The Origin and Role of Kinetochore Fibers During Mitosis

描述(申请人提供)：Khodjakov实验室最近表明，有丝分裂期间K-纤维的形成可以通过动粒固有的机制发生，而中心体负责K-纤维向纺锤体极点的定向。动粒介导的K-纤维形成的分子机制尚不清楚。我们提出的假设是检验K纤维形成的起始涉及通过中心体独立的机制捕获在动粒附近形成的短微管的假设。为了验证这一假设，我们将激光消融中心体以移除星形微管，同时通过Prc1的siRNA消除带间微管，Prc1是形成中间带所需的一种蛋白质。这一方法将使我们能够通过延时显微镜以前所未有的清晰度和精确度跟踪K纤维(通过a-微管蛋白/GFP表达标记)的形成。由K-纤维组成的微管的起源及其在K-纤维形成过程中的行为将通过固定在该过程不同阶段的细胞的EM断层扫描来确定。除了这个主要目标之外，我们的实验还将揭示中心体独立机制通过产生仅由K纤维组成的有丝分裂纺锤体来运作的程度。

DESCRIPTION (provided by applicant): 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.