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。最近分离的秀丽隐杆线虫突变体缺乏功能的VI类肌球蛋白（spe-15），强调了这种基于肌动蛋白的运动蛋白在发育过程中不对称细胞器分离的重要性。spe-15缺失的蠕虫是不育的，并且由于细胞器分配不当而表现出精子缺陷（Kelleher et al. 2000，提交）。这种突变表型的分子解释尚未阐明，但可以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。第一个目标是确定肌球蛋白VI在线虫精子发生过程中不对称细胞器分配中的可能作用。GFP-SPE-15融合构建体将用于挽救肌球蛋白VI缺失动物体内的spe-15，并研究spe-15在发育中的精细胞中的定位以及spe-15与细胞器的共定位。第二个目标是进一步分析肌球蛋白VI的结构和功能。SPE- 15的突变形式将被测试其拯救SPE- 15和与细胞器共定位的能力。非互补筛选将用于分离新的sp15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凝蛋白VI货物对接蛋白。广泛的遗传知识（包括基因组序列），生命周期短，体积小，身体透明，自受精，易于遗传操作，使秀丽隐杆线虫成为这些研究的选择系统。

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.