THE STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 分子马达蛋白在许多细胞内运输过程中起作用，包括细胞器运输、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。运动功能如何在发育背景下调节，以及运动功能障碍与许多医学问题（包括神经退行性疾病和癌症）的关系是当前研究活动的焦点。我们的工作集中在微管运动细胞质动力蛋白，以及关于这个单一的运动亚型如何完成多个任务的重要和未回答的问题。我们的目标将解决三个机制，可能调节动力蛋白的目标和/或活动。(1)首先，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和什么上。目的1包括生物化学和遗传学实验，以解决动力蛋白轻链和轻中间链如何影响动力蛋白的功能。(2)其次，动力蛋白亚基的翻译后修饰可能控制动力蛋白亚基的活性或结合亲和力。我们正在与John Yates博士合作确定动力蛋白亚基上磷酸化的体内位点，并将研究靶位点的意义。LIC亚基上的靶位点将被突变以模拟磷酸化或非磷酸化状态，并且将分析由表达突变亚基的转基因产生的表型。(3)在第三种机制中，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。以前的研究提供了证据表明，血影蛋白介导的附着dynactin和dynein的膜。我们的合作者Laura Ranum（UMN）最近发现脊髓小脑共济失调5型（SCA 5）是一种常染色体显性遗传性神经退行性疾病，由2-III血影蛋白（SPTBN 2）突变引起。在果蝇中，我们已经表明，突变体，而不是野生型，人的ss-III血影蛋白在神经元中表达的原因神经变性和粗糙的眼睛表型。一个目标是进行全基因组筛选，以恢复在致病过程中识别新基因的修饰基因座。第二个优先事项是确定是否在人类血影蛋白的突变，并在苍蝇的SS血影蛋白的相应突变，扰乱果蝇轴突囊泡运输。这些研究将有助于阐明SCA 5病理和神经退行性疾病的分子基础。公共卫生相关性：我们正在研究细胞内转运的分子基础。我们的工作集中在微管马达细胞质动力蛋白和机制，通过该马达可以完成不同的运输任务。细胞内转运的扰动涉及人类疾病，包括神经退行性疾病、癌症和出生缺陷。

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 including organelle transport, chromosome segregation, axonal transport, and signaling pathways. Motor dependent processes are critical for growth, proliferation, and the 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 and cancer, is a current focus of research activity. Our work is focused on the microtubule motor cytoplasmic dynein, and the important and unanswered questions regarding how this single motor isoform accomplishes multiple tasks. Our aims will address three mechanisms that potentially regulate dynein targeting and/or activity. (1) First, cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what, dynein is attached. Aim 1 includes biochemical and genetic experiments to address how the dynein light chain and light intermediate chain influence dynein functions. (2) Second, the posttranslational modification of dynein subunits might control dynein subunit activities or binding affinities. We are defining the in vivo sites of phosphorylation on dynein subunits in collaboration with Dr. John Yates and will study the significance of the target sites. The target sites on the LIC subunit will be mutated to mimic the phosphorylated or unphosphorylated state, and the phenotypes produced by transgenes that express the mutant subunits will be analyzed. (3) In a third mechanism, specific binding partners or effector proteins might mediate the targeting of the dynein motor to specific cargoes or locations. Previous studies have provided evidence that spectrin mediates the attachment of dynactin and dynein to membranes. Our collaborator, Laura Ranum (UMN), recently discovered that Spinocerebellar ataxia type 5 (SCA5), an autosomal dominant neurodegenerative disease, is caused by mutations in 2-III spectrin (SPTBN2). In Drosophila, we have shown that mutant, but not wild type, human ss-III spectrin expressed in neurons causes neurodegeneration and a rough eye phenotype. One goal is to conduct a genome-wide screen to recover modifier loci that identify novel genes in the pathogenic process. A second priority is to determine if mutations in human spectrins, and the corresponding mutations in fly ss spectrin, disrupt axonal vesicular transport in Drosophila. These studies will help to elucidate the molecular underpinnings of SCA5 pathology and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: We are studying the molecular basis of intracellular transport. Our work focuses on the microtubule motor cytoplasmic dynein and the mechanisms by which this motor can accomplish diverse transport tasks. Perturbations in intracellular transport are implicated in human diseases, including neurodegenerative disease, cancer, and birth defects.