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微管结构的适当组织，例如后期之前的双极纺锤体和后期期间的中央纺锤体（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，其中我们结合联合收割机结构，生物物理，化学和细胞生物学方法。在目前的建议，这是建立在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）获得结构的见解，在细胞分裂过程中的反平行微管交联。X射线晶体学，电子显微镜，和TIRF（全内反射荧光）显微镜分析将被用来研究如何PRC 1，一个广泛保守的非运动微管相关蛋白（MAP）所需的中央纺锤体组装，选择性交联反平行微管。我们还将分析PRC 1如何结合驱动蛋白-4来控制反平行微管重叠长度。(2)检查微管组织和功能如何在分裂细胞中调节。结构指导的诱变，体外测定和高分辨率显微镜将与化学抑制剂和基于RNAi/add-back的扰动相结合，以研究如何在两个不同水平上调节中心纺锤体组装和功能：（a）纳米尺度特征，如PRC 1识别的反平行微管重叠，和（B）纳米尺度空间活动模式，例如依赖于Aurora激酶的空间磷酸化梯度，Aurora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剂。(3)分析细胞分裂所必需的微管结构的机械特性。我们将使用双力校准的微针为基础的系统来检查中期纺锤体的粘弹性。化学和生物化学扰动将用于将关键蛋白质的已知生物化学和生物物理特性与纺锤体的微观力学联系起来。总之，我们的研究结果应该促进我们对关键蛋白质和调控线索（生物化学或机械）的理解，这些蛋白质和调控线索有助于我们基因组稳定繁殖所需的微管结构的自组织组装。由于细胞分裂错误可能导致的整个染色体丢失与人类的发育缺陷和疾病有关。我们的研究应该提供深入了解分子机制，确保细胞分裂完成没有错误。我们的研究还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。 公共卫生相关性：细胞分裂的错误与人类的发育缺陷和疾病有关。我们采取跨学科的方法来研究精确细胞分裂所需的分子机制。我们的发现有可能影响基于靶向细胞分裂的治疗策略的发展。

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 In the current proposal, which builds upon recent publications and unpublished preliminary data, we focus on the following three Aims: (1) Gain structural insights into antiparallel microtubule crosslinking during cell division.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. PUBLIC HEALTH RELEVANCE: Errors in cell division have been linked to developmental defects and disease in humans. We take an interdisciplinary approach to examine the molecular mechanisms needed for accurate cell division. Our findings have the potential to impact the development of therapeutic strategies based on targeting cell division.