Dissecting Induction of Cell Cleavage

剖析细胞分裂的诱导

• 批准号: 0090725
• 负责人: Dahong Zhang
• 金额: $ 32.2万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0090725&HistoricalAwards=false
• 关键词: Dissecting; Induction; Cell; Cleavage

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.

胞质分裂最终确保染色体和细胞质正确地分配成两个子细胞。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。乳沟的失败或不正确的定位可能导致癌症或出生缺陷。已知有丝分裂器限定细胞分裂平面。然而，由于我们对沟信号的来源和性质缺乏深入了解，目前尚不清楚有丝分裂器如何启动卵裂沟。有丝分裂器的每一部分，即星状体、中央纺锤体（微管阵列和纺锤体中间区）和染色体，已经发现能够在某些细胞类型中诱导分裂沟。然而，尚不确定哪一部分是信号的基本来源，以及所有部分是否一致行动。本项目的具体目标是：1）通过测试在所有其他纺锤体成分都不存在的情况下，用每个单独的纺锤体成分的犁沟诱导来确定哪个纺锤体成分是犁沟信号的基本来源; 2）区分纺锤体中间区的作用（或末期盘/中体）从微管分离（末期盘和剩余的微管阵列），并测试它们在犁沟起始中的独立作用。这些实验将联合收割机显微操作与数字增强偏振显微镜和落射荧光显微镜相结合，其中活细胞中的有丝分裂纺锤体根据需要被机械解剖和重新排列，这允许实时观察活细胞中的沟槽定位的结果。将显微操作到epifluorescence显微镜允许荧光标记的微管和肌动蛋白丝的直接微针，同时观察它们的动力学相对于收缩环的形成在沟启动。此外，显微操作的细胞将在感兴趣的时刻被固定，并进行共聚焦或EM显微镜染色，以更好地确定微管、肌动蛋白丝和胞质分裂中涉及的其他因子的分布。通过解剖的独立和/或重叠的作用，在这些严格的条件下，在犁沟起始的纺锤体成分，目前最有吸引力的模型，卵裂沟定位将被仔细检查。这些模型包括“星状刺激”的信号作用于赤道皮质，“极性松弛”的信号作用于极性皮质，和“赤道刺激”的信号作用于赤道皮质的纺锤体中间区（末期光盘和/或染色体）。该项目旨在提供新的见解，了解纺锤体微管的影响，无论是从紫苑或中央纺锤体，染色体和终末期盘上的组织肌动蛋白丝在犁沟定位。