Functional and Molecular Regulation of Actomyosin by Microtubules

微管对肌动球蛋白的功能和分子调节

• 批准号: 9630860
• 负责人: William Bement
• 金额: $ 27.89万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630860&HistoricalAwards=false
• 关键词: Functional; Molecular; Regulation; Actomyosin; Microtubules

9630860 Bement Directed cortical movement is a fundamental feature of a broad variety of biological phenomena, including cytokinesis, cell migration, and movement of developmental information in early embryos. Cortical flow is one of the most common mechanisms by which directed cortical movement is achieved. Cortical flow is manifest as movement of actin filaments, cell surface proteins and, in some cases, cortical organelles toward a particular site. It is well known that cortical flow is actin filament-dependent, but what regulates the magnitude and direction of the flow is unknown. Microtubules have been hypothesized to somehow regulate cortical flow, but uhether they stimulate or inhibit flow is contentious, largely because of technical difficulties entailed by study of cortical flow in most systems. The oocyte of the frog, Xenopus laevis, has been developed as a model for cortical flow. Xenopus oocytes treated with the phorbol ester, PMA, undergo rapid, synchronous cortical flow which is easily quantified. This cortical flow exhibits all of the hallmarks typical of cortical flow in other systems, including dependence on actomyosin, directed movement of cortical organelles and filamentous actin, and a requirement for free movement of cell surface proteins. The features of this system have permitted the parallel analysis of cortical flow and microtubule levels following treatments that promote polymerization or depolymerization of microtubules. Rates of cortical flow are inversely correlated with levels of microtubules, and this effect is mediated by the microtubules themselves rather than by microtubule dynamics or free tubulin. Microtubule levels are inversely correlated with levels of myosin-II filaments, the functional form of myosin II. Preliminary results have narrowed the possible mechanisms by which microtubules exert their inhibitory effect on cortical flow to three: 1, a transport-based mechanism, wherein microtubule motors transport factors that modulate myosin-II biochemistry to or from the cortex; 2, a competition-based mechanism wherein microtubules compete with actomyosin for binding sites in the cortex; or 3, a microtubule-associated protein (MAP)-based mechanism, wherein microtubules bind to and sequester a factor that promotes cortical flow and myosin-II filament formation. A combined microscopic, biochemical, and molecular approach will be used to distinguish between these three hypotheses. These will rely heavily both on the attributes of the intact Xenopus oocyte and the approaches possible using oocyte lysates . This model system permits analyses that are essentially impossible in most other models of cortical flow that are currently studied. The work will provide insights applicable to a number of distinct cellular and developmental phenomena, including cytokinesis, cell locomotion, and transport of developmental information during embryogenesis. ***

9630860定向皮质运动是多种生物学现象的基本特征，包括胞质分裂、细胞迁移和早期胚胎中发育信息的移动。皮层流动是实现定向皮质运动的最常见机制之一。皮质流表现为肌动蛋白细丝、细胞表面蛋白，在某些情况下，皮质细胞器向特定位置移动。众所周知，皮质流是肌动蛋白细丝依赖的，但调节流的大小和方向的因素尚不清楚。微管被假设以某种方式调节皮质血流，但它们究竟是刺激还是抑制血流存在争议，这主要是因为在大多数系统中研究皮质血流存在技术困难。非洲爪蛙的卵母细胞已被开发为皮质流动的模型。用佛波酯(PMA)处理的非洲爪哇卵母细胞，经历了快速、同步的皮质流动，这很容易量化。这种皮质流动表现出其他系统中典型的皮质流动的所有特征，包括对肌动蛋白的依赖，皮质细胞器和丝状肌动蛋白的定向运动，以及细胞表面蛋白自由运动的要求。该系统的特点使得在促进微管聚合或解聚的处理后，可以并行分析皮质流动和微管水平。皮质血流速度与微管水平呈负相关，这种影响是由微管本身而不是微管动力学或游离管蛋白介导的。微管水平与肌球蛋白II的功能形式肌球蛋白II细丝水平呈负相关。初步结果已将微管抑制皮质血流的可能机制缩小为三种：1，基于运输的机制，其中微管马达将调节肌球蛋白II生物化学的因子输送到皮质或从皮质传出；2，基于竞争的机制，其中微管与肌球蛋白竞争皮质中的结合部位；或3，基于微管相关蛋白(MAP)的机制，其中微管结合并隔离促进皮质流动和肌球蛋白II细丝形成的因子。我们将使用显微镜、生化和分子相结合的方法来区分这三种假说。这将在很大程度上依赖于完整的非洲爪哇卵母细胞的属性和使用卵母细胞裂解物的可能方法。这个模型系统允许进行在当前研究的大多数其他皮质血流模型中基本上不可能进行的分析。这项工作将为许多不同的细胞和发育现象提供适用的见解，包括细胞质分裂、细胞运动和胚胎发育过程中发育信息的传输。***