CYTOPLASMIC MOTILITY IN EARLY DEVELOPMENT

早期发育中的细胞质运动

• 批准号: 6351283
• 负责人: WILLIAM M SAXTON
• 金额: $ 22.31万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351283
• 关键词: Caenorhabditis elegans; SDS polyacrylamide gel electrophoresis; cell components; cell cycle; cell motility; double stranded RNA; early embryonic stage; immunoprecipitation; kinesin; microtubule associated protein; microtubules; nucleic acid probes; polymerase chain reaction; protein structure function; video microscopy; yeast two hybrid system

The goal of this project is to gain a better understanding of the mechanisms of microtubule-based movement in early embryos. We will identify and study the functions of all the microtubule motors present in early C. elegans embryos. This project is made feasible by key advances in the C. elegans system. First, the genome is almost completely sequenced and microtubule motor genes can be identified by database searches. Second, many of the mRNAs of C. elegans have been cloned as cDNAs, partially sequenced, and are available by mail. Third, virtually any single protein or combination of proteins can be eliminated during oogenesis via injection of inhibitory RNAs, an approach termed RNA-mediated interference (RNAi). The specific goals of the proposed research are as follows: 1) To identify all the microtubule motors in C. elegans. We have already identified 19 and expect to find a few more as the genome sequence is completed. 2) To use RNAi to screen the motors and identify those with unique functions in motility processes in early embryos. Of the 9 motors tested thus far, 3 have essential early functions. The RNAi phenotypes of those 3, and of other early motors we expect to identify in the remainder of the screen, will be studied in live embryos by video-DIC microscopy and in fixed embryos by immunolocalization of microtubules, centrosomes, DNA and other cell components. For further insights, we intend to develop new methods for fluorescence imaging of microtubule and chromosome behavior in live embryos. 3) To generate and use a panel of antisera that are specific for each of the C. elegans microtubule motors. These will be used to gain insight into the functions of motors through their distributions in fixed specimens. In addition they will identify motors that are present in embryos, but whose functions, as revealed by RNAi tests, may be obscured by redundancy. 4) To identify and study the functions of proteins that interact with the subset of microtubule motors that have important roles in early embryos. This will be done using immunoprecipitation, biochemistry, and yeast 2-hybrid analysis for identification of motor interacting proteins. Once identified, the functions of those proteins will be studied with the RNAi approach and immunolocalization. 5) To uncover functions for redundant embryonic motors by multiple motor knock-outs. Multiple knock-outs are straightforward with the RNAi approach.

该项目的目的是更好地了解 早期胚胎中基于微管运动的机制。 我们将 识别和研究所有微管电动机的功能 在早期的秀丽隐杆线虫胚胎中。 这个项目是通过钥匙可行的 秀丽隐杆线虫系统的进步。 首先，基因组几乎是 完全测序和微管运动基因可以通过 数据库搜索。 其次，秀丽隐杆线虫的许多mRNA是 克隆为cDNA，部分测序，可通过邮件获得。 第三， 几乎任何单一蛋白质或蛋白质的组合都可以是 通过注射抑制性RNA，在卵子发生过程中消除 方法称为RNA介导的干扰（RNAI）。 拟议研究的具体目标如下：1） 识别秀丽隐杆线虫中的所有微管电动机。 我们已经 确定19，并期望找到更多，因为基因组序列是 完全的。 2）使用RNAi筛选电动机并识别那些 早期胚胎的运动过程中的独特功能。 9 到目前为止，测试的电动机有3个具有必不可少的早期功能。 RNAi 这3的表型以及我们期望确定的其他早期电动机的表型 在屏幕的其余部分中，将在实时胚胎中研究 通过免疫定位的视频模式显微镜和固定胚胎中的固定胚胎 微管，中心体，DNA和其他细胞成分。 进一步 见解，我们打算开发新的方法，用于荧光成像 活胚胎中的微管和染色体行为。 3）生成和 使用针对每种秀丽隐杆线虫的特定的抗血小板 微管电动机。 这些将用于洞悉 电动机通过其分布在固定标本中的功能。 在 此外，他们还将确定胚胎中存在的电机，但是 RNAi测试所揭示的其功能可能被遮盖 冗余。 4）识别和研究蛋白质的功能 与具有重要作用的微管电动机的子集相互作用 在早期的胚胎中。 这将使用免疫沉淀完成， 生物化学和酵母2杂交分析，用于鉴定电机 相互作用的蛋白质。 一旦确定，这些蛋白质的功能 将使用RNAi方法和免疫定位研究。 5）到 通过多电动机发现冗余胚胎电动机的功能 淘汰赛。 RNAi的多个淘汰赛很简单 方法。