Stable spatial gradients of cytoskeleton assembly regulators

1. Stable spatial gradients of cytoskeleton assembly regulators David Odde University of Minnesota

2. Microtubule Structure

3. “Catastrophe” Length (µm) “Rescue” Time (minutes) Microtubule “Dynamic Instability” (DI) kc Vg Vs kr see VanBuren et al., PNAS USA (2002)

4. Microtubules in Mitosis

5. In animal cells: In yeast: 10-20 µm 1.5 µm ~1000 MTs ~40 MTs Mitotic Spindle Interpolar microtubule kinetochore spindle pole body spindle pole body kinetochoremicrotubule chromosome

6. Hypothesis Dynamic instability alone is sufficient to explain the observed MT length distribution in the yeast mitotic spindle

7. Results: Cse4p-GFP Distribution ? 2 µm Experimentally Observed Theoretically Predicted

8. “Catastrophe” Length (µm) “Rescue” Time (minutes) Microtubule “Dynamic Instability” (DI) kc Vg Vs kr

9. -0.4 -0.2 0 +0.4 μm +0.2 Point Spread Function (PSF) • A point source of light is spread via diffraction through a circular aperture • Modeling needs to account for PSF

10. Model-Convolution Original Fluorophore Distribution Simulated Image Obtained by Convolution of PSF and GWN with Original Distribution

11. Spindle Geometry

12. Results:Distribution of Cse4-GFP fluorescence Experimentally Observed Theoretically Predicted

13. QS SE QS x=0 x=L Results: Distribution of Cse4-GFP fluorescence

14. 1000 nm Results: DI Only Model

15. Results: DI Only Model

16. Alternative Models

17. k k* Surface reaction B-->A Homogeneous reaction A-->B MT Repellant Concentration MT Attractant X=L X=0 Position Microtubule Chemotaxis A: Phosphorylated Protein Stabilizes MTs B: Unphosphorylated Protein Destabilizes MTs Microtubule Immobile Kinase Mobile Phosphatase

18. Microtubule Chemotaxis:Op18 A: Op18-hi-P B: Op18-low-P Destabilizes MTs Chromatin Microtubule Immobile Plx1 Mobile PP2A Op18-low-P Concentration Op18-hi-P Position

19. Microtubule Chemotaxis: RanGTP A: RanGTP Stabilizes MTs B: RanGDP Chromatin Microtubule Immobile RCC1 Mobile RanGAP RanGDP Concentration RanGTP Position

20. Model for Chemotactic Gradients of Phosphoprotein State Fick’s Second Law with First-Order Homogeneous Reaction (A->B) B.C. 1: Surface reaction at x=0 (B->A) B.C. 2: No net flux at x=L Conservation of phosphoprotein

21. Predicted Concentration Profile If k= 1 s-1, D=10-11 m2/s, and L=10 µm, then g=3

22. Model Predictions: Effect of Homogeneous Reaction Rate

23. Model Predictions: Effect of Surface Reaction Rate

24. Microtubule Chemotaxis: RanGTP A: RanGTP Stabilizes MTs B: RanGDP Chromatin Microtubule Immobile RCC1 Mobile RanGAP RanGDP Concentration RanGTP Position

27. 1000 nm Results: Chemical Gradient and Polar Ejection Force Models

28. Figure 2 Right Half Spindle Left Half Spindle Cse4 Bleach @ end of simulation, mutant “Tension” model

29. Figure 4 Right Half Spindle Left Half Spindle Cse4 Bleach @ End of Simulation, wild-type, “Gradient-Only” Model

30. F F F F Mitotic Spindle Conclusion: Spatial gradients in MT DI parameter(s) may play a role in mediating budding yeast mitotis

31. Simulated Actin Filament Dendritic Branching Simulated Image of Actin Filament Dendritic Branching Y Y X Z X X Model-Convolution: Application to Dendritic Actin Filament Branching

32. Original Fluorophore Distribution Simulated Image Obtained by Model-Convolution of Original Distribution Image Obtained by Deconvolution of Simulated Image Potential Pitfalls of Deconvolution

33. Acknowledgements • Whitaker Foundation • National Science Foundation

34. Comparing Models to Microscopy Molecular Theory Molecular Reality Computer Simulation Fluorescence Microscope Model Predictions Microscopic Observations ???