The Micromechanics of Central Spindle Organization

描述(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世代中准确地分裂；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合，中期微管为两极结构，进入后期为中心纺锤体。这种结构在整个有丝分裂过程中都受到许多力的作用，必须提供稳定性，同时保持灵活性和对高度动态环境的顺应性。涉及的关键角色是微管、马达和非运动微管相关蛋白(或MAP)，它们的许多生化特性已经得到了广泛的研究。人们对机械力在调节主轴结构特性中所起的作用知之甚少。这项研究项目将利用单光束光学陷阱与双色TIRF(全内反射荧光)显微镜相结合，以对通过PRC1(人类非运动图谱)交联的微管结构施加已知大小和方向的力，并同时可视化该结构对机械施加的张力的反应。已知，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心纺锤体中间区。这种交叉桥梁如何应对体内细胞分裂过程中存在的力量，这个问题仍然没有答案。此外，特定蛋白质结构域和残基在促进组织稳定性/灵活性方面的作用尚不完全清楚。截短和突变结构的使用将有助于阐明蛋白质/微管单位的机械特性。已知的是，Prc1还向纺锤体中区招募蛋白质，如激动素和激动酶。一个这样的马达，kinesin-4，已经被证明与Prc1作为一个最小的蛋白质模块一起工作，以维持固定的中区长度。Kinesin-4是一种正端定向运动，可以抑制动态微管的生长。这种马达的招募和活动如何受到细胞分裂过程中产生的力量的调节，这个问题还没有答案。这个蛋白质模块将在体外重组，在那里将沿着微管施加力，并将测量两种蛋白质在中区的反应，以确定力在调节马达活动和中区重叠长度方面所起的作用。除了拟议的研究，研究金期间的一个重要组成部分将需要在洛克菲勒大学进行培训计划，包括课程作业、生物和临床研究的频繁研讨会，以及在研究实验室进行生化、细胞生物学和荧光成像技术的广泛教学。在该项目的整个过程中，将获得许多生物学和生物物理学方法和技术方面的杰出培训，从而在研究金期满时获得广泛的高度跨学科技能。

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.