Functional and Molecular Regulation of Actomyosin by Microtubules

小行星9630860 定向皮质运动是一个广泛的生物学现象，包括胞质分裂，细胞迁移和早期胚胎发育信息的运动的基本特征。 皮质流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丝、细胞表面蛋白以及在某些情况下皮质细胞器朝向特定位点的运动。 众所周知，皮质流是依赖于肌动蛋白的，但什么调节流量的大小和方向是未知的。 微管被假设以某种方式调节皮质流，但它们是刺激还是抑制流量是有争议的，主要是因为在大多数系统中研究皮质流所带来的技术困难。 卵母细胞的青蛙，非洲爪蟾，已被开发为皮质流的模型。 用佛波醇酯PMA处理的非洲爪蟾卵母细胞经历快速、同步的皮质流，这很容易量化。 这种皮质流表现出其他系统中皮质流的所有典型特征，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细胞表面蛋白自由运动的要求。 该系统的功能允许并行分析皮质流和微管水平的治疗，促进聚合或解聚的微管。 皮质流率与微管水平呈负相关，这种效应是由微管本身介导的，而不是由微管动力学或游离微管蛋白介导的。 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。 初步结果已经将微管对皮质流动发挥抑制作用的可能机制缩小到三种：1，基于运输的机制，其中微管马达将调节肌球蛋白-II生物化学的因子运输到皮质或从皮质运输; 2，基于竞争的机制，其中微管与肌动球蛋白竞争皮质中的结合位点;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，生物化学和分子的方法将被用来区分这三种假设。 这些将严重依赖于完整的非洲爪蟾卵母细胞的属性和可能使用卵母细胞裂解物的方法。 这个模型系统允许分析，基本上是不可能的，在大多数其他模型的皮质流，目前正在研究。 这项工作将提供适用于一些不同的细胞和发育现象，包括胞质分裂，细胞运动，并在胚胎发育过程中的发育信息的运输的见解。 ***

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. ***