cytoskeleton?

Cytoskeleton A dynamic structural and functional framework

Three types of filaments:A. IntermediateB. MicrotubulesC. Microfilaments

Cellular distribution of intermediate filaments and microtubules is similar

Polimerization: an exampleThree 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)

Functions of Microfilaments

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.

How is a filament built up?

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

Actin monomer

The filament

The polymerization...

~100 times faster in vivo than in vitro.

The 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)

Geometry of the Actin Filament

5,5 nm166o

Barbed end Pointed end

Again, a dynamic equilibrium exits and plays central role

Critical concentration

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

Cell Crawling

What kind of molecular motions are responsible for cell locomotion?

Movement

Subcellular, cellular levels Requires ATP (energy conservation) Cytoskeleton-mediated

Assembly and disassembly of cytoskeletal fibers (microfilaments and microtubules)

Motor proteins use cytoskeletal fibers (microfilaments and microtubules) as tracks

Push and pull!

Cell functions for actinCell functions for actin

Microtubules

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

Microtubules

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 see ‘search and capture’

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 (nucleation, capping, pl. Arp2/3, 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

Actin nucleation factors

What are they for?

The atomic model of Arp2/3The atomic model of Arp2/3(Andrea Alfieri)(Andrea Alfieri)

inactive stateinactive stateArp2

p34

p16p16

p20

Robinson et al., 2001. Crystal structure of Arp2/3 complex. Science. 294:1679-84.

p40

p21Arp3

The Arp2/3; active stateThe Arp2/3; active state

Volkmann, et al., 2001.Structure of Arp2/3 complex in its activated state and in actin filament branch junctions.

Science. 293:2456-9.

The cytoskeleton can be hijacked based on the use of Arp2/3!

Intracellular pathogens

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.

In vitro model

Formins(Manuelle Quinoud)

A proposed mechanism from S. Zigmond.

Motor proteins(why ‘motor’?)

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

The ATP hydrolysis cycle: an example

€

r =τon

τon+τoff

=τon

τtotal

The working cycle of motor proteins

€

v=δτon

€

τtotal=1V

€

τon=δv

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 =δVv

Processivity and the 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

Myosins

The superfamily

Diversity, adaptation, tuning

How do myosins work?

An example: the myosin in muscle cells

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

Cell functions for myosinsCell functions for myosins

Kinesins

Kinesin scheme

Single headed kinesins!?

Walking along the microtubules

Also remember processivity…

So, how does it all work together?

Pollard and Beltzner, Current Opinion in Structural Biology 2002, 12:768–774.

An example for actin cytoskeleton regulation

Thank You!