CYTOKINESIS AND CELL POLARITY IN C ELEGANS EMBRYOS

我们建议利用线虫早期胚胎强大的遗传学和令人印象深刻的细胞学特性来研究发育中动物的细胞质分裂及其与有丝分裂纺锤体定向的关系。早期线虫胚胎为这些研究提供了两个关键优势：(I)能够快速识别早期胚胎细胞中胞质分裂和有丝分裂纺锤体定位所需的基因，以及(Ii)能够高分辨率地显示大(约22 x 55微米)1-细胞期受精卵中功能重要蛋白的亚细胞定位。我们有三个长期目标：(1)使用遗传学和分子方法将胞质分裂定义为在早期胚胎中执行胞质分裂的一系列离散的分子相互作用。(2)确定早期胚胎细胞不对称分裂过程中胞质分裂终止与有丝分裂纺锤体定向机制之间的机制关系。(3)鉴定对胞质分裂和细胞不对称分裂产生重要作用的运动蛋白。这些研究将为人类病理学的分子基础提供重要的见解：细胞骨架/质膜相互作用已被证明与我们对癌症和其他重大疾病的理解有关，包括肌肉营养不良、耳聋和不孕症。在初步研究中，我们已经确定了一种名为Cyk-1的基因，它是细胞质分裂后期所必需的。这是在线虫中发现的第一个基因，它是早期胚胎胞质分裂所特需的。有趣的是，Cyk-1蛋白定位于胞质分裂后期分裂沟的前沿，在那里我们假设它连接了肌动蛋白和微管蛋白细胞骨架。虽然Cyk-1为鉴定发生在胞质分裂过程中的蛋白质/蛋白质相互作用提供了一个起点，但我们首先建议尽可能全面地鉴定胞质分裂和有丝分裂纺锤体定向所需的基因。为此，我们已经开始了对温度敏感的胚胎致死突变的大规模筛选，我们正在使用一种功能基因组学方法，其中包括使用一种最近发现的称为RNA干扰的技术。我们将从分子上克隆细胞质分裂和有丝分裂纺锤体定向所需的基因。通过使用遗传和分子上位性实验，并通过研究我们识别的不同蛋白质如何相互作用，我们将定义控制这些基本细胞和发育过程的分子相互作用和途径。

We propose to use the powerful genetics and the impressive cytological properties of the early C. elegans embryo to investigate cytokinesis and its relationship to mitotic spindle orientation in a developing animal. The early C. elegans embryo offers two key advantages for these studies: (i) the ability to rapidly identify genes required for cytokinesis and for mitotic spindle orientation in early embryonic cells, and (ii) the ability to visualize with high resolution the subcellular localization of functionally important proteins in the large (approximately 22 x 55 micron) 1-cell stage zygote. We have three long term goals: (1) To use genetic and molecular methods to define cytokinesis as a series of discrete molecular interactions that execute cytokinesis in the early embryo. (2) To determine the mechanistic relationship between the termination of cytokinesis and the mechanisms that orient mitotic spindles during asymmetric divisions in early embryonic cells. (3) To identify motor proteins important for cytokinesis and the generation of asymmetric cell divisions. These studies will provide significant insight into the molecular basis for human pathologies: cytoskeleton/plasma membrane interactions have proven relevant to our understanding of cancer and of other significant diseases, including muscular dystrophy, deafness, and sterility. In preliminary studies, we have identified a gene called cyk-1 that is required for a late step in cytokinesis. This is the first gene identified in C. elegans that is specifically required for cytokinesis in the early embryo. Intriguingly, the CYK-1 protein localizes to the leading edge of the cleavage furrow late in cytokinesis, where we hypothesize it bridges the actin and tubulin cytoskeleton. While CYK-1 provides a starting point for identifying functionally protein/protein interactions that occur during cytokinesis, we first propose to identify as comprehensively as possible the genes required for cytokinesis and mitotic spindle orientation. To this end, we have begun a large-scale screen for temperature-sensitive, embryonic-lethal mutants, and we are using a functional genomics approach that involves the use of a recently discovered technology called RNA interference. We will molecularly clone genes that are most specifically required for cytokinesis and mitotic spindle orientation. By using genetic and molecular epistasis experiments, and by examining how the different proteins we identify interact, we will define the molecular interactions and pathways that control these fundamental cellular and developmental processes.