Assessing the Contributions of Microtubule Dynamic Instability and Microtubule Ro

摘要 这项辅导性职业发展奖提案的PI在生物物理学理论方面拥有丰富的经验，并且 与实验者密切合作的悠久历史。然而，她缺乏直接的实验 事实证明，训练是她朝着长期职业目标--晋升--前进的严重限制 了解染色体运动和有丝分裂，以及相关的人类健康问题，通过 实验和理论相结合的方法。 这笔赠款将提供受保护的培训时间，以便让私人投资公司：(1)学习设计和实施前任- 周边。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)自学生物以补充物理训练 并允许她提出前沿的、与生物相关的研究问题。这将需要参与 在会议、研讨会、研讨会和杂志俱乐部。(3)在管理一项实验时发展她的专业知识- 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方面的培训。 该项目的研究部分将解决微管捕获动点(KCs)的问题。 在细胞分裂过程中。多年来，有丝分裂中KC捕获的主要机制被认为是微管搜索 以及捕获，其中动态MT从中心体向不同方向生长，并形成端接连接 和肯塔基州。然而，最近的工作发现，在旋转扩散的MTS上横向附着KC能够实现快速的KC 即使在MT动态显著降低的情况下也能捕获。以前的工作完全集中在MT动力学上 不稳定或旋转扩散，因此一直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：评估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 该提案被认为是第一个包括MT动态不稳定和MT和MT的KC捕获模型 KC扩散。将测量作为时间函数的丢失的Kcs和极点MT长度的百分比 从冷块恢复后将决定KC捕获的时间进程；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， 分开和一起，为KC捕获。 2：测量有丝分裂核极微管的动力学，并使用测量的参数 创建第二代动粒捕获模型。目标1中的研究需要拟合Key模型 MT动态不稳定性的参数。因为模型参数的不确定性导致模型的不确定性 对于可靠和准确的模型，最好使用MT动态不稳定参数的实测值。 参数，而不是依赖于估计或拟合。然而，有丝分裂中核极MTS的动态变化 以前没有进行足够详细的测量，无法在没有未知参数的情况下建立定量模型。 因为这些MT是短暂的，所以将比较几种活细胞成像方法来确定MT 动力学参数。 3：预测和测量微管动力学的变化如何影响着动粒的时间进程 抓捕。生物扰动，如疾病状态，可以导致MT动力学的改变，但它不是 了解这些更改如何影响KC Capture。目前的模型无法预测MT动力学如何 影响KC。该模型将被用来寻找参数空间中MT变化较小的“敏感”区域 动力学参数导致KC捕获的时间过程发生较大变化。多重实验 微扰可用于扰乱分裂酵母中的MT动态。结果将考验人们对 MT动力不稳定性和旋转扩散对KC俘获的贡献。 根据这笔赠款，国际和平组织将由J·理查德·麦金托什指导，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.pombe的细胞分裂。他将提供实验室空间和设备，以便 为实现这笔赠款的目标而设立的私募股权基金。这将是一个理想的训练环境，就像麦金托什最近所做的那样 虽然退休了，但仍有一个活跃的研究实验室。私家侦探是麻省理工学院的副教授 科罗拉多大学博尔德分校。科罗拉多州立大学博尔德分校的几项举措培养了强大的 跨学科研究环境。PI是生物前沿研究所的一部分，每月都会参加会议 和她来自多个部门的生物物理学同事。该机构致力于全力支持 当她学习细胞生物学技术时，她说。

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.