1. How can dynamic kinetochore movements result in stable kinetochore cluster positioning in metaphase?

2. EXPERIMENTS Dynamic Kinetochore Movements Metaphase Kinetochore Clustering ? COMPUTER SIMULATION Dynamic Kinetochore Movements Metaphase Kinetochore Clustering A Model for Regulation of Kinetochore Dynamics

3. A Model for Regulation of Kinetochore Dynamics Direct New Experimentation Develop Hypotheses for Mutant Phenotypes Account for Stochastic variation using quantitative analysis

4. Building a model: Budding Yeast Spindle Geometry

5. Length (µm) Time (minutes) “Catastrophe” “Rescue” A Stochastic Simulation: Kinetochore Microtubule “Dynamic Instability” VgVg VsVs kckc krkr

6. Evaluating Model Predictions: Model Convolution 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 … … Simulation Results Simulated Fluorescent Kinetochore and SPB Markers

7. Point Spread Function (PSF) A point source of light is spread via diffraction through a circular aperture Modeling needs to account for PSF -0.4-0.20+0.2 +0.4 μm

8. Simulated Fluorescent Kinetochore and Spindle Pole Body Markers Evaluating Model Predictions: Model Convolution Quantitative Microscope Point Spread Function Measured Background Noise Final Simulated Image

9. Can Microtubule Dynamic Instability Explain Kinetochore Congression in Budding Yeast? Experimentally Observed Theoretically Predicted ? 2 µm

10. Constant Parameters of Kinetochore Microtubule Dynamic Instability Sprague et al., Biophysical J., 2003 Catastrophe Frequency (kc) = Rescue Frequency (kr) UNIFORM DISTRIBUTION Unequal Catastrophe and Rescue Frequencies EXPONENTIAL DISTRIBUTION EXPERIMENTAL RESULTS: Peak in kinetochore fluorescence midway between poles and equator

11. Can only get peaks here Not here Right PoleLeft Pole Not here Constant Parameters of Kinetochore Microtubule Dynamic Instability

12. Spatial Gradient Model for Catastrophe Frequency

13. Spatial Gradient Model for Catastrophe Frequency Experimental Image E Catastrophe Gradient Catastrophe Gradient Simulated Image

14. Cse4-GFP Fluorescence Recovery After Photobleaching (FRAP) Experiment

15. Cse4-GFP FRAP Experiment: Simulation Results *Experimental data from Pearson et al., Curr Biol (2004)

16. Catastrophe Gradient-Tension Rescue Model 1 32

17. POLE Simulated Sister Kinetochore Position Tracking Catastrophe Gradient Model …Add Tension- Dependent Rescue POLE

18. Cse4-GFP FRAP Experiment: Simulation Results *Experimental data from Pearson et al., Curr Biol (2004)

19. Spatial Catastrophe Gradient Model with Tension-Dependent Rescue Frequency Experimental Image Simulated Image

20. GFP-Tubulin FRAP Experiment

21. Simulated kMT Dynamics Simulated Tubulin FRAP Recovery (Spindle-Half) GFP-Tubulin FRAP Experiment: Simulation Results *Experimental data from Maddox et al., Nature Cell Biol (2000) Tubulin FRAP Experiment Constrains Growth and Shrinking Velocities in Model

22. GFP-Tubulin FRAP by Spindle Position: Preliminary Simulation Results Tubulin FRAP by Spindle Position Experiment Constrains all Dynamic Instability Parameters in Model

23. What would the model predict for a mutant lacking tension at the kinetochore?

24. Mutant Spindles: Loss of Tension at the Kinetochore Spring Constant = 0

25. Mutant Cell Experiment: No Tension Between Sister Kinetochores EXPERIMENTALSIMULATION

26. CONCLUSION Metaphase kinetochore congression in budding yeast may be mediated by a catastrophe gradient, and depend on tension between sister kinetochores. SIMULATED METAPHASE CONGRESSION SIMULATED LOSS OF TENSION

27. A Model for Regulation of Kinetochore Dynamics Direct New Experimentation Develop Hypotheses for Mutant Phenotypes Account for Stochastic variation using quantitative analysis FUTURE DIRECTIONS

28. Extra slides

29. “Experiment-Deconvolution” vs. “Model-Convolution” Model Experiment Deconvolution Convolution

30. Steady-State “Metaphase” Spindle (Length 1.6-1.9 µm) Non-Steady State Early Metaphase Spindle (Length 1.1-1.5 µm) Quantitative Analysis of Spindle Fluorescence Images: Steady State Cse4-GFP Distribution Metaphase Reference Distribution

31. “Microtubule Chemotaxis” in a Chemical Gradient Immobile Kinase Mobile Phosphatase A: Phosphorylated Protein B: Dephosphorylated Protein k* Surface reaction B-->A k Homogeneous reaction A-->B Kinetochore Microtubules - + Immobile Kinase MT Destabilizer Position Concentration X=0 X=L

32. Loss of Tension at the Kinetochore Control Spindle (with Chromosome Replication) Replication Deficient Spindle Bipolar Attachment at KinetochoreMonopolar Attachment at Kinetochore