THE STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 分子运动蛋白在多种细胞内运输过程中发挥作用，包括细胞器的组织及其运输、染色体分离、轴突运输和信号传导途径。 运动依赖性过程对于细胞和组织的生长、增殖和分化至关重要。如何在发育背景下调节运动功能，以及运动功能障碍与神经退行性疾病、先天性染色体综合征和出生缺陷等众多医学问题的关系是当前研究活动的焦点。 我们的工作重点是微管运动细胞质动力蛋白，以及关于这种单一运动亚型如何完成多项任务的重要且尚未解答的问题。 动力蛋白如何靶向特定的货物和/或细胞位置和结构？ 我们的目标将解决可能有助于动力蛋白靶向的三种非排他性机制。 (1)首先，细胞质动力蛋白含有多个亚基。 各个亚基或亚基域可以指定动力蛋白连接的位置和内容。 为了检验这一假设，我们将询问轻质中间体和中间链多肽内的结构域是否赋予特定功能。 将使用诱变和分子遗传学方法来破坏结构域功能，并对突变表型进行表征。 (2)其次，动力蛋白亚基的翻译后修饰可能控制亚基是否能够以高亲和力结合货物。 与 John Yates 博士（斯克里普斯研究所）的合作将使用质谱方法确定动力蛋白复合物内亚基的磷酸化位点。随后，所识别的磷酸化位点将被突变以模拟相应亚基的磷酸化或非磷酸化状态。 将分析表达突变亚基的转基因产生的表型，以揭示动力蛋白磷酸调节的功能意义。 (3) 在第三种机制中，特定的结合伙伴或“效应子”？蛋白质可能介导动力马达瞄准特定的货物或位置。 我们将对上一时期确定的候选相互作用蛋白进行功能分析，并将继续对其他相互作用位点进行二次测试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Molecular motor proteins function in a multitude of intracellular transport processes that include the organization of organelles and their transport, chromosome segregation, axonal transport, and signaling pathways. Motor dependent processes are critical for the growth, proliferation, and differentiation of cells and tissues. How motor function is regulated in a developmental context, and the relationship of motor dysfunction to numerous medical problems including neurodegenerative disease, congenital chromosomal syndromes, and birth defects is a current focus of research activity. Our work is focused on the microtubule motor cytoplasmic dynein, and the important and unanswered question regarding how this single motor isoform accomplishes multiple tasks. How is dynein targeted to specific cargoes and/or cellular locations and structures? Our aims will address three non-exclusive mechanisms that potentially contribute to dynein targeting. (1) First, cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what, dynein is attached. To test this hypothesis we will ask whether domains within the light intermediate and the intermediate chain polypeptides confer specific functions. Mutagenesis and molecular genetic approaches will be used to disrupt domain function and the mutant phenotypes will be characterized. (2) Second, the posttranslational modification of dynein subunits might control whether subunits are competent to bind a cargo with high affinity. Collaboration with Dr. John Yates (Scripps Research Institute) will define the sites of phosphorylation on subunits within the dynein complex using a mass spectrometry approach. Subsequently, the phosphorylation sites identified will be mutated to mimic the phosphorylated or unphosphorylated state of the respective subunit. The phenotypes produced by transgenes that express the mutant subunits will be analyzed to reveal the functional significance of dynein phosphoregulation. (3) In a third mechanism, specific binding partners or ?effector? proteins might mediate the targeting of the dynien motor to specific cargoes or locations. We will pursue the functional analysis of candidate interacting proteins identified in the previous period and will continue with secondary tests on other interacting loci.