the cytoskeleton miklós nyitrai department of biophysics, university of pécs, pécs, hungary. embo...
TRANSCRIPT
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!