3.24.2010

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3.24.2010

  1. 1. 3.24.2010 – Mitosis<br /><ul><li>Identifying which stage of interphase is the cell in?
  2. 2. Label the cells with
  3. 3. Tritiated Thymidine
  4. 4. Bromodeoxyuridine (BRDU)
  5. 5. There is an antibody to detect BRDU in labeled DNA
  6. 6. Labels incorporated into the DNA
  7. 7. Tells how much DNA is in the cell during a stage
  8. 8. Stages of the Cell-Cycle
  9. 9. Precedes Mitosis
  10. 10. Has checkpoints to make sure cell is prepared in terms of size and chromosome copies.
  11. 11. Signaling at the Checkpoints
  12. 12. Can measure amount of DNA in the cell for measuring which step cell cycle is in
  13. 13. Labels used
  14. 14. Can be incorporated into DNA as it’s being copied.
  15. 15. To determine how much DNA is present
  16. 16. Radiography
  17. 17. Cells can be imaged and Trimidium label can be measured and labeled
  18. 18. Can determine which cells are at a certain stage
  19. 19. Majority of cells are in G1 phase > S phase > G2
  20. 20. Cell sorting can be done for separating the populations
  21. 21. FACS – fluorescent activated cell sorting
  22. 22. Can take labeled cells, run them through the FACS machine and separate the cells with the labels
  23. 23. BRDU label used primarily
  24. 24. What questions can be looked at?
  25. 25. What regulates the checkpoints?
  26. 26. What is happening at Mitosis?
  27. 27. Checkpoints
  28. 28. Between G1 and S: is environment favorable for entering DNA synthesis?
  29. 29. Can enter G0 stage until environment is favorable to continue to S phase
  30. 30. Cell can also undergo apoptosis if signals for ideal environment are not given at a certain time frame
  31. 31. G2 before Mitosis
  32. 32. Cell is looking for whether or not DNA has been replicated, and if so, correctly?
  33. 33. In Mitosis
  34. 34. Makes sure sister chromatids are attached and separate properly at mitotic spindle
  35. 35. Key Transition Points in the Cell Cycle
  36. 36. G1 S phase checkpoint – “restriction point” – requirements for S phase transition
  37. 37. Growth factors
  38. 38. Nutrients
  39. 39. Increase in cell size
  40. 40. Absence of DNA damage
  41. 41. Can enter G0
  42. 42. “G2 – M transition”
  43. 43. Cell size
  44. 44. DNA damage
  45. 45. DNA replication
  46. 46. Can enter G0 again and signal sequences can make cell go back to repair damage
  47. 47. Mitosis – “Metaphase-Anaphase transition”
  48. 48. Chromosome attachments to spindle so the chromatids can be pulled apart
  49. 49. What Did People do to determine that there were proteins that influenced cell splitting
  50. 50. Separate cells from different stages fused
  51. 51. The fused cell is called a heterokaryon (monoclonal antibodies in previous lectures) cells with two nuclei.
  52. 52. To fuse cells:
  53. 53. Can use viral infections
  54. 54. Chemical treatment
  55. 55. Electroporation (Electrical pulses)
  56. 56. Often used to put DNA into a cell as well
  57. 57. Then fuse separated cells
  58. 58. S and G1 fusion – G1 nucleus is activated to undergo DNA synthesis
  59. 59. M and G1 fusion – mitosis activated
  60. 60. Experimenting with oocytes to determine what was driving the cycle in fused cells
  61. 61. Stalled in Meiosis II
  62. 62. Cytoplasm injected and cell doesn’t divide
  63. 63. Side Note: Progesterone from female will move cell into mitosis and generate egg in resting phase. The signal begins to activate the completion of mitosis at the end of meiosis I
  64. 64. Fertilization would complete meiosis II, it would be the signal
  65. 65. Increased concentrations of certain proteins in mitosis
  66. 66. Saw two peaks in a graph
  67. 67. MPF = maturation promoting factor (green peak) (rose at onset of mitosis and fell very fast during mitosis)
  68. 68. Will mature the egg if being stalled in meiosis II aka. M phase promoting factor because it moved the cell into mitosis
  69. 69. Temperature Sensitive Yeast Experiments
  70. 70. Generated by random mutagenesis
  71. 71. Looked for mutations in mitosis at higher temps than what they normally grow at (ideal is 23 C for a normal cell)
  72. 72. At restrictive temp, some yeast cells will not move through mitosis, so the cell is stalled at some point in cell cycle.
  73. 73. Can see what isn’t happening at G1 and ask what protein is mutated or signaling molecule is absent that is not allowing the cell to move to S phase.
  74. 74. Can use for all stages of mitosis
  75. 75. Lee Hartwell
  76. 76. Used a particular yeast strain to identify mutations that didn’t allow mitosis to continue normally.
  77. 77. Wild Type yeast could divide into daughter cells normally wherease mutated ones did not divide into daughter cells
  78. 78. There was a mutation in cell cycle division (cDC2 cell division cycle)
  79. 79. Important for fission of mitotic event and without it, or mutated, the cells don’t divide
  80. 80. cDC2 is a protein that allows division of cells during mitotic events
  81. 81. without cDc2, the cells don’t divide.
  82. 82. We know it is involved in G2 to M phase transition point, but not sure of the specifics
  83. 83. CdC2 is involved in moving cell from G2 to M phase.
  84. 84. MPF is a cyclin-dependent kinase (CDK) because it doesn’t function until cyclin reaches a certain level.
  85. 85. Timothy Hunt and sea urchins
  86. 86. Found that there were cycling proteins through the period of the cell cycle and it peaked at the onset of mitosis. (G2 Mitosis)
  87. 87. Named it cyclin (also by Lee Hartwell at the same time) because it cycled to increase levels and dropped during the cell cycle
  88. 88. MPF and Cyclin
  89. 89. Both peak at onset of Mitosis
  90. 90. MPF is a Cdk – a cyclin dependent kinase because it doesn’t function until the cyclin concentrations reach a certain level
  91. 91. Cyclin increases during interphase
  92. 92. At G2, will activate Cdk as a complex
  93. 93. Complex is a functional protein that moves cell past G2 – M transition and into mitosis
  94. 94. Complex stays functional until cyclin levels drop
  95. 95. Relationhsip between CDk and Cyclin
  96. 96. Cdk is by itself in cell at all times, stable
  97. 97. As cyclin increases, more Cdk binds to it.
  98. 98. There are a series of inactivating phosphorylating events to activate complex
  99. 99. Kinases put Pi on Tyrosine and Threonine, which keeps complex inactive
  100. 100. Activation phosphorylation event: kinase comes in and Pi an activating site on cyclin.
  101. 101. Activating phosphatase after Cdk component is Pi’ed, is dephosphorylated and strips away inactivating Pi
  102. 102. Results is a cyclin Cdk complex as a functional protein
  103. 103. At G2/M transition, MPF is actually the cyclin Cdk complex which regulates G2 M.
  104. 104. Active MPF allows cell to move past G2 transition point and go into M phase
  105. 105. Table in text –
  106. 106. M-Cdk (cyclin Cdk complex)
  107. 107. Cyclin B (cyclin identified by Timothy Hunt) and Cdk is Cdk1
  108. 108. Cdk1 is CdC2 in yeast experiments
  109. 109. Between G2 and M
  110. 110. (((Future terminology)))
  111. 111. G2 M
  112. 112. M-cdk
  113. 113. Cyclin b and Cdk1
  114. 114. G1-Cdk – cyclin D and Cdk4 or Cdk6
  115. 115. G1/S-Cdk cyclin E
  116. 116. Ready to synthesize DNA
  117. 117. Makes sure ready to commit to S phase
  118. 118. S-Cdk
  119. 119. Commits cell to DNA synthesis
  120. 120. M-cdk is active between G2 and M
  121. 121. M-Cdk plays a part in the events between G2 and M
  122. 122. Nueclar envelope breaks down
  123. 123. G2 M
  124. 124. Lamins are phosphorylated by M-Cdk
  125. 125. Chromosomes must condense for mitosis
  126. 126. Condensin condenses them only when Pi’ed by M-Cdk
  127. 127. Recognition of amino acids on M-Cdk
  128. 128. Inactivating Kinase
  129. 129. Wee
  130. 130. Comes in first
  131. 131. Activating Kinase
  132. 132. MO15
  133. 133. Comes in next
  134. 134. Activating phosphatase (from wee and MO15)
  135. 135. Stimulated by recognizing Pi events on Cdk complex and remove inactivating Pi to alieve an active M-Cdk
  136. 136. As you make active M-Cdk, it will Pi the inactive phosphatase to make it active so there is a + feedback loop
  137. 137. More M-Cdk made, the more active phosphatase made to continue cycle.
  138. 138. Recognize amino acids to Pi on Cdk
  139. 139. Activating Phosphatase is sitmulated by recognizing Pi’ing events
  140. 140. As active M-Cdk is made, it will Pi the inactive phosphatase to make it active. Positive feedback.
  141. 141. Concentration of cyclin drops after transition to M phase, which inactives functional M-Cdk
  142. 142. Cyclin is ubiquiniated
  143. 143. Ubiquitin ligases which recognize a sequence on cyclin when it is in complex form
  144. 144. Cyclin binds to Cdk
  145. 145. Exposes site where ubiquitin can be placed onto cyclin
  146. 146. Ligases place ub. On cyclin and then ub acts as signal to degrade protein in proteosomes (organelles in cell that will target ub-linked proteins for degredation)
  147. 147. Cyclin is ubiquitinized by ub-ligases so that they are destroyed.
  148. 148. Once cyclin is lost, the M-Cdk is inactive and Cdk is left, which will stay constant
  149. 149. Ligases place ubiquitin on cyclin and that acts as a signal to degrade proteosomes (organelles in cell that target ubiquitin-linked proteins for degredation)
  150. 150. CDK IS FAIRLY CONSTANT THROUGHOUT CELL (concentration – but it may change as cell size increases)
  151. 151. CDK regulation
  152. 152. Important amino acids required to be Pi for Cdk to be functional
  153. 153. Tyrosine 15 and threonine 14 Pi’ed by Wee
  154. 154. Threonine at 161 Pi by MO15
  155. 155. Cdk Inhibitor (Cki)
  156. 156. Bind complex at the ATP binding site for the proteins
  157. 157. Wipes out abilities to be kinases
  158. 158. Laminine or condensin, so without binding to ATP, cannot function
  159. 159. Active MCdk cannot bind ATP and can’t use Pi from ATP to Pi target
  160. 160. Synthesis of Cyclin B
  161. 161. Increases during interphase
  162. 162. Interacts with Cdk 1
  163. 163. Activatin/inactivating Pi events
  164. 164. Inactivating Pi’s need to be removed
  165. 165. Activated phosphatase removes Pi events at Threonine 14/Tyrosinse 15 leaving only threonine 161 Pi’ed
  166. 166. Active M-Cdk complex!

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