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. ***
