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网络如何在微管蛋白水平上受到分子多样性的调节。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，在不同的环境中组织细胞。微管功能缺陷与许多人类疾病有关，包括神经疾病和癌症。因此，了解调节微管网络的机制对于了解缺陷如何导致疾病状态至关重要。我们理解中的一个主要缺陷是微管蛋白水平上的分子多样性-- 突变、同型表达、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不足。首先，我们开发了一个通过定向突变来操纵微管蛋白的遗传模型。其次，我们将我们的遗传模型扩展到互补系统，以分析CTTS如何影响体内的微管蛋白生化、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发动机和其他外部调节因子的影响，并可以确定这些影响对细胞网络的影响。 在这个项目中，我们将确定-和的羧基末端尾部基序(CTT)的作用 微管蛋白。CTTS支持与MT表面的马达和其他蛋白质的静电相互作用，在组织特定的微管蛋白同种类型中显示出序列多样性，并且是 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马达的活动来调节网络行为。我们将从三个方面验证这一假说：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如何调节进化保守的运动蛋白。3)确定CTTS在复杂的细胞微管网络--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，这些研究将为进一步研究微管蛋白翻译后修饰、等位基因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.