Mechanistic studies of a novel G-alpha nucleotide cycle

描述（由申请方提供）：在有丝分裂纺锤体中通过微管（MT）动力学进行的正确染色体分离是所有生物体细胞分裂的重要组成部分;该过程的异常可导致严重的发育异常。最近不同的遗传和生物化学证据表明，在细胞分裂过程中，适当的有丝分裂纺锤体功能所需的G蛋白α亚基的核苷酸循环的存在。该循环利用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GTP酶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的分子决定因素和时空动态，这种新的G-α调节周期及其对MT动态的影响。目的1是通过一系列的蛋白质生化研究来描述在细胞分裂中影响G-α亚基活性的新型G-α调节因子之间的功能相互作用。目的2详细介绍了开发和使用新型肽生物传感器的G-α核苷酸状态，以确定活性物种，调节MT动态和空间和时间动态的这一过程中活细胞。将采用一种新的G-α的GoLoco不敏感性突变来解决G-α/GoLoco相互作用对这些过程的必要性。目的3是映射RGS 14和G-α-i1蛋白的功能决定因素，直接调节MT动力学使用体外生物化学和细胞活力，MT网络和细胞周期转运的研究，从而测试我们最初的假设，即G-α和G-α调节器直接对微管蛋白和MT的协调作用可能代表有丝分裂过程中有丝分裂纺锤体功能的力发生器。这项研究计划将导致对G蛋白作用的多样性及其对微管动力学的影响的新认识，并进一步确定有丝分裂时微管力产生的精确分子机制的最终目标。这将有助于抗癌治疗的新药发现，因为目前许多抗增殖剂靶向微管动力学。这些研究也将提供洞察不对称细胞分裂的调节，因此，细胞极性和细胞命运的决定，具有特定的相关性神经规范，发育缺陷，和潜在的未来使用神经祖细胞干细胞治疗神经退行性疾病的机制。

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.