The Micromechanics of Central Spindle Organization

中心主轴机构的微观力学

• 批准号: 8203060
• 负责人: Scott Thomas Forth
• 金额: $ 5.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8203060
• 关键词: Adopted; Affect; Anaphase; Behavior; Binding; Biochemical; Biological; Biological Assay; Bundling; Cell Division Process; Cell division; Cells; Cellular biology; Chromosomes; Clinical Research; Color; Cytokinesis; Disease; Environment; Eukaryota; Exhibits; Failure; Fellowship; Fluorescence; Fluorescence Microscopy; Generations; Genetic; Genome; Growth; Human; Imaging Techniques; In Vitro; Instruction; Kinesin; Label; Learning; Length; Link; Literature; Maintenance; Malignant Neoplasms; Measurement; Measures; Mechanics; Mediating; Metaphase; Methodology; Methods; Microscope; Microtubule Polymerization; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Monitor; Motor; Motor Activity; Mutate; Nature; Phosphotransferases; Play; Plus End of the Microtubule; Polystyrenes; Process; Property; Protein Dynamics; Protein Family; Proteins; Publishing; Recruitment Activity; Regulation; Research; Research Project Grants; Role; Slide; Stress; Structure; Surface; Techniques; Tertiary Protein Structure; Therapeutic; Time; Training; Training Programs; Universities; Walking; Work; base; biological research; crosslink; design; dimer; flexibility; fluorescence imaging; in vivo; insight; instrument; link protein; member; optical traps; preference; reconstitution; response; skeletal; skills

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.

描述（由申请人提供）：为了传播我们的基因组，我们的细胞需要在许多世代中准确分裂；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，该组合在进入后期后采用双极构型和中央纺锤体。这种结构在整个有丝分裂过程中都受到了许多力，并且必须提供稳定性，同时保持灵活性和符合高度动机的环境。涉及的主要参与者是微管，电动机和非运动微管相关蛋白（或地图），并且已经对其许多生化特性进行了广泛的研究。关于机械力在调节纺锤体结构特性中的作用的知之甚少。 该研究项目将与两色TIRF（总内反射荧光）显微镜结合使用一个单束光学陷阱，以便在微管结构上施加已知大小和方向的力，该结构由PRC1（人类非运动图）交联的微管结构，并同时可视化这种结构的响应。众所周知，PRC1二聚体选择性地结合抗平行的微管，主要在后期的中心纺锤体中区域定位。这个跨桥如何应对整个细胞分裂的体内力量的响应的问题仍未得到解答。此外，尚不清楚特定蛋白质结构域和残基在有助于组织稳定性/灵活性中的作用。使用截短和突变的构建体的使用将有助于阐明蛋白质/微管单元的机械性能。 众所周知，PRC1可以募集蛋白质，例如驱动蛋白和激酶，从而将蛋白质募集到纺锤体中区。一种这样的电动机，即动力蛋白-4，已显示与PRC1一起用作最小蛋白质模块，以保持固定的中区长度。驱动蛋白-4是一个加号的定向电动机，可抑制动态微管的生长。该电动机的募集和活动是如何由细胞分裂过程中产生的力调节的问题。该蛋白质模块将在体外重构，在该蛋白质模块将沿微管施加力，并且将测量两种蛋白质在中区域的反应，以确定力在调节电动机活性和MIDZONE重叠的长度方面的作用。 除了拟议的研究外，研究金时期的重要组成部分还需要在洛克菲勒大学进行培训计划，包括课程，经常进行生物学和临床研究的研讨会以及研究实验室中的生化，细胞生物学和荧光成像技术的广泛指导。在该项目的整个过程中，将获得许多生物和生物物理方法和技术的出色培训，从而导致通过团契期结束，从而获得了广泛的高度跨学科技能。 公共卫生相关性：为了成功地传递我们的遗传信息，我们的细胞必须以无可挑剔的精度分裂；未能这样做是癌症等疾病的标志。在细胞分裂期间，有许多力将染色体拉入每个新细胞并将细胞机械骨骼网络推开。直接测量组成这些结构网络的组件的响应将提供有关细胞分裂功能的构建模块，维持许多世代分裂的忠诚度和稳定性的见解，并最终可能有助于指导潜在的癌症治疗剂的设计。