ELECTRON TOMOGRAPHY OF VERTEBRATE MITOTIC SPINDLES

脊椎动物有丝分裂纺锤体的电子断层扫描

• 批准号: 8362545
• 负责人: Daniel Needleman
• 金额: $ 3.19万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362545
• 关键词: Cell physiology; Cells; Cellular Structures; Centrosome; Chromosomes; Cluster Analysis; Computer software; Data; Enzymes; Freeze Substitution; Freezing; Funding; Grant; Hand; Metaphase; Methods; Microtubules; Mitotic spindle; Modeling; Motor; National Center for Research Resources; Preparation; Principal Investigator; Research; Research Infrastructure; Resources; Sampling; Software Tools; Source; United States National Institutes of Health; Work; cost; electron tomography; improved; reconstruction; research study; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Electron tomography of rapidly frozen, freeze-substitution fixed samples will be used to examine a significant fraction of the volume of metaphase spindles from U2OS cells, followed by comparable work on cells frozen and fixed during spindle formation. Our structural studies will employ improved methods for sample preparation (TR&D 3A) and both large-scale montages and multiple serial sections, assembled into 3D reconstructions (TR&D 5D). The trajectories of spindle microtubules (MTs) will initially be determined by hand modeling, though in subsequent work we hope to have software for automatic segmentation. The resulting models of MT trajectories will be studied with cluster analysis and several application-specific software tools to compare MT organization with the predictions from four distinct and popular models for spindle formation: the classic, centrosome-initiation model, the idea that spindle MTs self-organize as a result of MT-associated motor enzymes, the idea of MT initiation and/or stabilization in response to gradients that emanate from chromosomes, and the concept that a non/MT "matrix" guides the formation and organization of MTs. The data should eliminate or at least erode one or more of these models and will certainly provide baseline structural information for the interpretation of functional experiments on spindle formation and function that are being carried out in our labs and elsewhere.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 将使用快速冷冻、冷冻置换固定样品的电子断层扫描检查U2 OS细胞中期纺锤体体积的显著部分，然后对纺锤体形成期间冷冻和固定的细胞进行类似工作。 我们的结构研究将采用改进的样品制备方法（TR&D 3A）和大规模蒙太奇和多个连续切片，组装成3D重建（TR&D 5D）。纺锤体微管（MT）的轨迹最初将通过手工建模来确定，但在随后的工作中，我们希望有自动分割的软件。将使用聚类分析和几种特定于应用的软件工具来研究MT轨迹的结果模型，以将MT组织与四种不同且流行的纺锤体形成模型的预测进行比较：经典的中心体起始模型，即纺锤体MT由于MT相关运动酶而自组织的想法，MT起始和/或稳定的想法，响应于从染色体发出的梯度，以及非MT“基质”指导MT的形成和组织的概念。 这些数据应该消除或至少削弱这些模型中的一个或多个，并且肯定会为我们实验室和其他地方正在进行的关于纺锤体形成和功能的功能实验的解释提供基线结构信息。