The Micromechanics of Central Spindle Organization

描述（由申请人提供）：为了繁殖我们的基因组，我们的细胞需要在许多代中准确地分裂；细胞分裂过程中的错误与多种癌症有关。细胞分裂的基本结构是微管的自组织组装，在中期呈双极结构，进入后期呈中心纺锤体结构。这种结构在有丝分裂过程中受到许多力的影响，必须提供稳定性，同时保持灵活性和对高动力环境的适应性。微管、马达和非马达微管相关蛋白（MAPs）是其中的关键参与者，它们的许多生化特性已经被广泛研究。人们对机械力在调节主轴结构性能方面所起的作用知之甚少。本研究项目将利用单光束光学捕获与双色全内反射荧光（TIRF）显微镜相结合，以便在PRC1交联的微管结构（人类非运动MAP）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张力的响应。众所周知，PRC1二聚体选择性地结合反平行微管，并在后期主要定位于中央纺锤体中间区。在细胞分裂过程中，这种交叉桥如何对存在于体内的力量作出反应的问题仍然没有答案。此外，特定的蛋白质结构域和残基在促进组织稳定性/灵活性方面的作用尚不完全清楚。截断和突变结构的使用将有助于阐明蛋白质/微管单元的机制特性。众所周知，PRC1还能将蛋白，如激酶和激酶，募集到纺锤体中间区。其中一个马达，激酶-4，已被证明与PRC1一起作为一个最小的蛋白质模块来维持固定的中间区长度。Kinesin-4是一种正端定向马达，可抑制动态微管的生长。这个马达的招募和活动是如何被细胞分裂过程中产生的力所调节的，这个问题还没有答案。这个蛋白质模块将在体外重建，在那里，力将沿着微管施加，两种蛋白质在中间区域的反应将被测量，以确定力在调节马达活动和中间区域重叠长度方面所起的作用。除了拟议的研究之外，奖学金期间的一个重要组成部分将包括在洛克菲勒大学的培训计划，包括课程作业，频繁的生物和临床研究研讨会，以及在研究实验室中广泛的生物化学，细胞生物学和荧光成像技术指导。在这个项目的整个进展过程中，将获得许多生物和生物物理方法和技术方面的杰出训练，从而在研究金期间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. PUBLIC HEALTH RELEVANCE: In order to successfully pass along our genetic information, our cells must divide many times with impeccable precision; the failure to do so is a hallmark of diseases such as cancer. During cell division, there are many forces which arise to pull chromosomes into each new cell and push apart the skeletal network of the cell machinery. Directly measuring the response of the components that make up these structural networks to force will provide insights into how the building blocks of cell division function, maintain fidelity and stability over many generations of division, and ultimately may help guide the design of potential cancer therapeutics.