MOLECULAR INTERACTIONS IN MOVING AXONEMES

移动轴丝中的分子相互作用

• 批准号: 3303587
• 负责人: GIANNI PIPERNO
• 金额: $ 22.53万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303587
• 关键词: Chlamydomonas; actins; adenosine triphosphate; calcium binding protein; chemical binding; chemical structure; chimeric proteins; cilium /flagellum motility; complementary DNA; dynein ATPase; electron microscopy; genetic library; genome; hybridomas; immunoelectron microscopy; intermolecular interaction; laboratory mouse; microtubules; molecular cloning; nucleic acid sequence; phosphorylation; polymerase chain reaction; posttranslational modifications; protein sequence; protein structure; radionuclides; tissue /cell culture

Cilia and flagella are present in a wide variety of eukaryotic cells. Their disfunction is correlated with respiratory ailments or male sterility. This proposal has the following two long-term objectives: analyze the molecular and structural features of those axonemal components that are essential for the generation of axonemal movement and second, identify the molecular mechanisms of regulation that lead to the formation of specific bending patterns of cilia and flagella. Both objectives will be approached with the analysis of the inner dynein arms, because in vivo these structures are necessary and sufficient to generate both ciliary and flagellar types of movement in the absence of outer dynein arms. The organism used is the unicellular green alga Chlamydomonas reinhardtii. This organism allows for the analysis of the axoneme by a combination of approaches including genetics, electron microscopy and biochemical procedures. The comparison of Chlamydomonas wild-type axonemes with mutant axonemes lacking the outer dynein arms or part of the inner arms forms the basis of many experimental procedures outlined in this proposal. The comparison of inner dynein arms prepared from motile flagella with those from nonmotile flagella and analysis of defective inner arms in mutant axonemes, will be adopted to explain the function of the posttranslational modification of inner arm heavy chains. On this basis the long-term objective will be approached with the following specific aims: 1. complete the characterization of the structure and molecular composition of all forms of inner dynein arms present in Chlamydomonas axonemes; 2. develop a detailed analysis of the structures and the modifications of inner arm heavy chains and determine whether the modification of heavy chains is part of mechanism controlling axonemal motility; 3. characterize the axonemal components whose function is correlated with the restoration of flagellar activity of radial spoke-defective mutants; 4. determine whether axonemal actin and caltractin directly are involved in those changes of inner dynein arm activity that occur when Chlamydomonas axonemes pass from the ciliary to the flagellar type of motion.

纤毛和鞭毛存在于多种真核细胞中。 他们的功能障碍与呼吸系统疾病或男性有关 不育 该提案有以下两个长期目标： 这些轴丝成分的分子和结构特征， 轴丝运动的产生所必需的，第二，确定 分子调控机制，导致形成特定的 纤毛和鞭毛的弯曲模式。 这两个目标都将得到实现 与内部动力蛋白臂的分析，因为在体内， 结构是必要的和足够的，以产生纤毛和 鞭毛类型的运动在没有外部动力蛋白武器。 使用的生物体是单细胞绿色衣藻莱茵衣藻。 这种生物体允许通过以下组合来分析轴丝： 包括遗传学、电子显微镜和生物化学的方法 程序. 衣原体野生型和突变型轴丝的比较 缺乏外动力蛋白臂或部分内臂的轴丝形成 本提案中概述的许多实验程序的基础。 的 从运动鞭毛制备的内动力蛋白臂与从运动鞭毛制备的内动力蛋白臂的比较 和突变体内臂缺陷的分析 轴素，将被用来解释翻译后的功能 内臂重链的修饰。 在此基础上，将从以下方面实现长期目标 具体目标： 1.完成结构和分子组成的表征 所有形式的内部动力蛋白武器目前在衣原体轴丝; 2.对结构和修改进行详细分析， 内臂重链，并确定是否修改重链 链是控制轴丝运动机制的一部分; 3.表征其功能与以下相关的轴丝组分： 辐射状辐条缺陷突变体鞭毛活性的恢复; 4.确定轴丝肌动蛋白和钙牵引蛋白是否直接参与 当衣原体感染时， 轴丝从纤毛运动到鞭毛运动。