Structure and Function of Cytoplasmic Dynein

描述（由申请人提供）：分子马达蛋白在多种细胞内转运过程中发挥作用，包括细胞器的组织和转运、染色体分离、轴突转运和信号传导。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。在发育过程中如何调节运动功能，以及运动功能障碍与包括神经退行性疾病、先天性染色体综合征和出生缺陷在内的许多医学问题的关系是当前研究活动的焦点。我们的工作主要集中在微管运动细胞质动力蛋白，以及这个单一的运动亚型如何完成多个任务。动力蛋白如何靶向特定的货物和/或细胞位置？我们的目标将解决三个非排他性的机制，可能有助于动力蛋白的目标。(1)细胞质动力蛋白含有多个亚基。单个亚基或亚基结构域可以指定动力蛋白附着在哪里以及附着在什么货物上。为了检验这一假设，我们将询问轻链中间体和中间链多肽内的结构域是否赋予特定的功能。将使用诱变和分子遗传学方法来破坏结构域功能，并对突变体表型进行表征。(2)动力蛋白亚基的翻译后修饰可能控制亚基是否有能力以高亲和力结合货物。与John Yates博士（Scripps研究所）的合作将使用质谱法确定动力蛋白复合物内亚基上磷酸化的位点。随后，鉴定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。将分析由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)特异性结合伴侣或“效应”蛋白可能介导动力蛋白马达靶向特定的货物或位置。我们将继续对前一阶段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。

DESCRIPTION (provided by applicant): Molecular motor proteins function in a multitude of intracellular transport processes that include the organization and transport of organelles, chromosome segregation, axonal transport, and signaling. Motor dependent processes are critical for the growth, proliferation, and differentiation of cells and tissues. How motor function is regulated during development, 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 how this single motor isoform accomplishes multiple tasks. How is dynein targeted to specific cargoes and/or cellular locations? Our aims will address three non-exclusive mechanisms that potentially contribute to dynein targeting. (1) Cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what cargo, 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) The posttranslational modification of dynein subunits might control whether subunits are competent to bind a cargo with high affinity. A 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) Specific binding partners or "effector" proteins might mediate the targeting of the dynein 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.