Collaboration between actin nucleators - Spire and Cappuccino

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Project Summary/Abstract Cell polarity is essential to an array of processes, including transport of nutrients across epithelial layers, chemotaxis, and development. Thus, defects in polarization can have dire physiological consequences. The overarching goal of this proposal is to understand the role(s) of actin in polarity establishment in the developing egg. Cytoplasmic actin meshes are observed in oocytes of mouse, Drosophila, starfish, and C. elegans, which suggests meshes are a conserved feature that have diverged functionally, in some cases. The Drosophila oocyte is an ideal system in which to study the actin mesh because the ovaries are large, facilitating cell biological and biochemical examination in a genetically tractable model organism. The presence of the actin mesh and its correctly timed removal are critical to polarity establishment. Little is known about the mesh makeup beyond the two actin nucleators required to build it, Spire and Cappuccino, and a molecular motor that colocalizes with Spire on vesicles, myosin V. Our knowledge about the mesh is limited by two technical challenges: 1) several stages of oogenesis do not take place ex vivo and 2) many actin-binding proteins are essential to early oogenesis, diminishing the power of classical genetics to discover mesh components necessary during mid-oogenesis. We are developing an intra vital imaging platform to address both issues. Intra vital imaging will facilitate long-term, direct observation of the mesh and its disappearance within the animal. When combined with Crispr/Cas9- mediated gene editing and auxin-inducible-degradation, we will be able to observe changes in the mesh upon removal of specific proteins in a temporally controlled manner. We will use these tools to test the hypothesis that the mesh slows fluid flows to facilitate formation of a posterior anchoring structure that is necessary for polarity establishment and fails to form when fluid flows are prematurely accelerated, as is the case if the mesh is compromised. A comparable actin mesh, also built by Spire, Cappuccino, and myosin V, was recently discovered in a somatic cell, the dendritic melanocyte. Interestingly, in the mouse oocyte, the mesh moves vesicles towards each other and the periphery of the cell, whereas, in the melanocyte, the mesh moves vesicles apart, dispersing them throughout the cell. By combining in vivo experiments with in vitro reconstitution of the mesh, we will determine how the same set of proteins can drive vesicles in opposite directions in mouse oocytes and melanocytes and determine how the mesh is built in the Drosophila oocyte. Finally, we will examine the consequences of the recently-discovered interaction between Spir and myosin V, using complementary biochemical and genetic approaches. Coordination between an actin nucleator and an actin-based motor has exciting implications for how the mesh and other structures are built. Upon completion of this work, we will have established how the mesh, a structure essential to cell polarity, is built, identified previously unknown mesh components, and determined whether slow streaming is necessary for posterior anchoring. Now that we know that actin meshes are not exclusive to oocytes, the implications of our findings are even broader.