2. Label the cells with

3. Tritiated Thymidine

4. Bromodeoxyuridine (BRDU)

5. There is an antibody to detect BRDU in labeled DNA

6. Labels incorporated into the DNA

7. Tells how much DNA is in the cell during a stage

8. Stages of the Cell-Cycle

9. Precedes Mitosis

10. Has checkpoints to make sure cell is prepared in terms of size and chromosome copies.

11. Signaling at the Checkpoints

12. Can measure amount of DNA in the cell for measuring which step cell cycle is in

13. Labels used

14. Can be incorporated into DNA as it’s being copied.

15. To determine how much DNA is present

16. Radiography

17. Cells can be imaged and Trimidium label can be measured and labeled

18. Can determine which cells are at a certain stage

19. Majority of cells are in G1 phase > S phase > G2

20. Cell sorting can be done for separating the populations

21. FACS – fluorescent activated cell sorting

22. Can take labeled cells, run them through the FACS machine and separate the cells with the labels

23. BRDU label used primarily

24. What questions can be looked at?

25. What regulates the checkpoints?

26. What is happening at Mitosis?

27. Checkpoints

28. Between G1 and S: is environment favorable for entering DNA synthesis?

29. Can enter G0 stage until environment is favorable to continue to S phase

30. Cell can also undergo apoptosis if signals for ideal environment are not given at a certain time frame

31. G2 before Mitosis

32. Cell is looking for whether or not DNA has been replicated, and if so, correctly?

33. In Mitosis

34. Makes sure sister chromatids are attached and separate properly at mitotic spindle

35. Key Transition Points in the Cell Cycle

36. G1 S phase checkpoint – “restriction point” – requirements for S phase transition

37. Growth factors

38. Nutrients

39. Increase in cell size

40. Absence of DNA damage

41. Can enter G0

42. “G2 – M transition”

43. Cell size

44. DNA damage

45. DNA replication

46. Can enter G0 again and signal sequences can make cell go back to repair damage

47. Mitosis – “Metaphase-Anaphase transition”

48. Chromosome attachments to spindle so the chromatids can be pulled apart

49. What Did People do to determine that there were proteins that influenced cell splitting

50. Separate cells from different stages fused

51. The fused cell is called a heterokaryon (monoclonal antibodies in previous lectures) cells with two nuclei.

52. To fuse cells:

53. Can use viral infections

54. Chemical treatment

55. Electroporation (Electrical pulses)

56. Often used to put DNA into a cell as well

57. Then fuse separated cells

58. S and G1 fusion – G1 nucleus is activated to undergo DNA synthesis

59. M and G1 fusion – mitosis activated

60. Experimenting with oocytes to determine what was driving the cycle in fused cells

61. Stalled in Meiosis II

62. Cytoplasm injected and cell doesn’t divide

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. Fertilization would complete meiosis II, it would be the signal

65. Increased concentrations of certain proteins in mitosis

66. Saw two peaks in a graph

67. MPF = maturation promoting factor (green peak) (rose at onset of mitosis and fell very fast during mitosis)

68. Will mature the egg if being stalled in meiosis II aka. M phase promoting factor because it moved the cell into mitosis

69. Temperature Sensitive Yeast Experiments

70. Generated by random mutagenesis

71. Looked for mutations in mitosis at higher temps than what they normally grow at (ideal is 23 C for a normal cell)

72. At restrictive temp, some yeast cells will not move through mitosis, so the cell is stalled at some point in cell cycle.

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. Can use for all stages of mitosis

75. Lee Hartwell

76. Used a particular yeast strain to identify mutations that didn’t allow mitosis to continue normally.

77. Wild Type yeast could divide into daughter cells normally wherease mutated ones did not divide into daughter cells

78. There was a mutation in cell cycle division (cDC2 cell division cycle)

79. Important for fission of mitotic event and without it, or mutated, the cells don’t divide

80. cDC2 is a protein that allows division of cells during mitotic events

81. without cDc2, the cells don’t divide.

82. We know it is involved in G2 to M phase transition point, but not sure of the specifics

83. CdC2 is involved in moving cell from G2 to M phase.

84. MPF is a cyclin-dependent kinase (CDK) because it doesn’t function until cyclin reaches a certain level.

85. Timothy Hunt and sea urchins

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. Named it cyclin (also by Lee Hartwell at the same time) because it cycled to increase levels and dropped during the cell cycle

88. MPF and Cyclin

89. Both peak at onset of Mitosis

90. MPF is a Cdk – a cyclin dependent kinase because it doesn’t function until the cyclin concentrations reach a certain level

91. Cyclin increases during interphase

92. At G2, will activate Cdk as a complex

93. Complex is a functional protein that moves cell past G2 – M transition and into mitosis

94. Complex stays functional until cyclin levels drop

95. Relationhsip between CDk and Cyclin

96. Cdk is by itself in cell at all times, stable

97. As cyclin increases, more Cdk binds to it.

98. There are a series of inactivating phosphorylating events to activate complex

99. Kinases put Pi on Tyrosine and Threonine, which keeps complex inactive

100. Activation phosphorylation event: kinase comes in and Pi an activating site on cyclin.

101. Activating phosphatase after Cdk component is Pi’ed, is dephosphorylated and strips away inactivating Pi

102. Results is a cyclin Cdk complex as a functional protein

103. At G2/M transition, MPF is actually the cyclin Cdk complex which regulates G2 M.

104. Active MPF allows cell to move past G2 transition point and go into M phase

105. Table in text –

106. M-Cdk (cyclin Cdk complex)

107. Cyclin B (cyclin identified by Timothy Hunt) and Cdk is Cdk1

108. Cdk1 is CdC2 in yeast experiments

109. Between G2 and M

110. (((Future terminology)))

111. G2 M

112. M-cdk

113. Cyclin b and Cdk1

114. G1-Cdk – cyclin D and Cdk4 or Cdk6

115. G1/S-Cdk cyclin E

116. Ready to synthesize DNA

117. Makes sure ready to commit to S phase

118. S-Cdk

119. Commits cell to DNA synthesis

120. M-cdk is active between G2 and M

121. M-Cdk plays a part in the events between G2 and M

122. Nueclar envelope breaks down

123. G2 M

124. Lamins are phosphorylated by M-Cdk

125. Chromosomes must condense for mitosis

126. Condensin condenses them only when Pi’ed by M-Cdk

127. Recognition of amino acids on M-Cdk

128. Inactivating Kinase

129. Wee

130. Comes in first

131. Activating Kinase

132. MO15

133. Comes in next

134. Activating phosphatase (from wee and MO15)

135. Stimulated by recognizing Pi events on Cdk complex and remove inactivating Pi to alieve an active M-Cdk

136. As you make active M-Cdk, it will Pi the inactive phosphatase to make it active so there is a + feedback loop

137. More M-Cdk made, the more active phosphatase made to continue cycle.

138. Recognize amino acids to Pi on Cdk

139. Activating Phosphatase is sitmulated by recognizing Pi’ing events

140. As active M-Cdk is made, it will Pi the inactive phosphatase to make it active. Positive feedback.

141. Concentration of cyclin drops after transition to M phase, which inactives functional M-Cdk

142. Cyclin is ubiquiniated

143. Ubiquitin ligases which recognize a sequence on cyclin when it is in complex form

144. Cyclin binds to Cdk

145. Exposes site where ubiquitin can be placed onto cyclin

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. Cyclin is ubiquitinized by ub-ligases so that they are destroyed.

148. Once cyclin is lost, the M-Cdk is inactive and Cdk is left, which will stay constant

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. CDK IS FAIRLY CONSTANT THROUGHOUT CELL (concentration – but it may change as cell size increases)

151. CDK regulation

152. Important amino acids required to be Pi for Cdk to be functional

153. Tyrosine 15 and threonine 14 Pi’ed by Wee

154. Threonine at 161 Pi by MO15

155. Cdk Inhibitor (Cki)

156. Bind complex at the ATP binding site for the proteins

157. Wipes out abilities to be kinases

158. Laminine or condensin, so without binding to ATP, cannot function

159. Active MCdk cannot bind ATP and can’t use Pi from ATP to Pi target

160. Synthesis of Cyclin B

161. Increases during interphase

162. Interacts with Cdk 1

163. Activatin/inactivating Pi events

164. Inactivating Pi’s need to be removed

165. Activated phosphatase removes Pi events at Threonine 14/Tyrosinse 15 leaving only threonine 161 Pi’ed

166. Active M-Cdk complex!