1. Titration of the spindle assembly checkpoint in embryonic cells Danielle Miranda Advisor: Brad Shuster

2. Mitosis = DNA = Microtubules = Actin

3. The Spindle Assembly Checkpoint (SAC) ensures that cells don’t progress through mitosis if there are chromosome attachment errors The SAC senses microtubule attachment to kinetochores Loss of attachment or lack of tension activates the checkpoint Holds cell in mitosis until errors are corrected If error can’t be corrected, cells die by apoptosis

4. Checkpoint OffCheckpoint On Microtubule Kinetochore Mad1 Mad2 APC/C Cdc20 BubR1 Mps1 BubR1 Kinetochore Microtubule Mad1 Mad2 APC/C Cdc20 Mad2 BubR1 Mps1 BubR1 Bub3 Mad2 Mad1 Mps1 Bub3 SAC Proteins

5. The checkpoint can respond to a single unattached kinetochore in somatic cells Early embryos are “checkpoint challenged”: * In mammalian and early sea urchin embryos, spindle disruption results in a moderate delay in mitotic exit Why is this important? Spindle Checkpoint in Somatic vs Embryonic cells

6. Why are embryos “checkpoint challenged?” Two possibilities: 1. Checkpoint genes are not expressed in early embryos 2. The kinetochores to cytoplasmic volume ratio in a large cell is insufficient to arrest the cell in mitosis

7. Question: Are checkpoint genes expressed in early sea urchin embryos? BlastulaEgg Checkpoint Gene ++CyclinA (Control) +-Mps1 +-Bub3 +-Mad2b ++Mad2a +-Mad1

8. Is checkpoint responsiveness a function of cell size? Images of sea urchin early and late embryo cell spindles (see inset) taken at the same magnification

10. How do we evaluate the responsiveness of the checkpoint? Experiment 1 : (performed at 1 cell stage) Step 1: Experimentally induce polyspermy –ie increase chromosome # per unit vol cytoplasm Step 2: Induce the checkpoint by inhibiting microtubule polymerization Step 3: Flatten cells and measure time spent in mitotic arrest

11. WT +1 +3 WT +1 +3 WT +1 +3 WT +1 +3

12. Time spent in Mitosis

13. How do we evaluate the responsiveness of the checkpoint? Experiment 2 : (performed at 1 cell stage) Step 1: Inject cells with fluorescent histone –To visualize chromatin condensation Step 2: Treat cells with nocodazole –Depolymerize MT’s and activate checkpoint Step 3: Flatten cells and cut volume of cytoplasm –Compare cut and non-cut cells

14. Reducing cell volume increases the length of mitotic arrest

15. Summary mRNA’s for vital checkpoint genes are present in the early sea urchin embryo Increasing kinetochores to the cytoplasmic volume prolongs the SAC response Checkpoint responsiveness increases as cell volume is reduced

16. Future directions Increase N of our polyploidy experiments Perform additional cutting experiments Use dominant-negative mutants of Mad2 to disrupt checkpoint function in nocodazole- treated, polyploid eggs

17. Acknowledgements Dr. Shuster and the Shuster Lab John Pringle (Stanford University) Funding by the BRIDGES, MARC and SCORE programs