1. Cytoskeleton and locomotion www.dgci.sote.hu Lecture EPh 2014 Láng, Orsolya MD, PhD Dept. Genetics, Cell & Immunobiology, Semmelweis University

2. Main components of the eukaryotic cytoskeleton Microfilaments:  actin 7nm Microtubules:  tubulins (  25 nm Intermediate filaments:  lamin  cell specific prot. (e.g. vimentin) 8-12 nm + Associated proteins (e.g. motor proteins)

3. Filamentous structures in the cytoplasm of walled bacteria Microfilament like:  MreB Microtubule like:  FtsZ, TubZ Intermediate filament like:  Crescentin, Parm weak sequence homology but crystallography appreciate their striking structural properties and similarity to eukaryotic cytoskeletal elements Nature Cell Biology12, 731–733 (2010)

4. Tissue level:  muscle movement Cellular level:  determines shape of the cell  motility of the cells  cell adhesion  mitosis, meiosis Subcellular level:  anchors organelles  organization of organelles  provides tensile strength  movement of chromosomes  organizing cell polarity  Intracellular movement of vesicles - Endocytosis – clathrin-mediated endocytosis and phagocytosis Function of the cytoskeleton Dynamic Adaptable Stable Strong

5. Cytoskeletal filaments are dynamic and adaptable

6. Stability of cytoskeletal filaments

7. Strong cytoskeletal filaments Intermediate filaments- resistant to stretching forces

8. Microfilaments

9. ( Garland Science Molecular Biology of the Cell 2008) Molecular structure of actin F-actin G-actin

10. ATP ADP ADP ATP PiPi Polymerization - slow Depolymerization + - Drugs: cytochalasin – inhibition (cap at + end) phalloidin - stabilizer Polymerazition of actin

11. Treadmilling of actin filament Video: http://csls-text.c.u-tokyo.ac.jp/flash/0611_1.html

12. Molecular Biology of the Cell (© Garland Science 2008) Actin and accessory proteins

13. 3 groups:  banding and cross linking proteins  regulatory proteins: polymerization/depolymerization, severing proteins,capping proteins  Motor proteins - sliding on MF (myosin) - sliding on MF (myosin) Organisation of actin filaments Sliding Actin binding proteins (ABPs)

14. Molecular Biology of the Cell (© Garland Science 2008) Organisation of actin filaments

15. Molecular Biology of the Cell (© Garland Science 2008) Cross-linking proteins I. Contractile bundleParallel bundle

16. Molecular Biology of the Cell (© Garland Science 2008) Cross-linking proteins II. Gel-like network plasma membrane

17. Regulatory proteins -+ CofilinSeverinGelsolin   CAP39 SeverinGelsolinVillinCapZ Tropomodulin Profilin: G actin pool Thymosin: actin sequestring Formin: actin polymerizing protein

18. Actin polymerisation – moition I. (Lodish, H. et al. Mol. Cell Biol. 2000, 767) Acrosomal reaction

19. Listreia Monocytogenes infection Actin polymerisation – motion II.

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

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

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

23. Filamin – Membrane link filamin actin

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

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

26. Microvilli At bottom: (spectrin, myosin, intermediate filaments) It is called: terminal web

27. Motor proteins: myosins General structure:  Globular head and fibrillar tail  Heavy chains and light chains  Head: motor domain with ATP-ase activity  ADP- straight Direction: + end motors

28. Molecular Biology of the Cell (© Garland Science 2008) Myosin II molecule

29. 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))

30. Microtubules

31. Molecular Biology of the Cell (© Garland Science 2008) Molecular structure of tubulin dimers and microtubules GTP

32. Molecular Biology of the Cell (© Garland Science 2008) Polymerisation of microtubules Drugs:  Colchicine and colcemid– inhibition (binds subunit)  Taxol - stabilizer

33. Molecular Biology of the Cell (© Garland Science 2008) Polymerisation of microtubules Dynamic instability and treadmilling Video: http://csls-text.c.u-tokyo.ac.jp/flash/0612_1.html

34. Molecular Biology of the Cell (© Garland Science 2008) Role of  tubulin in nucleation of microtubules at - end

35.  Structural MAP-s - MT-assembly - links to MF and to IF (eg. tau, MAPs1 and MAPs2) (eg. tau, MAPs1 and MAPs2)  Motor proteins - sliding on MT - sliding on MT (e.g. kinesin and dynein family)  Enzymes, signal molecules - glycolytic enzymes - kinases Shape and polarity of the cell Vesicular transports Assembly of molecules Microtubule associated proteins (MAPs)

36. Structure of motor-proteins motor domain motor domain assoc. polypeptides assoc. polypeptides assoc. polypeptides „stalk” Kinesin MyozinDynein

37. Motor proteins + - dynein + kinesin light chain heavychain ?

38. + - kinesin dynein cAMP cAMP pigment cells

39. ATP ADP ATP ADP ADP ATP ADP ADP-Pi Kinesin

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

41. In cells: MTOC = Microtubule organizing center (Brinkley, B.R. Encyclop. Neurosci. 1987, 665)  -tubulin 9x3 microtubules A,B,C 2 centrioles at a right angle Organisator: Gamma-tubulin in pericentriolar matrix - end of forming microtubule

42. Molecular Biology of the Cell (© Garland Science 2008) Centrosome

43. Cilia cilia flagellum Paramecium

44. A B dynein-arms nexintubulin (13 ill. 11 protofilaments) Structure of cilia

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

46. Role of the dynein arms in beating cilia Telescopic effect Beating

47. Microtubules of mitotic spindle and kinetochore

48. How motor proteins can organise the position of cell organelles (ER, Golgi) ? (Hirokawa, N. Science 1998, 279:519

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

50. Intermediate filaments

51. Molecular Biology of the Cell (© Garland Science 2008) Structure of an intermediate filaments Monomer Parallel dimer Antiparallel tetramer Protofilaments Intermedaite filaments

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

53. Role of intermedier filaments Buffer against external mechanical stress Tissue specificity !!! Epithel – keratin Connective tissue Muscles Neuroglia Neurones(axon) - neurofilaments } vimentin-like Exception: Nucleus – lamines (A,B,C) →(lamina fibrosa) Vimentin Desmin Glial protein

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

55. Molecular Biology of the Cell (© Garland Science 2008) Cross bridges are formed by non-helical C terminus few cross bridges Axon of a neuron Neurofilament (NF-H) Glial cell Glial filaments Axon cross-section

56. Cell locomotion/ movement Cellular level:  Ciliar movement  Amoeboid mocevent  Mesenchymal migration Collective migration Tissue level:  muscle movement – skeletal muscle

57. Cilia - flagellum

58. Amoeboid movement chemoattractant

59. Composition of thick filament in a sarcomer

60. Composition of sarcomer   Working unit bordered by two Z-lines/disc  Z line –  -actinin + desmin  Thin filament –  -helical actin molecules + tropomyosins + troponin  Thick filament – myosin II molecules + MBP (myosin binding proteins)  Other proteins: titin, distrophin Troponin: Tn-T binds tropomyosin Tn-C binds Ca 2+ (4 Ca 2+ /mol = calmodulin) Tn-I inhibitor

62. Skeletal muscle - Sarcomere

63. Skeletal muscle - Sarcoplasmic reticulum

64. X-linked recessive inheritance Duschenne muscular distrophy

65. Useful videos and linkes: http://csls-text.c.u-tokyo.ac.jp/active/06_01.html http://www.microscopyu.com/moviegallery/livecellimaging/index.html http://www.cellmigration.org/science/