Collaborative Research: motor-driven pattern formation during cell division

合作研究：细胞分裂过程中电机驱动的模式形成

• 批准号: 1041173
• 负责人: Linda Wordeman
• 金额: $ 59.26万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1041173&HistoricalAwards=false
• 关键词: Collaborative; Research; motor; driven; pattern

Abstract Intellectual Merit: During cell division, the cytoskeleton reorganizes itself rapidly to align and separate chromosomes, and then cleave the cell in two. Cell division itself is mediated by contractile proteins, whose assembly is controlled by the signaling protein Rho. The mitotic apparatus, which sorts the chromosomes, is based on microtubules, and somehow provides the spatial information to pattern Rho activity, and hence contractile protein assembly, in space and time. A long-standing hypothesis proposes that molecular motors carry signals along microtubules of the mitotic apparatus to tell the cell surface where to contract. This project will directly test this hypothesis by investigating the quantitative conditions under which motors of the kinesin family can translate the information immanent within the rapidly-changing microtubule array into a pattern of Rho signaling at the cell surface. To do this, the investigators will create hybrids between motor proteins and photoactivatable fluorescent proteins and measure their biochemical properties. They will measure motor motility parameters by observing single motors attaching to and moving along single microtubules in vitro. These same hybrid motor probes will be introduced into cells, and live imaging will be used to measure their dynamic behavior during cell division at high resolution. Meanwhile, advanced 3D computer simulations will be developed to predict how these same quantified agents should behave in the context of the whole cell, either normally or when the geometry of the microtubule array is experimentally altered; in turn, assays in live cells will test these predictions. By combining live-cell imaging, single-molecule measurement, and computer simulation of hypothetical outcomes, the project will produce an account of the physically-plausible conditions under which the cell's toolkit of molecular motors and cytoskeletal assembly regulators could add up to a mechanism for robust spatial pattern formation during cell division. Broader impacts: This project will train undergraduate and graduate students in high-resolution live-cell microscopy. The research also involves development of a sophisticated agent-based computer simulation program that is expected to have broad application to many other fundamental problems in cell biology, beyond the specific research goals of this project. This software and computer code will be made freely available to other researchers. Likewise, the research is expected to produce several useful molecular probes that will facilitate broader studies of the behavior of the cytoskeleton by other researchers. These probes will be distributed freely to researchers using Addgene.org, a non-profit plasmid repository which makes constructs available for the cost of shipping. Finally, this research will generate, as by-products of the specific experiments conducted, numerous microscope images and videos which the investigators will make freely available via both lab websites and public collections for educational and other research use.

摘要 智力优点：在细胞分裂过程中，细胞骨架快速自我重组，排列和分离染色体，然后将细胞一分为二。细胞分裂本身是由收缩蛋白介导的，而收缩蛋白的组装则由信号蛋白 Rho 控制。对染色体进行排序的有丝分裂装置基于微管，并以某种方式提供空间信息来模拟 Rho 活动，从而在空间和时间上进行收缩蛋白组装。一个长期存在的假设提出，分子马达沿着有丝分裂装置的微管携带信号，告诉细胞表面在哪里收缩。该项目将通过研究驱动蛋白家族马达将快速变化的微管阵列中固有的信息转化为细胞表面 Rho 信号传导模式的定量条件来直接检验这一假设。为此，研究人员将创建运动蛋白和光激活荧光蛋白之间的杂交体，并测量它们的生化特性。他们将通过观察体外附着在单个微管上并沿着单个微管移动的单个电机来测量运动参数。这些相同的混合电机探针将被引入细胞中，并使用实时成像以高分辨率测量它们在细胞分裂过程中的动态行为。同时，将开发先进的 3D 计算机模拟来预测这些相同的量化试剂在整个细胞的背景下应如何表现，无论是正常情况还是当微管阵列的几何形状通过实验改变时；反过来，活细胞检测将检验这些预测。通过结合活细胞成像、单分子测量和假设结果的计算机模拟，该项目将描述物理上合理的条件，在这些条件下，细胞的分子马达和细胞骨架组装调节器的工具包可以形成一种机制细胞分裂过程中稳健的空间格局形成。更广泛的影响：该项目将为本科生和研究生提供高分辨率活细胞显微镜方面的培训。该研究还涉及开发一种复杂的基于代理的计算机模拟程序，该程序预计将广泛应用于细胞生物学中的许多其他基本问题，超出该项目的具体研究目标。该软件和计算机代码将免费提供给其他研究人员。同样，该研究预计将产生几种有用的分子探针，这将有助于其他研究人员对细胞骨架行为进行更广泛的研究。这些探针将免费分发给使用 Addgene.org 的研究人员，Addgene.org 是一个非盈利性质粒存储库，可通过运输费用提供构建体。最后，这项研究将产生大量的显微镜图像和视频，作为具体实验的副产品，研究人员将通过实验室网站和公共收藏免费提供这些图像和视频，以供教育和其他研究使用。