Assessing the Contributions of Microtubule Dynamic Instability and Microtubule Ro

评估微管动态不稳定性和微管 Ro 的贡献

• 批准号: 8677173
• 负责人: Meredith Betterton
• 金额: $ 14.65万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8677173
• 关键词: Address; Affect; Aneuploidy; Animal Model; Biochemistry; Biological; Biological Factors; Biology; Biophysics; Cell division; Cells; Cellular biology; Centrosome; Chromosome Segregation; Chromosomes; Collaborations; Colorado; Commit; Complement; Congenital Abnormality; Data; Development; Diffuse; Diffusion; Disease; Educational process of instructing; Educational workshop; Environment; Equipment; Eukaryota; Fission Yeast; Fostering; Foundations; Future; Generations; Genetic; Goals; Grant; Health; Human; Image; Institutes; Institution; Interdisciplinary Study; Journals; K-Series Research Career Programs; Kinetochores; Laboratories; Lateral; Lead; Learning; Length; Life; Malignant Neoplasms; Measurement; Measures; Mentors; Microtubule Polymerization; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular Biology; Motion; Nuclear; Physics; Process; Proteins; Recording of previous events; Recovery; Relative (related person); Research; Role; Rotation; Safety; Scientist; System; Techniques; Testing; Time; Training; Uncertainty; Universities; Work; Writing; base; career; cellular imaging; daughter cell; design; experience; meetings; neglect; physical model; prevent; professor; public health relevance; research study; responsible research conduct; segregation; symposium; theories; tool

Summary The PI of this Mentored Career Development Award proposal has significant experience in biophysics theory, and a long history of working closely in collaboration with experimentalists. However, her lack of direct experimental training has proved to be a severe limitation in her progress toward her long-term career objective: to advance the understanding of chromosome motions and mitosis, and related human-health issues, through an integrated experimental and theoretical approach. This grant will provide protected training time in order for the PI to: (1) Learn to design and conduct ex- periments. This will require practical laboratory training in a range of techniques in biophysics, cell biology, molecular biology, genetics, and biochemistry. (2) Educate herself in biology to complement her physics training and to allow her to formulate cutting-edge, biologically relevant research questions. This will require participation in conferences, workshops, seminars, and journal clubs. (3) Develop her expertise as she manages an experi- mental laboratory. This will require training in lab safety, grantwriting, and the responsible conduct of research. The research component of the project will address the capture of kinetochores (KCs) by microtubules (MTs) in cell division. For years the primary mechanism of KC capture in mitosis was believed to be microtubule search and capture, in which dynamic MTs grow in different directions from centrosomes and make end-on attachments with KCs. However, recent work found that lateral KC attachment to rotationally diffusing MTs enabled rapid KC capture even with significantly reduced MT dynamics. Previous work has focused exclusively on MT dynamic instability or rotational diffusion and therefore has been unable to compare the two mechanisms and determine their relative importance. The specific aims are: 1: Evaluate the importance of microtubule dynamic instability and rotational diffusion to kinetochore capture using quantitative imaging and a first-generation model. The preliminary model developed for this proposal is believed to be the first model of KC capture that includes both MT dynamic instability and MT and KC diffusion. Measurements will be made of the fraction of lost KCs and polar MT lengths as a function of time after recovery from cold block will determine the time course of KC capture; the data will be used to fit unknown parameters in the model. The model will allow assessment the importance of MT rotation and dynamic instability, separately and together, for KC capture. 2: Measure the dynamics of mitotic nuclear polar microtubules and use the measured parameters to create a second-generation kinetochore capture model. The study in Aim 1 requires fitting key model parameters for MT dynamic instability. Because uncertainty in model parameters leads to uncertainty in model predictions, for a reliable and accurate model it is best to use measured values of MT dynamic instability pa- rameters, rather than relying on estimates or fits. However, dynamics of nuclear polar MTs in mitosis have not previously been measured in sufficient detail to build a quantitative model without unknown parameters. Because these MTs are short-lived, several live-cell imaging approaches will be compared to determine MT dynamics parameters. 3: Predict and measure how alterations in microtubule dynamics affect the time course of kinetochore capture. Biological perturbations such as disease states can lead to alterations in MT dynamics, but it is not understood how these alterations affect KC capture. Current models are not able to predict how MT dynamics affect KC. The model will be used to find "sensitive" regions of parameter space where small changes in MT dynamics parameters lead to relatively large changes in the time course of KC capture. Multiple experimental perturbations are available to perturb MT dynamics in fission yeast. The results will test the understanding of the contributions of MT dynamic instability and rotational diffusion to KC capture. The PI will be mentored under this grant by J. Richard McIntosh, an esteemed scientist whose work has focused on cell division in the model organism S. pombe. He will provide lab space and equipment to allow the PI to fulfill the aims of this grant. This will be an ideal training environment, as McIntosh has recently retired from teaching, though still maintains an active research lab. The PI is an associate professor at the University of Colorado, Boulder. There are several initiatives in place at CU Boulder that have fostered a strong interdisciplinary research environment. The PI is part of the Biofrontiers Institute, and attends monthly meetings with her biophysics colleagues from a range of departments. The institution is committed to fully supporting the PI as she learns techniques in cell biology.

总结 本指导职业发展奖提案的PI在生物物理学理论方面具有丰富的经验， 与实验学家密切合作的悠久历史。然而，她缺乏直接的实验 事实证明，培训严重限制了她实现长期职业目标的进程： 通过一个综合的方法来了解染色体运动和有丝分裂，以及相关的人类健康问题， 实验和理论方法。 该补助金将提供受保护的培训时间，以便PI：（1）学习设计和进行前， 实验这将需要在生物物理学、细胞生物学， 分子生物学遗传学和生物化学(2)自学生物学以补充物理训练 并让她能够提出前沿的、与生物学相关的研究问题。这将需要参与 参加会议、讲习班、研讨会和期刊俱乐部。(3)发展她的专业知识，因为她管理的经验- 心理实验室这将需要在实验室安全，授权和负责任的研究行为的培训。 该项目的研究部分将解决微管（MT）捕获动粒（KC）的问题 在细胞分裂中。多年来，有丝分裂中KC捕获的主要机制被认为是微管搜索 和捕获，其中动态MT从中心体向不同方向生长并进行末端附着 与KC然而，最近的研究发现，旋转扩散型MT的横向KC附着使得快速KC 即使在MT动态显著降低的情况下也能捕获。以前的工作主要集中在MT动态上 不稳定性或旋转扩散，因此无法比较这两种机制并确定 其相对重要性。具体目标是： 1：评价微管动态不稳定性和旋转扩散对动粒的重要性 使用定量成像和第一代模型进行捕获。为此开发的初步模型 建议被认为是第一个模型的KC捕获，包括MT动态不稳定性和MT， KC扩散。将测量作为时间函数的丢失KC和极性MT长度的分数 从冷块恢复后，将确定KC捕获的时间过程;数据将用于拟合未知 模型中的参数。该模型将允许评估MT旋转和动态不稳定性的重要性， 单独或一起抓捕KC 2：测量有丝分裂核极微管的动力学，并使用测量的参数来 创建第二代动粒捕获模型。目标1中的研究需要拟合关键模型 MT动态不稳定性参数。因为模型参数的不确定性导致模型的不确定性 预测，对于一个可靠和准确的模型，最好使用MT动态不稳定性参数的测量值。 参数，而不是依赖于估计或拟合。然而，在有丝分裂中，核极性MT的动力学 先前没有被足够详细地测量以建立没有未知参数的定量模型。 由于这些MT是短暂的，几种活细胞成像方法将进行比较，以确定MT 动力学参数 3：预测和测量微管动力学的改变如何影响动粒的时间过程 捕获.生物扰动，如疾病状态，可导致MT动力学的改变，但它不是 了解这些改变如何影响KC捕获。目前的模型无法预测MT动力学如何 影响KC。该模型将用于寻找参数空间的“敏感”区域，其中MT的微小变化 动力学参数导致KC捕获的时间过程中相对较大的变化。多个实验 干扰可用于干扰裂变酵母中的MT动力学。结果将测试的理解 MT动力学不稳定性和旋转扩散对KC捕获的贡献。 PI将由J. Richard McIntosh指导，他是一位受人尊敬的科学家，他的工作 重点研究了模式生物S.粟酒他将提供实验室空间和设备， PI以实现该补助金的目标。这将是一个理想的训练环境，就像麦金托什最近 从教学中退休，但仍然保持着活跃的研究实验室。PI是一名副教授， 科罗拉多大学博尔德分校。CU Boulder有几项举措，这些举措促进了一个强大的 跨学科的研究环境。PI是Biofrontiers研究所的一部分，并参加每月会议 和她来自不同部门的生物物理学同事一起。该机构致力于全面支持 PI，因为她学习细胞生物学技术。