1. The cytoskeleton (December 6, 2006)

2. 1. What is the cytoskeleton? 2. Filament types, and polymerization 3. Motor proteins

3. Cytoskeleton A dynamic framework Three types: A. Intermediate B. Microtubules C. Microfilaments Cellular distribution of intermediate filaments and microtubules is similar

4. Polimerization Three phases: 1. Lag phase: nucleation 2. Elongation 3. Equilibrium

5. Equilibrium 1. Dynamic equilibrium 2. Dynamic unstability: slow elongation followed by rapid (catastrophic) depolymerisation 3. ‘Tread-milling’

6. -Intrinsic flexibility -Thermal (entropy) flexibility (persistence length) A = persistence length F Z = end-to-end distance L c = contour length Polymer mechanics Bending stiffness: F Longitudinal stiffness: F Torsion: F Mechanism: The direction of force:

7. Microfilaments (actin)

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

9. Globular (G-) actin MW: 43 kDa, 375 aa, 1 bound ATP or ADP Subdomains (4) Actin monomer 1 4 3 2 nucleotide

10. 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)

11. 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

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

13. Cell Crawling

14. Growth of Filopodia

16. Motility with actin polymerization Intracellular pathogens

17. Biophysical methods to study the cytoskeleton -Fluorescence spectroscopy -Fluorescence microscopy -Atomic force microscopy -EPR spectroscopy -Calorimetry -In vitro motility assays …etc

18. Microtubules

19. Subunit: tubulin MW: ~50 kD,  - és  -tubulin -> heterodimer 1 bound GTP or GDP; Microtubuls  

20. 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

21. Intermediate filaments

22. The monomer is not globular, a fiber! Tissue specific IF types Nuclear laminsA, B, C lamins (65-75kDa) Vimentin typeVimentin (54kDa) Desmin (53kDa) Peripherin (66kDa) KeratinsType I (acidic) (40-70kDa) Type II (neutral/basic) (40-70kDa) Neuronal IFneurofilament proteins (60-130kDa)

23. The subunit of filaments: „coiled-coil” dimer Vimentin dimer

24. Polymerisation of IF protofilamentum filamentum 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

25. 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 („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 effects a. Induce depolymerization („severing”, pl. gelsolin) b. Stabilizing (pl. profilin, tropomiozin) 3. Motor proteins

26. Motor proteins

27. 1.They can bind to specific filament types 2. They can travel along filaments 3. They hydrolyze ATP Motor proteins

28. 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 Types of motor proteins

29. 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

31. 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) Common properties N C

33. The working cycle of motor proteins Duty ratio: In vitro sliding velocity: Cycle time:Attached time: attached  on detached  off ATP cycle power stroke back stroke attachment detachment  = working distance  =working distance (or step size); V=ATPase activity; v=In vitro sliding velocity

34. Duty ratio 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.  =working distance or step size V=ATPase activity v=in vitro motility velocity

36. Kinesin scheme

37. How to follow polymerisation? Pyrene fluorescence Monomer filament fluorescence

38. Dia3 Dia1 Elongációs sebesség The effect of Formin FH2 FH2 decreased the rate of polymerisation.

39. F-actin myosin mikroscop cover slit In vitro motility assay

40. Laser tweezer Micro bead Laser tweezer

41. 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..

42. Movement of a migrating Keratocyte (Vic. Small).

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

44. 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.

46. 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

47. Az ATP hidrolízis ciklusa

49. Other cell functions for actin and myosin

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

51. 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.