Regulation of Microtubule Function by Tubulin Carboxy-terminal Tails

 描述（由申请人提供）：本项目的目标是了解微管蛋白水平上的分子多样性如何调控微管网络。微管蛋白组装成动态微管聚合物，与相关的马达蛋白形成网络，以在不同的环境中组织细胞。微管功能的缺陷与许多人类疾病有关，包括神经病和癌症。因此，了解调控微管网络的机制对于理解缺陷如何导致疾病状态至关重要。我们理解的一个主要缺陷是微管蛋白水平的分子多样性-- 突变，同种型表达，翻译后修饰--导致网络水平的变化。 我的实验室现在准备通过克服微管领域的两个历史障碍来解决这一问题。首先，我们已经开发了一个遗传模型操纵微管蛋白的定向突变。其次，我们将我们的遗传模型扩展到互补系统，以分析CTTs如何影响微管蛋白生物化学，MT运动活性和MT功能。通过应用这些协同方法，我们可以区分对内在微管动力学的影响与对电机和其他外在调节器的影响，并且我们可以确定这些影响对细胞网络的后果。 在这个项目中，我们将确定羧基末端尾基序（CTTs）的作用， 微管蛋白CTT支持与MT表面的马达和其他蛋白质的静电相互作用，在组织特异性微管蛋白同种型中表现出序列多样性，并且是主要位点 翻译后修饰我们假设CTTs通过促进微管动力学和进化上不同类别的MT马达的活性来调节网络行为。我们将从三个方面来检验这一假设：1）确定CTTs如何直接影响微管蛋白组装成微管聚合物。2)确定CTTs如何调节进化上保守的驱动蛋白马达。3)定义CTTs在复杂细胞微管网络-有丝分裂纺锤体组装中的分子作用。这项工作的结果将为微管网络的分子调控提供新的见解。此外，这些研究将为今后研究微管蛋白翻译后修饰、同种型表达和疾病相关等位基因的作用奠定基础。

 DESCRIPTION (provided by applicant): The goal of this project is to understand how microtubule networks are regulated by molecular diversity at the level of tubulin proteins. Tubulin proteins assemble into dynamic microtubule polymers that form networks with associated motor proteins to organize the cell in diverse contexts. Defects in microtubule function are linked to numerous human diseases, including neuropathies and cancer. Understanding the mechanisms that regulate microtubule networks is therefore critical for understanding how defects contribute to the disease state. A major deficit in our understanding is how molecular diversity at the level of tubulin proteins -- mutations, isotype expression, posttranslational modifications -- lead to changes at the level of the network. My laboratory is now poised to address this deficit by overcoming two historical barriers in the microtubule field. First, we have developed a genetic model for manipulating tubulin proteins by directed mutation. Second, we extend our genetic model to complementary systems for analyzing how CTTs affect tubulin biochemistry, MT motor activity, and MT function in vivo. By applying these synergistic approaches, we can distinguish effects on intrinsic microtubule dynamics from effects on motors and other extrinsic regulators, and we can determine the consequences of these effects for a cellular network. In this project, we will determine the roles of carboxy-terminal tail motifs (CTTs) of - and  tubulin. CTTs support electrostatic interactions with motors and other proteins at the MT surface, exhibit sequence diversity across tissue-specific tubulin isotypes, and are major sites of post-translational modification. We hypothesize that CTTs regulate network behavior by promoting microtubule dynamics and the activity of evolutionarily distinct classes of MT motors. We will test this hypothesis in three aims: 1) Determine how CTTs directly affect the assembly of tubulin proteins into microtubule polymers. 2) Determine how CTTs regulate evolutionarily conserved kinesin motors. 3) Define the molecular roles of CTTs in the assembly of a complex cellular microtubule network - the mitotic spindle. The results of this work will provide new insight into the molecular regulation of microtubule networks. Furthermore, these studies will set the stage for future investigations of the roles of tubulin posttranslational modifications, isotyp expression, and disease-associated alleles.