Mechanistic studies of a novel G-alpha nucleotide cycle

描述(由申请人提供)：在有丝分裂纺锤体上通过微管(MT)动力学进行适当的染色体分离是所有生物细胞分裂的重要组成部分；这一过程的异常可能导致严重的发育异常。最近不同的遗传和生化证据表明，在细胞分裂过程中，G蛋白α亚基存在一个核苷酸循环，这是正常的有丝分裂纺锤体功能所必需的。这个循环使用了在后生动物中保守的G-α调节蛋白，包括GoLoco基序核苷酸解离抑制剂、含有RGS结构域的GTPase加速蛋白和鸟嘌呤核苷酸交换因子RIC-8。我们的长期目标是确定这种新的G-α调节周期背后的分子决定因素和时空动力学及其对MT动力学的影响。目的1是通过一系列的蛋白质生化研究，描述在细胞分裂过程中影响G-α亚单位活性的新的G-α调节因子之间的功能相互作用。目的2详细介绍新型G-α核苷酸多肽生物传感器的开发和应用，以确定在活细胞中调节MT动态的活性物种以及这一过程的空间和时间动力学。一种新的对G-α不敏感的GoLoco突变将被用来解决G-α/GoLoco相互作用对这些过程的必要性。目的3是利用体外生化和细胞活力、MT网络和细胞周期传递研究，定位直接调控MT动力学的RGS14和G-α-I1蛋白的功能决定因素，从而检验我们最初的假设，即G-α和G-α调节剂直接作用于微管蛋白和MTS可能是有丝分裂过程中纺锤体功能的力生成器。这一研究计划将使人们对G蛋白作用的多样性及其对微管动力学的影响有一个新的理解，并进一步确定有丝分裂时微管力产生的精确分子机制。这将促进抗癌治疗的新药发现，因为目前许多抗增殖剂针对的是微管动力学。这些研究还将深入了解不对称细胞分裂的调节，从而深入了解细胞极性和细胞命运决定的机制，这些机制与神经规范、发育缺陷以及神经前体干细胞作为神经退行性疾病治疗的潜在未来用途具有特定的相关性。

DESCRIPTION (provided by applicant): Proper chromosomal segregation by microtubule (MT) dynamics at the mitotic spindle is an essential component of cell division in all organisms; aberration of this process can lead to severe developmental abnormalities. Recent disparate genetic and biochemical evidence suggests the existence of a nucleotide cycle for G-protein alpha subunits required for proper mitotic spindle function during cell division. This cycle employs G-alpha regulatory proteins conserved across metazoa, including GoLoco motif nucleotide dissociation inhibitors, RGS-domain-containing GTPase-accelerating proteins, and the guanine nucleotide exchange factor RIC-8. Our long-term objective is to define the molecular determinants and spatiotemporal dynamics that underlie this novel G-alpha regulatory cycle and its effects on MT dynamics. Aim 1 is to delineate the functional interplay between the novel G-alpha regulatory factors that impinge on G-alpha subunit activity in cell division via a series of protein biochemical studies. Aim 2 details development and use of novel peptide biosensors for G-alpha nucleotide state to determine the active species that modulates MT dynamics and the spatial and temporal dynamics of this process in live cells. A novel GoLoco-insensitivity mutation to G-alpha will be employed to address the necessity of the G-alpha/GoLoco interaction to these processes. Aim 3 is to map the functional determinants of RGS14 and G-alpha-i1 proteins that directly modulate MT dynamics using in vitro biochemistry and cell viability, MT network, and cell cycle transit studies, thereby testing our initial hypothesis that the coordinated action of G-alpha and G-alpha regulators directly on tubulin and MTs might represent the force generator in mitotic spindle function during mitosis. This research program will lead to a new understanding of the diversity of G-protein action and its impact on microtubule dynamics, and further the ultimate goal of defining the precise molecular mechanisms of microtubule force generation at mitosis. This should facilitate new drug discovery for anticancer therapeutics, as many current anti-proliferative agents target microtubule dynamics. These studies will also afford insight into the regulation of asymmetric cell division and, consequently, mechanisms of cell polarity and cell-fate determination that have specific relevance to neural specification, developmental defects, and the potential future uses of neuroprogenitor stem cells as treatment for neurodegenerative disorders.