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

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

3. So, what is the cytoskeleton?

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

5. Polimerization: an example Three phases: 1. Lag phase: nucleation 2. Elongation 3. Equilibrium

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

7. - 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:

8. Microfilaments (actin)

9. 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.

10. How is a filament built up?

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

12. The filament The polymerization... ~100 times faster in vivo than in vitro.

13. 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)

14. Geometry of the Actin Filament

15. Barbed end Pointed end Again, a dynamic equilibrium exits and plays central role Critical concentration

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

17. What kind of molecular motions are responsible for cell locomotion?

18. 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

19. Push and pull!

20. Cell functions for actin

21. Microtubules

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

23. ~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’

24. Intermediate filaments

25. 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)

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

27. 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

28. 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

29. Actin nucleation factors What are they for?

30. The atomic model of Arp2/3 (Andrea Alfieri) inactive state Arp2 p34 p16 p20 Robinson et al., 2001. Crystal structure of Arp2/3 complex. Science. 294:1679-84. p40 p21 Arp3

31. 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.

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

33. Intracellular pathogens

34. 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

35. Formins (Manuelle Quinoud) A proposed mechanism from S. Zigmond.

36. Motor proteins (why ‘motor’?)

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

38. 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

39. 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

40. 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

41. The ATP hydrolysis cycle: an example

42. 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

43. Processivity and the 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

44. Myosins

45. The superfamily

46. Diversity, adaptation, tuning

47. How do myosins work?

48. An example: the myosin in muscle cells

49. The head group of the myosin walks toward the plus end of the actin filament. Cell functions for myosins

50. Kinesins

51. Kinesin scheme Single headed kinesins!?

52. Walking along the microtubules Also remember processivity…

53. So, how does it all work together?

54. Pollard and Beltzner, Current Opinion in Structural Biology 2002, 12:768–774. An example for actin cytoskeleton regulation

55. Thank You!