Functional and Molecular Regulation of Actomyosin by Microtubules

9630860 Bement定向皮质运动是多种生物现象的基本特征，包括细胞分裂、细胞迁移和早期胚胎发育信息的运动。皮层流动是实现定向皮层运动的最常见机制之一。皮层流动表现为肌动蛋白丝、细胞表面蛋白，在某些情况下，皮层细胞器向特定部位移动。众所周知，皮层流动依赖于肌动蛋白丝，但是什么调节了流动的大小和方向尚不清楚。微管被假设以某种方式调节皮层流动，但它们是否刺激或抑制流动是有争议的，主要是因为在大多数系统中研究皮层流动所带来的技术困难。非洲爪蟾（Xenopus laevis）的卵母细胞已被开发为皮质流动的模型。经磷酯（PMA）处理的爪蟾卵母细胞发生快速、同步的皮质流动，且易于量化。这种皮质流动表现出其他系统中皮质流动的所有典型特征，包括对肌动球蛋白的依赖，皮质细胞器和丝状肌动蛋白的定向运动，以及对细胞表面蛋白自由运动的要求。该系统的特点允许在促进微管聚合或解聚的处理后并行分析皮质流和微管水平。皮层血流速率与微管水平呈负相关，这种影响是由微管本身介导的，而不是由微管动力学或自由微管蛋白介导的。微管水平与肌球蛋白-II细丝（肌球蛋白-II的功能形式）水平呈负相关。初步结果将微管抑制皮层血流的可能机制缩小为三种：1，一种基于运输的机制，其中微管运动将调节肌球蛋白- ii生化的因子运输到皮层或从皮层运输；2，一种基于竞争的机制，其中微管与肌动球蛋白竞争皮层中的结合位点；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。结合微观，生化和分子的方法将被用来区分这三种假设。这些将在很大程度上依赖于完整爪蟾卵母细胞的属性和使用卵母细胞裂解物的可能方法。该模型系统允许分析在目前研究的大多数其他皮质流模型中基本上是不可能的。这项工作将为许多不同的细胞和发育现象提供见解，包括细胞分裂、细胞运动和胚胎发生期间发育信息的运输。＊＊＊

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. ***