A Motor-Based Tension Sensor and Spindle Mechanobiology

在细胞分裂过程中，染色体中遗传物质的两个副本被称为“纺锤体”的细胞结构分开。纺锤体是一系列重叠的细丝，它们感知并传播分离染色体所需的力量。这些力量被认为主要是由细丝的快速生长和收缩以及沿着细丝移动的特殊蛋白质产生的，这种蛋白质被称为马达蛋白。尽管对细胞分裂的研究已经进行了一个多世纪，对马达蛋白质的研究也已经进行了几十年，但纺锤体中承载负荷和产生力量所需的关键元素还没有被确定。细丝中的力从未被测量过。我们也不知道在细胞分裂过程中，力在空间和时间上是如何变化的。然而，我们确实知道，这些力量未能分离染色体会导致有机体发育和健康的严重问题。关于导致细胞分裂的力量的新知识将对理解细胞分裂的功能产生重大影响，包括分裂细胞与其他细胞(如干细胞)的不同之处，以及癌细胞异常分裂的基础。这一新信息可能导致调节细胞分裂或逆转疾病状态的新方法。该项目的更广泛影响包括编写学习材料，向高中生介绍生物物理学，这是生物物理学会最近加强K-12科学教育的努力的一部分。PI将使用学会在2016年3月生物物理周期间公布的与研究相关的主题(例如，扩散、荧光、光学显微镜)制定的教案。光显微镜课程计划使用了一个小木制显微镜，将在国家研讨会上与教学计划一起交给教师。目标是创造一种遗传编码的分子张力传感器，来测量纺锤体中马达蛋白质所承受的负荷，并确定负荷在分裂过程中的变化。新的主轴传感器由Kinesin-14马达蛋白组成，并插入了先前报道的张力传感器模块，该模块可产生随力变化的荧光信号，并可通过共聚焦显微镜检测到。供体光漂白试验将被用来测量在分裂过程中马达蛋白所承担的负荷。传感器也将由突变的马达构建，以测试马达通过滑动和交联微管产生张力的假设，从而机械地抵抗相反方向的滑移力。传感器上的力将在正常分裂期间和被突变体扰动、破坏微管动力学和延长有丝分裂的发育变化后确定。将解决的主要问题包括形成和延长纺锤体所需的绝对力，在有丝分裂过程中和不同细胞条件下力量如何变化，以及纺锤体张力在分裂过程中变化的影响。该项目将创造新的研究工具，能够感知和报告纺锤力的分子张力传感器，并将促进对纺锤体功能和染色体分布的分子机制的理解。

During cell division, the two copies of the genetic material in the chromosomes are separated by a cellular structure call the "spindle". The spindle is an array of overlapping filaments that sense and propagate the forces needed to separate the chromosomes. The forces are thought to be produced primarily by rapid filament growth and shrinking, together with specialized proteins, known as motor proteins that move along the filaments. Despite more than a century of work on cell division and decades of work on motor proteins, the critical elements of the spindle that are needed to bear loads and produce forces have not been identified. The forces in the filaments has never been measured. Neither do we know how the forces change spatially and over time during cell division. We do know, however, that failure of these forces to separate the chromosomes causes severe problem with development of an organism and with health. New knowledge regarding the forces causing cell division will have a large impact on understanding how cell division function, including how dividing cells differ from other cells, such as stem cells, and the basis of abnormal division in cancer cells. This new information could lead to new ways of regulating cell division or reversing disease states. Broader impacts of this project include the creation of study materials to introduce biophysics to high school students, as part of recent efforts by the Biophysical Society to enhance K-12 science education. The PI will use lesson plans developed on topics related to the research (e.g., Diffusion, Fluorescence, Light Microscopy) that were made public by the Society during Biophysics Week in March 2016. The Light Microscopy Lesson Plan uses a small wooden microscope that will be given to teachers at national workshops with the lesson plan.The goal is to create a genetically encoded molecular tension sensor to measure loads borne by a motor protein in the spindle and determine how the loads change during division. The new spindle sensor consists of a kinesin-14 motor protein with an insertion of a previously reported tension sensor module, which produces a fluorescence signal that varies with force and is detectable by confocal microscopy. Donor photobleaching assays will be used to measure loads borne by the motor protein during division. Sensors will also be constructed from mutant motors to test the hypothesis that the motor produces tension by both sliding and crosslinking microtubules, mechanically resisting oppositely-directed sliding forces. Forces across the sensor will be determined during normal division and after perturbation by mutants, disrupted microtubule dynamics, and developmental changes that extend mitosis. Major questions that will be addressed include the absolute forces required to form and elongate the spindle, how forces vary during mitosis and under different cellular conditions, and the effects of changes in spindle tension during division. This project will create new research tools, molecular tension sensors capable of sensing and reporting spindle forces, and will advance understanding of molecular mechanisms underlying spindle function and chromosome distribution.