Structure and Function of Cytoplasmic Dynein

描述（由申请人提供）：分子运动蛋白在多种细胞内运输过程中起作用，包括细胞器运输、染色体分离、轴突运输和信号通路。运动依赖过程对细胞和组织的生长、增殖和分化至关重要。运动功能在发育过程中是如何调节的，以及运动功能障碍与许多医学问题的关系，包括神经退行性疾病和癌症，是当前研究活动的重点。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异构体如何完成多重任务的重要和未解决的问题。我们的目标是解决可能调节动力蛋白靶向和/或活性的三种机制。(1)首先，细胞质动力蛋白包含多个亚基。单个亚基或亚基结构域可以指定动力蛋白附着的位置和部位。目的1包括生化和遗传实验，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会控制动力蛋白亚基的活性或结合亲和力。我们正在确定动力蛋白亚基磷酸化的体内位点，并将研究目标位点的意义。LIC亚基上的靶位点将发生突变，以模拟磷酸化或未磷酸化状态，并且将分析表达突变亚基的转基因产生的表型。(3)在第三种机制中，特定的结合伙伴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。先前的研究已经提供证据表明，谱蛋白介导动力蛋白和动力蛋白在膜上的附着。我们的合作者Laura Ranum （UMN）最近发现脊髓小脑性共济失调5型（SCA5）是一种常染色体显性神经退行性疾病，是由2-III谱蛋白（SPTBN2）突变引起的。在果蝇中，我们已经证明突变型，而不是野生型，在神经元中表达的人类ss-III谱蛋白会导致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位点。第二个重点是确定人类幽灵蛋白的突变，以及果蝇幽灵蛋白的相应突变，是否会破坏果蝇的轴突囊泡运输。这些研究将有助于阐明SCA5病理和神经退行性疾病的分子基础。

DESCRIPTION (provided by applicant): 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 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.