Motor Protein Dynamics and Mitotic Mechanisms

描述（由申请人提供）：我们的基因组通过细胞分裂的稳定繁殖取决于复杂和动态的微管结构的适当组织，如后期前的双极纺锤体和后期的中央纺锤体（或中间区）。我们的长期目标是确定这些细胞骨架结构的组装和功能所需的分子机制。为了实现这一目标，我们采取跨学科的方法，其中我们结合了结构，生物物理，化学和细胞生物学方法。在当前的提案中，基于最近的出版物和未发表的初步数据，我们专注于以下三个目标：(1)获得细胞分裂过程中反平行微管交联的结构见解。x射线晶体学、电子显微镜和TIRF（全内反射荧光）显微镜分析将用于检查PRC1（一种广泛保守的非运动微管相关蛋白（MAP），用于中央纺锤体组装）如何选择性地交联反平行微管。我们还将分析PRC1如何结合激酶-4来控制反平行微管重叠长度。(2)研究细胞分裂过程中微管的组织和功能是如何调控的。结构导向诱变、体外实验和高分辨率显微镜将结合化学抑制剂和基于RNAi/add-back的扰动来研究中央纺锤体组装和功能如何在两个不同的水平上受到调节：(a)纳米尺度的特征，如PRC1识别的反平行微管重叠；(b)微米尺度的空间活动模式，如依赖于极光激酶的空间磷酸化梯度，极光激酶是成功细胞分裂所需的几个关键过程的广泛保守的调节因子。(3)分析细胞分裂所必需的微管结构的力学特性。我们将使用双力校准微针为基础的系统来检查中期主轴的粘弹性特性。化学和生化扰动将被用于将关键蛋白质的已知生化和生物物理特性与纺锤体的微观力学联系起来。总之，我们的发现应该促进我们对关键蛋白质和调控线索（生化或机械）的理解，这些线索有助于微管结构的自组织组装，这是我们基因组稳定繁殖所必需的。由于细胞分裂错误可能导致整个染色体丢失，这与人类的发育缺陷和疾病有关。我们的研究应该提供洞察分子机制，确保细胞分裂完成无误。我们的研究也应该有助于寻找治疗药物的新靶点，并影响靶向细胞分裂所需蛋白质的药物的开发。

DESCRIPTION (provided by applicant): The stable propagation of our genomes through cell division depends on the proper organization of complex and dynamic microtubule-based structures, such as the bipolar spindle prior to anaphase and the central spindle (or midzone) during anaphase. Our long-term goal is to determine the molecular mechanisms required for the assembly and function of these cytoskeletal structures. To achieve this goal we take an interdisciplinary approach in which we combine structural, biophysical, chemical and cell biological methods. In the current proposal, which builds upon recent publications and unpublished preliminary data, we focus on the following three Aims: (1) Gain structural insights into antiparallel microtubule crosslinking during cell division. X-ray crystallography, electron microscopy, and TIRF (total internal reflection fluorescence) microscopy assays will be used to examine how PRC1, a widely conserved non-motor microtubule associated protein (MAP) required for central spindle assembly, selectively crosslinks antiparallel microtubules. We will also analyze how PRC1 binds kinesin-4 to control antiparallel microtubule overlap length. (2) Examine how microtubule organization and function are regulated in dividing cells. Structure-guided mutagenesis, in vitro assays, and high-resolution microscopy will be combined with chemical inhibitor and RNAi/add-back based perturbations to examine how central spindle assembly and function are regulated at two different levels: (a) Nanometer-scale features, such as antiparallel microtubule overlap, recognized by PRC1, and (b) Micron-scale spatial activity patterns, such as a spatial phosphorylation gradient that depends on Aurora kinase, a widely conserved regulator of several key processes required for successful cell division. (3) Analyze the mechanical properties of microtubule-based structures essential for cell division. We will use a dual force-calibrated microneedle-based system to examine the viscoelastic properties of the metaphase spindle. Chemical and biochemical perturbations will be used to link the known biochemical and biophysical properties of key proteins to the spindle's micro-mechanics. Together, our findings should advance our understanding of key proteins and regulatory cues (biochemical or mechanical) that contribute to the self-organized assembly of microtubule structures required for the stable propagation of our genomes. Whole chromosome loss, which can arise due to errors in cell division, has been linked to developmental defects and diseases in humans. Our research should provide insight into the molecular mechanisms that ensure cell division is completed without error. Our studies should also contribute to finding new targets for therapeutic agents and impact the development of drugs that target proteins needed for cell division.