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: 8848090
• 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/8848090
• 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; 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; 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; quantitative imaging; research study; responsible research conduct; segregation; symposium; temporal measurement; theories; tool

DESCRIPTION (provided by applicant): 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 experiments. 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 experimental laboratory. This will require training in lab safety, grant writing, 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 parameters, 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.

描述（由申请人提供）：本指导职业发展奖提案的 PI 在生物物理学理论方面拥有丰富的经验，并且长期与实验学家密切合作。然而，事实证明，她缺乏直接的实验训练，这严重限制了她实现长期职业目标的进程：通过综合实验和理论方法，增进对染色体运动和有丝分裂以及相关人类健康问题的理解。 这笔赠款将提供受保护的培训时间，以便 PI 能够： (1) 学习设计和进行实验。这将需要生物物理学、细胞生物学、分子生物学、遗传学和生物化学等一系列技术的实际实验室培训。 (2) 进行生物学教育，以补充她的物理训练，并让她能够提出前沿的、生物学相关的研究问题。这 需要参加会议、讲习班、研讨会和期刊俱乐部。 (3) 在管理实验实验室时发展她的专业知识。这需要实验室安全、资助写作和负责任的研究行为方面的培训。 该项目的研究部分将解决细胞分裂中微管 (MT) 捕获动粒 (KC) 的问题。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，并与 KC 进行末端附着。然而，最近的研究发现，即使 MT 动态显着降低，横向 KC 附着到旋转扩散 MT 也能实现快速 KC 捕获。之前的工作只关注 MT 动态不稳定性或旋转扩散，因此无法比较这两种机制并确定它们的相对重要性。具体目标是： 1：使用定量成像和第一代模型评估微管动态不稳定性和旋转扩散对动粒捕获的重要性。为该提案开发的初步模型被认为是第一个 KC 捕获模型，其中包括 MT 动态不稳定性以及 MT 和 KC 扩散。从冷块恢复后，将测量损失的 KC 和极性 MT 长度随时间变化的比例，从而确定 KC 捕获的时间过程；数据将用于拟合模型中的未知参数。该模型将允许单独和共同评估 MT 旋转和动态不稳定性对于 KC 捕获的重要性。 2：测量有丝分裂核极微管的动力学，并使用测量的参数创建第二代着丝粒捕获模型。目标 1 的研究需要拟合 MT 动态不稳定性的关键模型参数。由于模型参数的不确定性会导致模型预测的不确定性，因此对于可靠且准确的模型，最好使用 MT 动态不稳定参数的测量值，而不是依赖于估计或拟合。然而，之前尚未对有丝分裂中核极性 MT 的动态进行足够详细的测量，无法在参数未知的情况下建立定量模型。由于这些 MT 的寿命很短，因此将比较几种活细胞成像方法以确定 MT 动态参数。图 3：预测并测量微管动力学的变化如何影响动粒捕获的时间过程。诸如疾病状态之类的生物扰动可能会导致 MT 动力学的改变，但尚不清楚这些改变如何影响 KC 捕获。当前模型无法预测 MT 动态如何影响 KC。该模型将用于查找参数空间的“敏感”区域，其中 MT 动态参数的微小变化会导致 KC 捕获的时间过程发生相对较大的变化。多种实验扰动可用于扰动裂殖酵母中的 MT 动力学。结果将测试对 MT 动态不稳定性和旋转扩散对 KC 捕获的贡献的理解。 PI 将接受 J. Richard McIntosh 的指导，他是一位受人尊敬的科学家，其工作重点是模式生物粟酒裂殖酵母的细胞分裂。他将提供实验室空间和设备，使 PI 能够实现这笔赠款的目标。这将是一个理想的培训环境，因为麦金托什最近从教学中退休，但仍然保留着一个活跃的研究实验室。该 PI 是科罗拉多大学博尔德分校的副教授。科罗拉多大学博尔德分校采取了多项举措，培育了强大的跨学科研究环境。该 PI 是生物前沿研究所的一部分，每月与来自多个部门的生物物理学同事参加会议。该机构致力于全力支持 PI 学习细胞生物学技术。