The Micromechanics of Central Spindle Organization

描述（由申请人提供）：为了繁殖我们的基因组，我们的细胞需要准确地分裂许多代;细胞分裂过程中的错误与各种癌症有关。细胞分裂的基本结构是微管的自组织聚集，中期微管呈双极结构，进入后期微管呈纺锤体结构。这种结构在整个有丝分裂过程中受到许多力的作用，必须提供稳定性，同时保持灵活性和对高动力环境的顺应性。涉及的关键参与者是微管，马达和非马达微管相关蛋白（或MAP），它们的许多生化特性已被广泛研究。关于机械力在调节纺锤体结构特性中所起的作用，人们所知甚少。 该研究项目将利用单光束光阱结合双色TIRF（全内反射荧光）显微镜，以便在由PRC 1（人类非运动MAP）交联的微管结构上施加已知大小和方向的力，同时可视化该结构对机械施加的张力的响应。据了解，PRC 1二聚体选择性地结合反平行微管和本地化主要在中央纺锤体中间区在后期。这个跨桥如何响应整个细胞分裂过程中体内存在的力的问题仍然没有答案。此外，特定蛋白质结构域和残基在促进组织稳定性/灵活性中的作用尚不完全清楚。使用截短和突变的构建体将有助于阐明蛋白质/微管单元的机械性质。 PRC 1还已知将蛋白质如驱动蛋白和激酶募集到纺锤体中间区。一种这样的马达，驱动蛋白-4，已被证明与PRC 1一起作为最小的蛋白质模块来维持固定的中间区长度。Kinesin-4是一种正末端定向马达，抑制动态微管的生长。这个马达的募集和活动如何被细胞分裂过程中产生的力调节的问题还没有答案。该蛋白质模块将在体外重构，其中力将沿着微管施加沿着，并且将测量两种蛋白质在中间区的响应，以确定力在调节马达的活性和中间区重叠的长度中所起的作用。 除了拟议的研究，奖学金期间的一个重要组成部分将需要在洛克菲勒大学的培训计划，包括课程作业，在生物和临床研究频繁的研讨会，并在生物化学，细胞生物学和荧光成像技术的研究实验室广泛的指令。在本项目的整个过程中，将获得许多生物和生物物理方法和技术方面的出色培训，从而在研究金期结束时获得广泛的高度跨学科的技能。

DESCRIPTION (provided by applicant): In order to propagate our genome, our cells need to divide accurately over many generations; errors in the cell division process are linked to a wide variety of cancers. The fundamental structure of cell division is the self-organized assemblage of microtubules which adopts a bipolar configuration in metaphase and a central spindle upon entry into anaphase. This structure is subjected to numerous forces throughout mitosis, and must provide stability while remaining flexible and compliant to the highly motive environment. The key players involved are microtubules, motors, and non-motor microtubule-associated proteins (or MAPs), and many of their biochemical properties have been studied extensively. Much less is known about the role mechanical force plays in regulating the spindle's structural properties. This research project will utilize a single-beam optical trap in conjunction with two-color TIRF (total internal reflection fluorescence) microscopy in order to exert a force of known magnitude and direction on a microtubule structure that is cross-linked by PRC1 (a human non-motor MAP) and simultaneously visualize the response of this structure to the mechanically applied tension. It is known that PRC1 dimers selectively bind anti-parallel microtubules and localize predominantly at the central spindle midzone in anaphase. The question of how this cross-bridge responds to the forces present in vivo throughout cell division is still unanswered. Additionally, the role of specific protein domains and residues in contributing to organizational stability/flexibility is not fully known. The use of truncated and mutated constructs will help elucidate the mechanistic properties of the protein/microtubule unit. PRC1 is also known to recruit proteins, such as kinesins and kinases, to the spindle midzone. One such motor, kinesin-4, has been shown to work together with PRC1 as a minimal protein module to maintain a fixed midzone length. Kinesin-4 is a plus-end directed motor, which inhibits the growth of dynamic microtubules. The question of how this motor's recruitment and activity is modulated by the forces generated during cell division is unanswered. This protein module will be reconstituted in vitro, where force will be applied along the microtubules and the response of both proteins at the midzone will be measured in order to determine the role that force plays in regulating both the motor's activity and the length of the midzone overlap. In addition to the proposed research, a significant component of the fellowship period will entail a training program at Rockefeller University consisting of coursework, frequent seminars in biological and clinical research, and extensive instruction in biochemical, cell biology, and fluorescence imaging techniques in the research lab. Throughout the progression of this project, outstanding training in many biological and biophysical methodologies and techniques will be acquired, resulting in the attainment of a broad range of highly interdisciplinary skills by the conclusion of the fellowship period.