THE FORMATION AND FUNCTION OF MICROTUBULES

微管的形成和功能

• 批准号: 3270108
• 负责人: RAYMOND E STEPHENS
• 金额: $ 18.55万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270108
• 关键词: Bivalvia; Echinoidea; adenosinetriphosphatase; binding proteins; cell differentiation; cell membrane; chemical structure function; cilium; dynein ATPase; egg /ovum; electrofocusing; electron microscopy; flagellum; freeze etching; gel electrophoresis; genetic regulation; glycoproteins; high performance liquid chromatography; membrane proteins; microtubules; nonmammalian vertebrate embryology; protein biosynthesis; protein reconstitution; protein sequence; protein structure; radiotracer; saltwater environment; sea urchins; thermodynamics; tissue /cell culture; tritium; tubulin

The role of microtubules in development and the mechanism of microtubule-based motility will be approached through a detailed study of the chemistry of tubulin and its interactions with itself and with other proteins. The classes and amounts of tubulin in cells during development, the state of tubulin pools before, during, and after microtubule assembly into spindles or cilia, tubulin association with accessory proteins to form specific organelles such as basal bodies or ciliary axonemes, the association of tubulin with membranes, and the interaction of tubulin with dynein are basic questions that this project has been exploring. There will be four major aims during this project period. First, studies of reverse-phase HPLC separation and analysis of tubulin subunits will be extended by systematically varying stationary-phase and solvent conditions, performing separations with new detergent-modified systems, exploring co- chromatography to detect protein-protein interaction, and separating large organelle-specific peptides in an attempt to define distinguishing regions of the tubulin molecules. Second, further studies on tektins, a new class of tubulin-associated proteins, will involve fractionation of ciliary remnants, nearly devoid of tubulin, to ascertain the location of tektin filaments, a systematic study of the reassembly properties of tektin subunits and the reassembly of tubulin on tektin filaments in an attempt to produce doublet microtubules and, finally, an investigation of possible spindle tektin synthesis at prophase. Third, studies on ciliary membrane tubulin will be continued in order to determine what chemical differences distinguish membrane from axonemal tubulin. We will study protein topology with membrane probes, determine tubulin interaction with specific lipids, and study the possible interaction of tubulin-containing membranes with cytoplasmic proteins. Finally, studies of a neuronally-controlled dynein from mussel gill lateral cilia will involve a thorough characterization of both the inner and outer arm dynein molecules and a 10S phosporylated entity that co-extracts with dynein, an analysis of the specificity of phosphorylation of 3 presumptive light chains, and a determination of possible dynein-associated kinases and phosphatases.

微管在发育中的作用及其机制 基于微管的运动将通过详细的 微管蛋白的化学性质及其与自身相互作用的研究 以及其他蛋白质。 微管蛋白的种类和含量 细胞在发育过程中，微管蛋白池之前的状态， 在微管组装成纺锤体或纤毛期间和之后， 微管蛋白与辅助蛋白结合形成特异性 细胞器，例如基底体或纤毛轴丝， 微管蛋白与膜的结合以及相互作用 微管蛋白和动力蛋白是这个项目的基本问题 探索。 该项目将有四个主要目标 时期。 一、反相HPLC分离研究 微管蛋白亚基的分析将通过系统地扩展 改变固定相和溶剂条件，执行 使用新的洗涤剂改性系统进行分离，探索共同 色谱法检测蛋白质-蛋白质相互作用，以及 分离大的细胞器特异性肽，试图 定义微管蛋白分子的区别区域。 第二， 对 tektins（一类新的微管蛋白相关蛋白）的进一步研究 蛋白质，将涉及睫状残余物的分级，几乎 缺乏微管蛋白，以确定 tektin 丝的位置，a tektin 亚基重组特性的系统研究 以及微管蛋白在tektin丝上的重新组装，试图 产生双联微管，最后，研究 可能在前期合成纺锤体tektin。 三、研究 将继续睫状膜微管蛋白以确定 膜与轴丝的化学差异是什么 微管蛋白。 我们将用膜探针研究蛋白质拓扑结构， 确定微管蛋白与特定脂质的相互作用，并研究 含微管蛋白的膜可能与 细胞质蛋白。 最后，神经元控制的研究 来自贻贝鳃侧纤毛的动力蛋白将涉及彻底的 内臂和外臂动力蛋白分子的表征 以及与动力蛋白共提取的 10S 磷酸化实体， 3个假定的磷酸化特异性分析 轻链，以及可能的动力蛋白相关的测定 激酶和磷酸酶。