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.

概括 这项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，并且 与实验者合作紧密合作的悠久历史。但是，她缺乏直接的实验 事实证明，培训对她长期职业目标的进步有一个严重的限制：促进 通过综合 实验和理论方法。 该赠款将提供受保护的培训时间，以便PI：（1）学会设计和执行 杂种。这将需要在生物物理学，细胞生物学的一系列技术中进行实践实验室培训 分子生物学，遗传学和生物化学。 （2）教育生物学以补充她的物理培训 并允许她提出尖端，生物学相关的研究问题。这将需要参与 在会议，研讨会，研讨会和期刊俱乐部。 （3）在管理实验时发展她的专业知识 精神实验室。这将需要在实验室安全，授予写作和负责任的研究中进行培训。 该项目的研究部分将解决微管（MTS）捕获动力学（KC） 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 并捕获，其中动态MTS从中心体朝着不同的方向生长并进行最终附件 与KCS。然而，最近的工作发现，侧向旋转散射MT的侧向附着启用了快速KC 即使有大大减少的MT动态捕获。以前的工作仅专注于MT动态 不稳定性或旋转扩散，因此无法比较两种机制并确定 他们的相对重要性。具体目的是： 1：评估微管动态不稳定性和旋转扩散对动力学的重要性 使用定量成像和第一代模型捕获。为此开发的初步模型 据信，建议是KC捕获的第一个模型，其中包括MT动态不稳定性和MT和MT和 KC扩散。将测量丢失的KC和极地MT长度随时间的函数进行测量 从冷块中恢复后，将确定KC捕获的时间过程；数据将用于适合未知 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， 分别一起捕获KC。 2：测量有丝分裂核极性微管的动力学，并使用测量参数 创建第二代动力学捕获模型。 AIM 1中的研究需要合适的关键模型 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 预测，对于可靠且准确的模型，最好使用MT动态不稳定性PA-的测量值 拉列特，而不是依靠估计或拟合。但是，有丝分裂中核极性MT的动力学已有 以前没有足够详细地测量没有未知参数的定量模型。 由于这些MT是短暂的，因此将比较几种活细胞成像方法以确定MT 动力学参数。 3：预测和衡量微管动力学的变化如何影响动力学的时间过程 捕获。诸如疾病状态之类的生物扰动可能导致MT动态的改变，但不是 了解这些改变如何影响KC捕获。当前模型无法预测MT动力学的方式 影响KC。该模型将用于查找参数空间的“敏感”区域，其中MT的小变化 动力学参数导致KC捕获时间过程的相对较大变化。多次实验 扰动可用于裂变酵母中的MT动态。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。 PI将由尊敬的科学家J. Richard McIntosh的赠款指导，其工作已经 专注于模型生物体中的细胞分裂。他将提供实验室空间和设备以允许 PI实现这笔赠款的目标。这将是理想的培训环境，因为麦金托什最近有 从教学中退休，尽管仍然维持一个活跃的研究实验室。 PI是 科罗拉多大学博尔德大学。 Cu Boulder有几项举措，这些举措促进了强大的 跨学科研究环境。 PI是Biofrontiers Institute的一部分，并参加每月会议 她的生物物理学同事来自各个部门。该机构致力于充分支持 PI在学习细胞生物学方面的技术时。