The Origin and Role of Kinetochore Fibers During Mitosis

有丝分裂过程中着丝粒纤维的起源和作用

• 批准号: 7113291
• 负责人: Christopher B O'Connell
• 金额: $ 4.4万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-12 至 2009-06-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7113291
• 关键词: bioimaging /biomedical imaging; cell component structure /function; cell cycle; centromere; cytoskeleton; electron microscopy; green fluorescent proteins; laser capture microdissection; light microscopy; microtubules; mitotic spindle apparatus; molecular /cellular imaging; postdoctoral investigator; small interfering RNA; time resolved data

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.

描述（由申请人提供）：Khodjakov实验室最近表明，有丝分裂期间K-纤维的形成可以通过着丝粒固有的机制发生，而中心体负责K-纤维朝向纺锤体极的方向。负责kinetochore介导的K-纤维形成的分子机制仍然未知。我们建议是测试的假设，K-纤维形成的启动涉及捕获的短微管，通过中心体独立的机制在动粒附近形成。为了验证这一假设，我们将激光烧蚀中心体以去除星形微管，同时通过PRC 1的siRNA消除中间区形成所需的蛋白质的区间微管。这种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-纤维的形成（通过α-微管蛋白/GFP表达标记）。包含K-纤维的微管的起源及其在K-纤维形成期间的行为将通过在该过程的各个阶段固定的细胞的EM断层扫描来确定。除了这个主要目标，我们的实验将揭示在何种程度上中心体独立的机制通过产生只由K-纤维组成的有丝分裂纺锤体来运作。