2.19.2010<br /><ul><li>Microtubule polymerization slide 9, 10, 11
Nucleation – the limiting step.
Non covalent binding of tubulin monomers
Dimers add to plus end of microtubule.
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  1. 1. 2.19.2010<br /><ul><li>Microtubule polymerization slide 9, 10, 11
  2. 2. Nucleation – the limiting step.
  3. 3. Non covalent binding of tubulin monomers
  4. 4. Dimers add to plus end of microtubule.
  5. 5. Polymerization is favored at the plus end, minus is depolymerization favored
  6. 6. Plateau phase
  7. 7. Equilibrium when dimmers add and come off equally.
  8. 8. GTP bound dimer
  9. 9. Both alpha and beta have GTP bound to them.
  10. 10. Microtubules can be stabilized.
  11. 11. Plus ends carry the GTP and they form a ‘cap’ when GTP is bound, which stabilizes the microtubule ofr a short time.
  12. 12. Cap allows growth in plus direction
  13. 13. The cap will continue to move down the length of the tubule until it reaches minus end and hydrolysis of GTP takes place.
  14. 14. Treadmilling
  15. 15. Theory of dynamic instability
  16. 16. Capping of microtubules with GTP bound dimmers, which stabilizes, but a catastrophic event can take place at the plus end, which can provide a source for tubulin for other tubules elsewhere.
  17. 17. If the GTP bound dimer concentration is high, elongation is favored, if low, then depolymerization is favored
  18. 18. Loss of cap results in GTP bound tubulin and rapid catastrophe occurs.
  19. 19. Growing end unwinds
  20. 20. Shrinkage can be stopped by increased concentration of tubulin dimers bound to GTP.
  21. 21. Provides a source of GDP dimers to be regenerated into GTP bound dimmers.
  22. 22. Shrinkage allows exchange of GDP and GTP dimers for other microtubules.
  23. 23. GEFS squeeze out GDP and then GTP is favored
  24. 24. GAPs – GTPase Activate Proteins for making GTP GDP
  25. 25. Tubulin can be an intracellular signal
  26. 26. Where are microtubules?
  27. 27. Centrosomes
  28. 28. A microtubule organizing center
  29. 29. Nerve cells have stabilized microtubules that is responsible for the orientation of them along the axon
  30. 30. Ciliated epithelial cells have basal bodies (similar to centrosomes) that attach to minus end of tubules and extend toward the apical end
  31. 31. Marginal bundle of microtubule – plus and minus are randomly assorted
  32. 32. Centrosome is made of a pair of centrioles
  33. 33. Centrioles are microtubules
  34. 34. Arranged in triplets, linked together.
  35. 35. 9 triplets make centriole
  36. 36. they contain ‘gamma-tubulin ring complex’ that make up the circular features around the peri-centriole-complex, a ton of proteins around the centrosomes to hold them. They stabilize microtubule growing filaments by binding the minus end. As long as there is enough GTP bound dimers to add on.
  37. 37. the grip proteins bind up the minus end of a microtubule, and that allows the tubule to grow rapidly at the plus end as long as there are GTP bound dimers. The grip proteins allows the orientation to take place to drive mitosis
  38. 38. Microtouble organizing centers – MTOCs
  39. 39. Nerve cells are polar w/long axons and short dendrites, so we have stabilized microtubules that have centrosomes that are responsible for the polarity. The dendrites don’t have a polarity or MTOC so the microtubules line up in opposite if not random ways.
  40. 40. Apical and basal ends, plus end at end, apical, and MTOCS attach to minus end and stabilize minus end of MT
  41. 41. Basal bodies help orient polarity of epithelial cells.
  42. 42. MT help organize the polarity of the cell
  43. 43. Motor proteins
  44. 44. Kinesin – move toward the plus end
  45. 45. Drive movement of organelles and proteins along tubules.
  46. 46. Dynein – toward the minus end
  47. 47. Drive movement of organelles and proteins along tubules.
  48. 48. Globular heads that link up to MT that requires ATP hydrolysis and a walking motion occurs. They have an ATP binding site, which specifically bind onto B tubulin subunit.
  49. 49. Light chain interacts with cargo, and heads have atp binding sites, which bind to beta tubulin subunits during movement.
  50. 50. Have different types for different cargo
  51. 51. Growth factors are taken in by receptor mediated endocytosis and can be moved by motor proteins into the cell
  52. 52. Slow and fast transport (don’t worry too much)
  53. 53. Microtoubules – slide 17
  54. 54. Important for where organelles are in the cell.
  55. 55. Tubules associate with proteins of endoplasmic reticulum and help shape/form in cytosplasm
  56. 56. Some proteins can cap the tubules and determine the structure of the cell.
  57. 57. Shape/movement are important aspects as well.
  58. 58. Polarity affects function of cell
  59. 59. MT can be capped and stabilized
  60. 60. Unstable MT can remain dynamic
  61. 61. Slide 18
  62. 62. 2 types of microubules that form outer doublet that surrounds central pair of microtubules. A and B form outer doublet.
  63. 63. Same tubules in center
  64. 64. Outer doublets are held together by stabilizing proteins, such as nexin, and the inner dynein arm, which allows outer tubule doublet to slide along one another and yet be held together to create a whip-like movement, such as cilia or cilia-like structures.
  65. 65. Slide 19
  66. 66. Linked by dynein motor proteins that bind ATP, and with ATP the motor proteins shift the microtubules in separate directions.
  67. 67. Or linking proteins that use shift of tubules, to allow the tubules to bend. When ATP is hydrolyzed, one shift ups and one shifts down for a bending, which creates movement.
  68. 68. Actin Filaments – Slide 20
  69. 69. Found just underneath plasma membrane
  70. 70. Made of actin monomers instead of tubulin monomers, such as microtubules.
  71. 71. Nucleating proteins
  72. 72. Sequestering monomer proteins
  73. 73. Bundling proteins that bind actin filaments.
  74. 74. Cross-linking proteins
  75. 75. Two actin filaments form a helical shape.
  76. 76. Polymerization of actin is bound by ATP. Actin can be bound by ATP or ADP
  77. 77. ATP: stable and addition is favored
  78. 78. ADP bound actin undergoes conformational change and associates with other actin monomers less tightly and falls off actin filament.
  79. 79. Works like tubulin, but ATP instead of GTP is involved
  80. 80. Types of Actin Filaments and associated proteins:
  81. 81. Nucleating proteins
  82. 82. Allow places for new actin filaments to grow
  83. 83. Bundling proteins
  84. 84. Bind individual actin filaments and align them so that filopodia can function
  85. 85. Filopodia are fingerlike projects at the edge of the cell. Protrude out of the membrane. Provide an ability to feel out in space and find things to take up for movement or adhesion with neighboring cells.
  86. 86. Bundling in filopodia is important for elongation.
  87. 87. Motor proteins
  88. 88. Allow for movement
  89. 89. Myosin I helps actin slide along itself
  90. 90. Capping
  91. 91. No longer polymeize
  92. 92. Cross-linking
  93. 93. Form a matrix and increase stability under plasma membrane instead of paralleling
  94. 94. Severing proteins
  95. 95. Sever actin
  96. 96. Microtubules-specific drugs
  97. 97. Phalloidin – tagged with fluorescent dyes to visualize actin filaments.
  98. 98. Used in cell cultures
  99. 99. Cytochalasin – caps filament plus
  100. 100. Taxol, colchicine – stabilize microtubules (and prevents polymerization)
  101. 101. Myosin proteins are used to stabilize actin in cells.
  102. 102. In the head, there is an actin binding domain and tail region hangs off end and regulated by other binding proteins.
  103. 103. Slide 25 – muscular cells.
  104. 104. Sarcomere will shorten as actin filaments are broguh together.
  105. 105. Cell pinching is driven by actin-myosin association.
  106. 106. Myosin-I is important for allowing actin-filament dynamics to take place at peripheral edge of membrane. Can move to distal end of cell membrane