The Khodjakov lab has recently shown that formation of K-fibers during mitosis can occur via mechanisms intrinsic to the kinetochores, while centrosomes are responsible for the orientation of K-fibers toward spindle poles. Molecular mechanisms responsible for the kinetochore-mediated K-fiber formation remain unknown. We propose is to test the hypothesis that initiation of K-fiber formation involves capture of short microtubules that form in the vicinity of the kinetochore via centrosome-independent mechanisms. To test this hypothesis, we will laser-ablate centrosomes to remove astral microtubules, and simultaneously eliminate interzonal microtubules by siRNA of PRC1, a protein required for formation of the midzone. This approach will allow us to follow formation of K-fibers (labeled via a-tubulin/GFP expression) by time-lapse microscopy with unprecedented clarity and precision. The origin of microtubules comprising K-fibers and their behavior during K-fiber formation will be determined by EM tomography of cells fixed at various stages of the process. In addition to this primary goal, our experiments will reveal the extent to which centrosome-independent mechanisms operate by generating mitotic spindles comprised only of K-fibers. ? ? ?
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