Dissecting Induction of Cell Cleavage

细胞质分裂最终确保染色体和细胞质正确地分裂成两个子细胞。在动物细胞中，这是通过形成分裂沟来实现的，分裂沟将分离的染色体之间的有丝分裂（或减数分裂）纺锤体一分为二。卵裂沟的失败或不正确的定位可能导致癌症或出生缺陷。我们知道有丝分裂器决定细胞的分裂面。然而，由于我们对卵裂沟信号的来源和性质缺乏深入的了解，有丝分裂装置如何启动卵裂沟尚不清楚。有丝分裂装置的每一部分；即紫苑，中央纺锤体（微管阵列和纺锤体中间区）和染色体，已被发现能够在某些细胞类型中诱导卵裂沟。然而，尚不确定哪一部分是信号的主要来源，以及所有部分是否一致行动。该项目的具体目标是：1)在没有其他所有主轴成分的情况下，通过测试每个主轴成分的犁沟感应，确定哪个主轴成分是犁沟信号的主要来源；2)通过将中心纺锤体分解为离散的部分（末相盘和剩余的微管阵列），并测试它们在沟槽形成中的独立作用，区分纺锤体中间区（或末相盘/中体）和微管的作用。这些实验将显微操作与数字增强偏振显微镜和荧光显微镜相结合，其中活细胞中的有丝分裂纺锤体被机械地解剖并按需要重新排列，从而可以实时观察活细胞中沟槽定位的结果。显微操作纳入到荧光显微镜允许直接微针荧光标记的微管和肌动蛋白细丝，同时观察其动力学方面的收缩环形成在犁沟起始。此外，微处理细胞将在感兴趣的时刻被固定，并在共聚焦或电镜下染色，以更好地确定微管、肌动蛋白丝和其他参与细胞分裂的因素的分布。在这些严格的条件下，通过剖析独立和/或重叠的纺锤体成分在犁沟形成中的作用，将仔细研究目前最有吸引力的解理犁沟定位模型。这些模型包括由紫苑作用于赤道皮层的信号引起的“星体刺激”，由紫苑作用于极地皮层的信号引起的“极性松弛”，以及由纺锤体中间区（末期盘和/或染色体）作用于赤道皮层的信号引起的“赤道刺激”。该项目旨在为理解纺锤体微管（来自紫苑或中央纺锤体）、染色体和末期盘在沟定位过程中对肌动蛋白丝组织的影响提供新的见解。

Cytokinesis ultimately ensures the proper partition of chromosomes and cytoplasm into two daughter cells. In animal cells, this is achieved by the formation of a cleavage furrow that bisects the mitotic (or meiotic) spindle between segregated chromosomes. Failure in, or improper positioning of the cleavage furrow may lead to cancer or birth defects. It is known that the mitotic apparatus defines the cell cleavage plane. However, it is not clear how the mitotic apparatus initiates the cleavage furrow due to our lack of in-depth understanding about the source and nature of the furrow signal. Each part of the mitotic apparatus; namely asters, central spindle (microtubule arrays and spindle midzone), and chromosomes, has been found capable of inducing a cleavage furrow in certain cell types. Yet it is uncertain which part is the essential source of the signal and whether all parts act in concert. The specific aims of this project are to: 1) determine which spindle constituent is the essential source of furrow signal by testing furrow induction with each single spindle constituent in the absence of all the others; 2) distinguish the role of spindle midzone (or telophase disc/midbody) from microtubules by dissecting the central spindle into discrete parts (the telophase disc and the remaining microtubule arrays), and testing their independent role in furrow initiation. These experiments combine micromanipulation with digital-enhanced polarization microscopy and epifluorescence microscopy, in which mitotic spindles in living cells are mechanically dissected and rearranged as desired, which allows real-time observation of the resulting effect on furrow positioning in living cells. Incorporation of micromanipulation into epifluorescence microscopy permits direct microneedling of fluorescently-labeled microtubules and actin filaments while observing their dynamics with respect to contractile ring formation during furrow initiation. In addition, micromanipulated cells will be fixed at moments of interest and stained for confocal or EM microscopy to better determine the distribution of microtubules, actin filaments and other factors involved in cytokinesis. Through dissecting independent and/or overlapping roles of the spindle constituents in furrow initiation under these stringent conditions, the currently most attractive models for cleavage furrow positioning will be scrutinized. These models include "astral stimulation" by signals from the asters acting on equatorial cortex, "polar relaxation" by signals from the asters acting on polar cortex, and "equatorial stimulation" by signals from the spindle midzone (telophase disc and/or chromosomes) acting on the equatorial cortex. The project aims to provide new insights towards understanding of the impact of spindle microtubules, either from asters or the central spindle, chromosomes, and the telophase disc on the organization of actin filaments during furrow positioning.