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Cytoskeleton - Locomotion

Cytoskeleton - Locomotion

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Cytoskeleton - Locomotion

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  1. Cytoskeleton - Locomotion Kohidai, Laszlo MD, PhD Med. habil., Assoc. Professor Dept. Genetics, Cell & Immunobiology, Semmelweis University http://gsi.semmelweis.hu Lecture ED 2015

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

  3. Cytoskeleton • Microfilaments (actin) • Microtubuli (tubulin) • Intermedier filaments • Motor proteins • Actin and microtubule associated proteins

  4. Microfilaments Microtubuli Intermedier filaments

  5. SLIDING Globular proteins Ca2+ ATP Motor proteins Fibrillar proteins

  6. Microfilaments

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

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

  9. Other actin homologues ((Roeben A et al. J Mol. Biol2006, 358, 145)

  10. Comparison of homologues • Polymerization in both forms • Opposite chirality !!! ((Wickstead and Gull J Cell. Biol2011, 194, 513)

  11. Moving cytoplasm Stationary (cortical) cytoplasm Plasma membrane Actin filaments Cell-wall Chloroplasts Cyclosis • Transitional connections between actin and myosin • Ca2+, temperature- and pH-dependent (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

  12. „Fountain” mechanism Ca2+-dep. requires ATP Mono- Poly- Lobo- podial Filo- Reticulo- Formation of pseudopodium stress-fibrillums integrins

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

  14. Migrating keratinocyte 15 mm/sec Formation of lobopodium microtubuli actin-network

  15. - + Regulator proteins of actin polymerisation gCAP39 Severin Gelsolin Villin CapZ Tropomodulin  Cofilin Severin Gelsolin

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

  17. local actin polymerization • speed: 10 mm/min • high ability to transmit • in tissues Listeria monocytogenes actin (Fred Soo & Julie TheriotLaboratory

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

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

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

  21. Profilin-mechanism Tb4 =thymosin b4 Proline-rich protein (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

  22. Filamin – Membrane link filamin actin

  23. Structure of focal contact actin filament a actinin vinculin + paxillin talin integrin fibronectin

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

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

  26. ATP - ADP Pi Myosin head Ca2+-dependent phosphorylation and its effect on the 3D strcture light chain heavy chain a helix myosin I. 150 kD monomer myosin I I. 260 kD Head: - ATP-ase - motor dimer

  27. Distribution of myosines in the migrating Dyctiosteliumand in dividing cell myosin I. (green) myosin II. (red) (Fukui, Y. Mol. Cell Biol 2000, 785))

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

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

  30. Microtubules

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

  32. Comparison of homologues • Polymerization in both forms • Monomers build helical structure vs. dimers build tubulus ((Wickstead and Gull J Cell. Biol2011, 194, 513)

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

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

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

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

  37. specific region of the cortex MTOC = Microtubulus organizing center g-tubulin ((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

  38. 24 nm a-bdimer Protofilaments alphatubulin betatubulin Network of microtubuli Fibroblast

  39. Cilia cilia flagellum Paramecium

  40. tubulin (13 ill. 11 protofilaments) A B dynein-arms nexin

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

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

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

  44. Microtubules of mitotic spindle and kinetochore

  45. Arrangement of actin during cell-division

  46. Intermedier filaments

  47. Crescentin

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

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

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