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The c y toskeleton Mikl ós Nyitrai Department 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?.
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The cytoskeleton Miklós Nyitrai Department 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
A dynamic structural and functional framework Three types of filaments: A. Intermediate B. Microtubules C. Microfilaments Cytoskeleton • Cellular distribution of intermediate filaments and microtubules is similar
Polimerization: an example Three 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’
Torsion: Bending stiffness: Longitudinal stiffness: F F F A = persistence length F Lc = contour length Z = end-to-end distance Polymer mechanics The direction of force: Mechanism: - Intrinsic flexibility -Thermal (entropy) flexibility (persistence length)
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.
Actin monomer Globular (G-) actin MW: 43 kDa, 375 aa, 1 bound ATP or ADP Subdomains (4)
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)
Again, a dynamic equilibrium exits and plays central role Pointed end Barbed end Critical concentration
Cell Crawling Migrating melanocyte expressing GFP-tagged actin.(Vic. SMALL).
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
Microtubules Subunit: tubulin MW: ~50 kD, - és -tubulin -> heterodimer 1 bound GTP or GDP; a b
Microtubules ~25nm thick, tube shape 13 protofilaments Right hand, short helix Left hand, long helix Stiff polymer chain (persistence length: a few mm!) Structural polarization: + end: rapid polymerization, - end: slow polymerization GTP-cap see ‘search and capture’
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 monomer is not globular, a fiber!
The subunit of filaments: „coiled-coil” dimer Vimentin dimer
protofilamentum filamentum Polymerisation of IF Polymerised in cell lack 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 filaments 1. Actin-associated (e.g. myosin) 2. MT- associated (e.g. Tau protein) 3. IF- associated B. According to the binding site 1. 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 effects a. Induce depolymerization („severing”, pl. gelsolin) b. Stabilizing (pl. profilin, tropomiozin) 3. Motor proteins
Actin nucleation factors What are they for?
The atomic model of Arp2/3(Andrea Alfieri) Arp3 p34 p21 p20 p16 Arp2 p40 inactive state • Robinson et al., 2001. Crystal structure of Arp2/3 complex. Science. 294:1679-84.
The 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!
In vitro model 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.
Formins (Manuelle Quinoud) A proposed mechanism from S. Zigmond.
Motor proteins (why ‘motor’?)
Motor proteins • They can bind to specific filament types • 2. They can travel along filaments • 3. They hydrolyze ATP
Types of motor proteins 1. Actin-based: myosins Conventional (miozin II) and nonconventional myosins Myosin families: myosin I-XVIII 2. Microtubule based motors a. Dynein Flagellar and cytoplasmic dyneins. MW~500kDa They 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 based DNA and RNA polymerases They move along a DNA and produce force
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
N C Common properties 1. Structure N-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, function In principle: cyclic function and work Motor -> binding to a filament -> force -> dissociation -> relaxation 1 cycle requires 1 ATP hydrolysis They can either move (isotonic conditions) or produce force (isometric conditions)
ATP cycle attached ton detached toff The working cycle of motor proteins = working distance power stroke attachment detachment back stroke In vitro sliding velocity: Duty ratio: Attached time: Cycle time: d=working distance (or step size); V=ATPase activity; v=In vitro sliding velocity
Processivity and the duty ratio d=working distance or step size V=ATPase activity v=in vitro motility velocity Processive motor: r->1 pl. kinesin, DNA-, RNA-polimerase the motor is attached to the track in most of the working cycle Nonprocessive motor: r->0 pl. conventional myosin A motor protein can produce force in the pN range.
Cell functions for myosins The head group of the myosin walks toward the plus end of the actin filament.