SLIDING FILAMENT MECHANISMS IN MITOTIC SPINDLES

有丝分裂纺锤体中的滑动丝机制

• 批准号: 6498547
• 负责人: INGRID BRUST-MASCHER
• 金额: $ 5.21万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6498547
• 关键词: Caenorhabditis elegans; Drosophilidae; antibody; cell cycle; confocal scanning microscopy; crosslink; cytogenetics; cytoskeletal proteins; dynein ATPase; embryo /fetus; fluorescence microscopy; histogenesis; immunoelectron microscopy; intracellular transport; laboratory rabbit; microfilaments; microinjections; microtubule associated protein; microtubules; mitotic spindle apparatus; mutant; protein purification; protein structure function; spindle pole body; technology /technique development

The overall aim of the project is to learn how multiple microtubule sliding motors contribute to the morphogenesis of the mitotic spindle in Drosophila syncytial blastoderm-stage embryos. These embryos are amenable to biochemical, genetic and cytological analysis of mitotic mechanisms, and previous work done in this laboratory showed how three mitotic motors, KLP61F, Ncd, and cytoplasmic dynein, cooperate fo position spindle poles during spindle assembly, maintenance and elongation. We want to further test the hypothesis that KLP61F and Ncd, located on interpolar microtubule bundles, and dynein localized on the cell cortex, cooperate to drive a sliding filament mechanism that precisely positions spindle poles. To do this we will use techniques already developed in the laboratory, including the purification and analysis of native motor proteins, the microinjection of function- blocking antibodies and mutant proteins, and the high resolution analysis of spindle pole positioning by time-lapse confocal microscopy of living embryos. We will augment these approaches with the use of Fluorescence Photobleaching, Fluorescence Photoactivation or Fluorescent Speckle Microscopy to directly visualize microtubule- microtubule and microtubule-cortical actin sliding in living embryos.

该项目的总体目标是了解多个微管滑动马达如何促进果蝇合胞胚层阶段胚胎有丝分裂纺锤体的形态发生。这些胚胎适合对有丝分裂机制进行生化、遗传和细胞学分析，该实验室之前所做的工作表明，三种有丝分裂马达（KLP61F、NCd 和细胞质动力蛋白）如何在纺锤体组装、维持和伸长过程中合作定位纺锤体极。我们想要进一步检验这样的假设：位于极间微管束上的 KLP61F 和 Ncd 以及位于细胞皮层上的动力蛋白合作驱动精确定位纺锤体极点的滑动丝机制。为此，我们将使用实验室已开发的技术，包括天然运动蛋白的纯化和分析、功能阻断抗体和突变蛋白的显微注射，以及通过延时共聚焦显微镜对纺锤体极定位进行高分辨率分析。活胚胎。我们将通过使用荧光光漂白、荧光光活化或荧光散斑显微镜来增强这些方法，以直接可视化活体胚胎中微管-微管和微管-皮质肌动蛋白的滑动。