A Role for Myosin VI in Asymmetric Organelle Segregation

细胞骨架在许多发育过程中起着关键作用，从细胞运动到细胞成分的不对称分离。最近分离到一个C。缺乏功能性VI类肌球蛋白（SPE-15）的线虫突变体突出了这种基于肌动蛋白的马达蛋白在发育期间的不对称细胞器分离中的重要性。spe-15缺失的蠕虫是不育的，由于细胞器分配不当而表现出精子细胞缺陷（Kelleher等，2000年提交）。这种突变表型的分子解释尚未阐明，但可以提供进一步了解细胞骨架和非常规肌球蛋白在发育过程中的作用。这项建议包括两个主要目标。第一个目标是确定可能的作用，肌球蛋白VI在精子发生过程中的不对称细胞器分区在秀丽隐杆线虫。GFP-SPE-15融合构建体将用于在肌球蛋白VI缺失动物中拯救SPE-15，以及研究SPE-15在发育精母细胞中随时间的定位以及SPE-15与细胞器的共定位。第二个目的是进一步分析肌球蛋白VI的结构和功能。将测试SPE- 15的突变形式拯救SPE-15和与细胞器共定位的能力。将进行非互补筛选以分离新的spe-15等位基因，包括温度敏感突变体。酵母双杂交筛选将用于鉴定潜在的肌球蛋白VI货物对接蛋白。丰富的遗传学知识（包括基因组序列），短的生命周期，小的体积，身体透明，自我受精，以及易于遗传操作，使C。elegans是这些研究的首选系统。

The cytoskeleton plays a critical role in many developmental processes, from cell motility to asymmetric segregation of cellular components. The recent isolation of a C. elegans mutant lacking a functional class VI myosin (spe-15) highlights the importance of this actin-based motor protein in asymmetric organelle segregation during development. Worms with a deletion in spe-15 are sterile and exhibit spermatid defects due to improper organelle partitioning (Kelleher et al. 2000, submitted). The molecular explanation for this mutant phenotype has not been elucidated, but could offer further insight into roles for the cytoskeleton and unconventional myosins in developmental processes. This proposal encompasses two major objectives. The first objective is to define the possible roles for myosin VI in asymmetric organelle partitioning during spermatogenesis in C, elegans. GFP-SPE-15 fusion constructs will be used to rescue spe-15 in myosin VI deletion animals, as well as to study SPE-15 localization over time in developing spermatocytes and co- localization of SPE-15 with organelles. The second objective is to further analyze the structure and function of myosin VI. Mutated forms of SPE- 15 will be tested for their ability to rescue spe-15 and to co-localize with organelles. Noncomplementation screens will be performed to isolate new spe-15 alleles, including temperature sensitive mutants. A yeast two-hybrid screen will be used to identify potential myosin VI cargo docking proteins. The vast genetic knowledge available (including the genome sequence), short life cycle, small size, body transparency, self- fertilization, and ease of genetic manipulation make C. elegans the system of choice for these studies.