Collaborative Research: motor-driven pattern formation during cell division

智力优势：在细胞分裂过程中，细胞骨架会迅速重组，对齐并分离染色体，然后将细胞分裂成两半。细胞分裂本身是由收缩蛋白介导的，收缩蛋白的组装由信号蛋白Rho控制。对染色体进行分类的有丝分裂装置以微管为基础，以某种方式提供空间信息，以模式Rho的活动，从而在空间和时间上收缩蛋白质组装。一个由来已久的假说提出，分子马达沿着有丝分裂装置的微管携带信号，告诉细胞表面哪里收缩。该项目将通过研究驱动蛋白家族的马达可以将快速变化的微管阵列中固有的信息转化为细胞表面的Rho信号模式的量化条件，直接检验这一假设。为了做到这一点，研究人员将创造马达蛋白和可光激活的荧光蛋白之间的杂交，并测量它们的生化特性。他们将通过观察单个电机附着在单个微管上并在体外沿着单个微管移动来测量运动参数。这些相同的混合马达探头将被引入细胞中，并将使用实时成像来测量它们在高分辨率细胞分裂期间的动态行为。与此同时，先进的3D计算机模拟将被开发出来，以预测这些相同的量化试剂在整个细胞的环境中应该如何表现，无论是正常情况下还是当微管阵列的几何形状被实验改变时；反过来，活细胞中的分析将检验这些预测。通过结合活细胞成像、单分子测量和假想结果的计算机模拟，该项目将产生一个物理上可信的条件，在这些条件下，细胞的分子马达和细胞骨架组装调节器工具箱可以加在一起，形成细胞分裂期间强大的空间模式形成机制。更广泛的影响：这个项目将培训本科生和研究生使用高分辨率活细胞显微镜。这项研究还包括开发一个复杂的基于代理的计算机模拟程序，预计该程序将广泛应用于细胞生物学中的许多其他基本问题，超出了该项目的具体研究目标。这些软件和计算机代码将免费提供给其他研究人员。同样，这项研究预计将产生几个有用的分子探针，有助于其他研究人员对细胞骨架行为的更广泛研究。这些探针将免费分发给使用Addgene.org的研究人员，Addgene.org是一个非营利性的质粒库，它使构建物可以通过运输费用获得。最后，这项研究将产生大量显微镜图像和视频，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，用于教育和其他研究用途。

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.