1. Cytoskeleton - Locomotion www.dgci.sote.hu Kohidai, Laszlo MD, PhD Med. habil., Assoc. Professor Dept. Genetics, Cell & Immunobiology, Semmelweis University Ecelctive Course / 2012

2. Main functions of cytoskeleton Determines the shape of the cell Determines the shape of the cell Anchores organelles Anchores organelles Movement of organelles Movement of organelles Tensile strength Tensile strength Movement of chromosomes Movement of chromosomes Polarity Polarity Motility Motility

3. Cytoskeleton Microfilaments (actin) Microfilaments (actin) Microtubuli (tubulin) Microtubuli (tubulin) Intermedier filaments Intermedier filaments Microtubule associated proteins (MAP-s) Microtubule associated proteins (MAP-s) Motor proteins Motor proteins

4. Microfilaments Microtubuli Intermedierfilaments

5. SLIDING Globular proteins Fibrillar proteins Ca 2+ ATP Motor proteins

6. Microfilaments

7. Polymerization of actin ATP ADP ADP ATP PiPiPiPi Polymerization - slow Depolymerization + - cytochalasin – inh. phalloidin - stabilizer

8. Actin - still in Prokaryots ! ((Ent et al. Nature 2001,413, 39)

9. Cyclosis Movingcytoplasm Stationary(cortical)cytoplasm Actin filaments Chloroplasts Cell-wall Plasma membrane (Lodish, H. et al. Mol. Cell Biol. 2000, 767) Transitional connections between actin and myosin Transitional connections between actin and myosin Ca 2+, temperature- and pH-dependent Ca 2+, temperature- and pH-dependent

10. „Fountain” mechanism Ca 2+ -dep. requires ATP integrins stress-fibrillums Formation of pseudopodium Mono-Poly- Lobo- podial Filo-Reticulo-

11. Cross-linking proteins of actin contractile bundle  actinin – in stress fibr. „tight” parallel bundle fimbrin – in filopodium gel-like network filamin - cortex

12. Migrating keratinocyte 15  m/sec actin-network microtubuli Formation of lobopodium

13. Regulator proteins of actin polymerisation -+ CofilinSeverinGelsolin   CAP39 SeverinGelsolinVillinCapZ Tropomodulin

14. Actin polymerisation – acrosomal-reaction (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

15. Listeria monocytogenes local actin polymerization local actin polymerization speed: 10  m/min speed: 10  m/min high ability to transmit high ability to transmit in tissues in tissues actin (Fred Soo & Julie Theriot Laboratory)

16. Model of actin nucleation WASP = Wiscott-Aldrich syndr. prot.

17. Structure of cortical region (Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)

18. Actin – membrane links F-Actin Integrin Filamin Profilin - G-aktin Myozin I. Arp2/3 membrane

19. Proline-rich protein Profilin-mechanism (Lodish, H. et al. Mol. Cell Biol. 2000, 767) T  4 =timozin  4 T  4 = timozin  4

20. Filamin – Membrane link filamin actin

21. actin filament  actinin vinculin paxillin talin integrin fibronectin Structure of focal contact +

22. Thrombocyte Muscle Epithel A plasma membrane – cortex links Spectrintetramer Glycophorin Ankyrin ((Lux SE, 1979 Nature 281:426)

23. E - - + + + Adhesion plaque Electromagnetic field induces the transformation induces the transformation of cytoskeleton and of cytoskeleton and formation of pseudopodia

24. Myosin myosin I. 150 kD myosin I I. 260 kD head heavy chain  helix light chain ATP - ADP PiPiPiPi Head: - ATP-ase - motor - motor Ca 2+ -dependent phosphorylation Ca 2+ -dependent phosphorylation and its effect on the 3D strcture dimer monomer

25. Distribution of myosines in the migrating Dyctiostelium and in dividing cell myozin I. (green) myozin II. (red) (red) (Fukui, Y. Mol. Cell Biol 2000, 785))

26. + - Main types of interactions between the globular and fibrillar components of cytoskeleton of cytoskeleton membrane

27. Non-treated F-actin blocked MT-blocked

28. Microtubules

29. FtsZ Tubulin Tubulin – still in Prokaryotes ! (Margolin Laboratory, University of Texas)

30. Polymerization of tubulin Protofilament (strait) Protofilament (curved) GTP GTP GTP GTP GDP GDP GDP GDP Polymerization - fast Depolymerization

31. Dynamics of microtubule-assembly incorporation balanced release + - Nucleation Elongation

32. Role of  -tubulin in nucleation (Wiease et al. Curr.Opin.Struct.Biol. 1999, 9, 250)

33. Interphase cell Dividing cell Neuron centrosome Basal body Cilla spindle centrosome axon Microtubular systems systems in the cells in the cells - Centrosome - Cilia / flagellum - Mitotic system - Vesicular transport

34. MTOC = Mikrotubul organizing center specialized region of the cortex ((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)  -tubulin

35. Network of microtubuli Protofilaments dimer ab dimer tubulin a tubulin tubulin b tubulin 24 nm Fibroblast

36. Cilia cilia flagellum Paramecium

37. A B dynein-arms nexintubulin (13 ill. 11 protofilaments)

38. ATP-independent binding ATP-dependent hydrolisis The arm moves toward the - pole Composition of dynein-arms

39. The role of dynein arms in beating of cilia „Telescoping” Bending Proteolysis

40. Molecules composing the cilia 70%  and  tubulin 70%  and  tubulin dynein arms dynein arms outer - 9 polypeptides - ATP-ase inner – composition varies radial spokes - 17 polypeptides radial spokes - 17 polypeptides more than 250 types of molecules

41. Microtubules of mitotic spindle and kinetochore

42. Arrangement of actin during cell-division

43. Intermedier filaments

44. Mechanical characterization of cytoskeleton components deformation force actin filament intermedier filament i.e. vimentin microtubule = rupture

45. Role of intermedier filaments Buffer against external mechanical stress Tissue specificity Epithel – keratin Connective tissue MusclesNeuroglia Neurones - neurofilaments } vimentin Nucleus – lamines (lamina fibrosa)

46. Structure of intermedier filamentums (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

47. H 2 N- - COOH keratins vimentin neurofilam. prot. nuclear prot  helical domain Domain structures of intermedier filamentums

48. Intermedier filaments Keratin filaments Vimentin-like filaments ! They DO NOT co-polymerise !

49. Microvilliactin myosin I. villin „terminal web” a rigid bundle composed by 20-30 actin mol.s a rigid bundle composed by 20-30 actin mol.s actin + on the apical part actin + on the apical part villin is the linker molecule of actins villin is the linker molecule of actins „terminal web” = intermed.fil. + spectrin „terminal web” = intermed.fil. + spectrin myosin I. and calmodulin anchore to the surface membrane myosin I. and calmodulin anchore to the surface membrane

50. SEM structure of microvilli „terminal web” actin bundle linker molecules

51. Intermedier filaments The number of protein cross-links between the intermedier filaments varies in different tissues intermedier filaments varies in different tissues Neuro-filaments – many cross-linkers Glial filaments – few cross-linkers – few cross-linkers

52. Microtubuli associated proteins (MAP-s)

53. Groups of MAP-s Structural MAP-s Structural MAP-s - MT-assembly - MT-assembly - links to MF and to IF - links to MF and to IF Motor proteins Motor proteins - sliding on MT - sliding on MT Enzymes, signal molecules Enzymes, signal molecules - glycolytic enzymek - kinases Shape and polarity of the cell Membrane transports Assembly of molecules

54. Motor-proteins

55. Structure of motor-proteins motor domain motor domain asszoc. polypeptides asszoc. polypeptides asszoc. polypeptides „stalk” Kinesin MyozinDynein

56. Motor proteins kinesin dynein microtubule light chain heavychain kinesin dynein + -

57. + - kinesin dynein cAMP cAMP pigment cells

58. ATP ADP ATP ADP ADP ATP ADP ADP-Pi Kinesin

59. MT-motor proteins and the transported elements (Hirokawa, N. Science 1998, 279:519

60. Dynein – membrane relations (Hirokawa, N. Science 1998, 279:519)

61. There are other mechanisms over sliding …

62. Locomotion – with spasmoneme of Vorticella

63. Spasmoneme spring Contracts 40% in few msecs Velocity: 8 cm˛/sec Negativecharges Neutralization with Ca 2+

64. Actin spring in sperm of horse- shoe crab Limulus polyphemus The extension does not involve a myosin The extension does not involve a myosin motor or actin polymerization motor or actin polymerization The bundle is crystalline in its coiled and The bundle is crystalline in its coiled and uncoiled states uncoiled states acrosome actin bundle !

65. Signalling mechanisms – In a nutshell –

66. Polymerization of microtubules - Significance of motor proteins

67. Ameboid movement and microtubules

68. Complex effects of focal adhesion kinase (FAK)

69. LIM domains of proteins

70. Central role of vimentin in signalling mechanisms of cytoskeleton

71. Phosphatases and cell migration