SLIDING FILAMENT MECHANISMS IN MITOTIC SPINDLES

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期胚胎有丝分裂纺锤体的形态发生。这些胚胎适合于有丝分裂机制的生化、遗传和细胞学分析，该实验室先前的工作表明，在纺锤体组装、维护和伸长过程中，KLP61F、Ncd和细胞质动力蛋白这三个有丝分裂马达如何合作定位纺锤极。我们希望进一步验证位于极性微管束上的KLP61F和Ncd以及位于细胞皮层上的动力蛋白共同驱动滑动丝机制以精确定位纺锤杆的假设。为了做到这一点，我们将使用实验室已经开发的技术，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，以及通过活胚胎的延时共聚焦显微镜对纺锤杆定位的高分辨率分析。我们将使用荧光光漂白、荧光光激活或荧光斑点显微镜来增强这些方法，直接观察活胚胎中微管-微管和微管-皮质肌动蛋白滑动。

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.