Molecular Mechanics and Regulation of Centrosomes

描述(申请人提供)：中心体是细胞中重要的机械中枢，它维持与微管的承重连接，并协调微管阵列以支持细胞运动、核定位、有丝分裂纺锤体组装和染色体分离。影响中心体维持或调节微管附着能力的突变与基因组不稳定性的增加和癌症的进展有关。尽管中心体-微管相互作用在体内发挥着重要的作用和相当大的作用力，但在任何生物体中，从未有人对中心体与微管的连接进行过机械研究。因此，这些相互作用可能在多大程度上受到机械调控也从未被研究过。在与阿斯伯里、戴维斯和阿加德实验室的一个合作项目中，这项工作旨在揭示中心体维持、感知和响应微管传递的力的分子相互作用。中心体-微管的相互作用将在体外重建，并结合使用单分子激光捕获、全内反射荧光(TIRF)显微镜和分子细胞生物学技术进行单独询问。通过将Asbury实验室的生物物理专业知识与Davis和Agard实验室的生化和结构专业知识相结合，这项提案将比以往任何时候都更准确和直接地确定特定分子成分在维持和调节微管与中心体连接方面的作用。揭示中心体是如何维持、感知和响应机械力的，将有助于揭示这些途径如何在癌症等人类疾病中失败，并最终可能导致识别新的化疗靶点。

DESCRIPTION (provided by applicant): Centrosomes are vital mechanical hubs in cells that sustain load-bearing attachments to microtubules and orchestrate arrays of microtubules to support cell motility, nuclear positioning, mitotic spindle assembly, and chromosome segregation. Mutations affecting the ability of centrosomes to sustain or regulate attachments of microtubules are correlated with an increase in genome instability and progression of cancers. Despite this important role and the considerable forces that centrosome-microtubule interactions are thought to sustain in vivo, microtubule attachments to centrosomes have never been mechanically investigated in any organism. Consequently, the extent to which these interactions may be mechanically regulated has also never been investigated. In a collaborative project with the Asbury, Davis, and Agard labs, this work aims to reveal the molecular interactions by which centrosomes sustain, sense, and respond to forces transmitted by microtubules. Centrosome-microtubule interactions will be reconstituted in vitro and individually interrogated using a combination of single molecule laser trapping, total internal reflection fluorescence (TIRF) microscopy, and molecular cell biology techniques. Through the combination of biophysical expertise in the Asbury lab with the biochemical and structural expertise of the Davis and Agard labs, this proposal will determine the role of specific molecular components in sustaining and regulating attachments of microtubules to centrosomes more precisely and directly than has ever been done before. Revealing how centrosomes sustain, sense, and respond to mechanical forces will shed light on how these pathways fail in human diseases such as cancer and may ultimately lead to the identification of new chemotherapeutic targets.