Motor Protein Dynamics and Mitotic Mechanisms

描述(申请人提供)：我们基因组通过细胞分裂的稳定繁殖依赖于复杂和动态的基于微管的结构的适当组织，例如后期之前的两极纺锤体和后期的中心纺锤体(或中间区)。我们的长期目标是确定这些细胞骨架结构组装和功能所需的分子机制。为了实现这一目标，我们采取了跨学科的方法，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，建立在最近的出版物和未发表的初步数据的基础上，我们专注于以下三个目标：(1)获得对细胞分裂过程中反平行微管交联的结构见解。X射线结晶学、电子显微镜和TIRF(全内反射荧光)显微镜分析将用于研究PRC1是如何选择性地交联反平行微管的。PRC1是一种广泛保守的非运动微管相关蛋白(MAP)，用于中心轴组装。我们还将分析PRC1如何与kinesin-4结合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节的。结构导向突变、体外试验和高分辨率显微镜将结合化学抑制剂和RNAi/Add-back扰动来研究中心纺锤体的组装和功能如何在两个不同的水平上受到调控：(A)纳米级特征，例如反平行微管重叠，由PRC1识别；(B)微米级空间活动模式，例如依赖于Aurora激酶的空间磷酸化梯度，后者是成功细胞分裂所需几个关键过程中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性能。我们将使用双力校准的基于微针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来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机制联系起来。总之，我们的发现应该会促进我们对关键蛋白质和调控信号(生化或机械)的理解，这些信号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。由于细胞分裂错误而导致的整个染色体丢失与人类的发育缺陷和疾病有关。我们的研究应该为确保细胞分裂无误完成的分子机制提供洞察力。我们的研究还应该有助于为治疗剂找到新的靶点，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准确的细胞分裂所需的分子机制。我们的发现有可能影响基于细胞分裂靶向的治疗策略的发展。

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 In the current proposal, which builds upon recent publications and unpublished preliminary data, we focus on the following three Aims: (1) Gain structural insights into antiparallel microtubule crosslinking during cell division.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.