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

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

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

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Building a model: Budding Yeast Spindle Geometry

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

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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

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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

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

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

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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

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

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Spatial Gradient Model for Catastrophe Frequency

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

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Cse4-GFP Fluorescence Recovery After Photobleaching (FRAP) Experiment

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

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Catastrophe Gradient-Tension Rescue Model 1 32

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

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

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

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GFP-Tubulin FRAP Experiment

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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

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

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

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

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Mutant Cell Experiment: No Tension Between Sister Kinetochores EXPERIMENTALSIMULATION

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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

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

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Extra slides

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

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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

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“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

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

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