Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped shape despite microtubule turnover and internal stresses. This ellipsoidal architecture, in which microtubule minus-ends are focused into two poles, is essential to the spindle’s function of accurately segregating chromosomes. In this work, I ask how the spindle forms and holds its steady-state shape. I report that the molecular motor dynein and the microtubule binding-protein NuMA are essential for mammalian spindles to reach and hold a steady-state geometry. In their absence, the kinesin-5 Eg5 powers a turbulent microtubule network that can drive flow of cytoplasmic organelles and whole-cell movement. Dynein and NuMA were previously known to be essential for spindle pole formation, but we did not know their contribution to shape stabilization at the whole-spindle scale—nor did we know how and where they pull on microtubules to build poles. Using quantitative live imaging and laser ablation, I show that dynein pulls specifically on microtubule minus-ends, rapidly transporting them towards poles. Dynein localization to microtubule minus- ends depends on NuMA, which recruits the dynein adaptor dynactin to minus-ends. Contrary to previous models, NuMA localization to minus-ends is independent of dynein and involves a C-terminal region outside its canonical microtubule-binding domain. Thus, NuMA serves as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles. This microtubule end-clustering compacts the spindle microtubule network to a defined geometry and suppresses network turbulence, maintaining a steady-state spindle shape over long timescales.
|Commitee:||Agard, David A., Reiter, Jeremy F., Vale, Ronald D.|
|School:||University of California, San Francisco|
|School Location:||United States -- California|
|Source:||DAI-B 80/05(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Biophysics|
|Keywords:||Dynein, Microtubules, Mitosis, Self-organization, Spindle, Spindle mechanics|
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