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Stable spatial gradients of cytoskeleton assembly regulators

Stable spatial gradients of cytoskeleton assembly regulators. David Odde University of Minnesota. Microtubule Structure. “Catastrophe”. Length (µm). “Rescue”. Time (minutes). Microtubule “Dynamic Instability” (DI). k c. V g. V s. k r. see VanBuren et al., PNAS USA (2002).

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Stable spatial gradients of cytoskeleton assembly regulators

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  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

  25. 1000 nm Results: Chemical Gradient and Polar Ejection Force Models

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

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

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

  29. 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

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

  31. Acknowledgements • Whitaker Foundation • National Science Foundation

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

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