CAREER: Dissecting the Role of Mechanical Forces in the Regulation of Cytoskeletal Dynamics during Mitosis

细胞蛋白和核酸在纳米尺度上的生物力学特性对于调节包括细胞分裂在内的关键细胞过程至关重要。特别是，姐妹染色体上的着丝粒在有丝分裂过程中被拉伸，这种拉伸产生张力，这对于确保染色体正确分离成两个子细胞是重要的。然而，对于有丝分裂着丝粒拉伸张力是如何建立和维持的，缺乏一个完整的机制理解。这项工作的总体目标是研究有丝分裂过程中有丝分裂微管动力学如何产生和响应张力，并开始剖析张力在哺乳动物细胞有丝分裂过程中建立着丝点和染色体定位中的作用。这项工作将最终导致更好地理解蛋白质和核酸如何作为纳米机械系统在细胞分裂过程中调解适当的染色体分离。要验证的总体假设是，微管是细胞骨架聚合物，在有丝分裂过程中负责排列和分离重复的染色体，可以通过产生和响应张力来自我调节其动力学。假设和总体目标将通过追求以下具体目标来解决。目的1：确定在出芽酵母有丝分裂过程中，体内张力是否调节着丝点微管动力学以控制染色体排列；目的2：确定纯化的体外微管产生的张力是否能够内在地自我调节微管动力学；目的3：区分哺乳动物细胞有丝分裂中重要的反极力。智力优势：提出的研究将开发工具，以更好地了解细胞分裂过程中内部产生的有丝分裂纺锤体力的起源和作用。具体来说，微管是有丝分裂纺锤体中的一个关键元素，因此负责产生和响应调节细胞分裂正常进程的力。由于不受控制的细胞分裂是癌症的一个标志，许多癌症治疗干预措施是通过破坏微管动力学来起作用的，对力和微管动力学之间关系的解剖有可能导致更合理的癌症治疗方法。该研究将利用物理原理和先进的显微镜来评估活细胞内部的力，然后利用这些方法来剖析这些力与有丝分裂过程中基于微管的染色体分离之间的关系。此外，细胞和分子尺度的生物物理重构和计算建模将用于直接测试假设，并为解释实验结果提供框架。所产生的结果有望用于更好地理解细胞分裂的基本生物物理学，并为潜在的癌症治疗提供定量数据。更广泛的影响：提出的工作对推进细胞有丝分裂过程中分子力作用的定量理解具有重要的更广泛的影响，这在细胞生物学和癌症研究中具有重大意义。将定量工程方法、计算机编程和物理原理整合到生物学基本问题的研究中，已被证明可以提高生命科学研究人员的生产力和洞察力。该提案的一个强大推力旨在将定量工程方法灌输到细胞生物学教育和研究中，无论是在本科还是研究生阶段，这将有助于培养多样化的，具有全球竞争力的STEM劳动力。特别是，教育目标与研究计划相结合，以(1)在研究生生涯的早期阶段向细胞生物学研究生介绍定量工程方法；(2)为生物学研究生和本科生建立课程和研究机会，将定量工程方法应用到他们当前的学习中，并随后应用到他们未来的职业生涯中；(3)资助本科生和少数族裔学生在细胞生物学或生物医学工程实验室进行定量研究项目。

1350741GardnerThe biomechanical properties of cellular proteins and nucleic acids at the nanoscale are critical for regulating key cellular processes, including cell division. In particular, the centromeres on sister chromosomes are stretched apart during mitosis, and this stretching generates a tension force, which is important for ensuring proper chromosome segregation into two daughter cells. However, an integrated mechanistic understanding for how mitotic centromere stretching tension is established and maintained is lacking. The overall objective of the proposed work is to study how mitoticmicrotubule dynamics generate and respond to tension forces during mitosis, and to begin to dissect the role of tension forces in establishing kinetochore and chromosome positioning during mammalian cell mitosis. This work will ultimately lead to a better understanding of how proteins and nucleic acids work as a nanomechanical system to mediate proper chromosome segregation during cell division. The overarching hypothesis to be tested is that microtubules, which are the cellular cytoskeletal polymers responsible for aligning and segregating duplicated chromosomes during mitosis, can self-regulate their dynamics by both generating and responding to tension forces. The hypothesis and overall objective will be addressed by pursuing the following specific aims.Aim 1: Determine whether in vivo tension forces regulate kinetochore microtubule dynamics to control chromosome alignment during budding yeast mitosis; Aim 2: Determine whether tension generated by purified in vitro microtubules can intrinsically self-regulate microtubule dynamics; and Aim 3: Distinguish important anti-poleward forces in mammalian cell mitosis.Intellectual Merit: The proposed research will develop tools to better understand the origin and role of internally generated mitotic spindle forces during cell division. Specifically, microtubules are a key element in the mitotic spindle, and are thus responsible for generating and responding to forces which regulate the proper progression of cell division. Because uncontrolled cell division is a hallmark of cancer, and many cancer therapeutic interventions work by disrupting microtubule dynamics, dissection of the relationship between force and microtubule dynamics has the potential to lead to more rational approaches in cancer treatment. The proposed research will make use of physical principles and advanced microscopy to evaluate forces inside of living cells, and then use these methods to dissect the relationship between these forces and microtubule-based chromosome segregation during mitosis. In addition, biophysical reconstitution and computational modeling at both cellular and molecular scales will be used to directly test the hypotheses, and to provide a framework for interpreting experimental results. The generated results are expected to be applicable both in better understanding the fundamental biophysics of cell division, and in providing quantitative data on potential cancer therapies.Broader Impacts: The proposed work has significant broader impacts on advancing the quantitative understanding of the role of molecular forces during cell mitosis, which has substantial implications in cell biology and cancer research. The integration of quantitative engineering methods, computer programming, and physical principles into the study of fundamental problems in biology has been demonstrated to increase the productivity and insights available to researchers in the life sciences. A strong thrust of this proposal is aimed at instilling quantitative engineering approaches into Cell Biology education and research, at both the undergraduate and graduate levels, which will contribute to development of a diverse, globally competitive STEM workforce. In particular, the education aims are integrated with the research proposal to (1) introduce quantitative engineering approaches to cell biology graduate students at the earliest point in their graduate careers; (2) build coursework and research opportunities for both graduate and undergraduate biology students to implement quantitative engineering methods into their current studies, and subsequently into their future careers; and (3) fund undergraduate and underrepresented minority students to perform quantitative research projects in Cell Biology or Biomedical Engineering laboratories.