A Motor-Based Tension Sensor and Spindle Mechanobiology

点击翻译按钮获取中文摘要

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.