Cells have evolved mechanisms to establish spatial order of their constituent organelles and provide direction for transport of cellular components. This is most apparent in the need for cell polarity and the ability to segregate organelles appropriately during cell division. We have been using the budding yeast to study how the actin cytoskeleton brings about this cellular organization. We have shown that bundles of polarized actin filaments, nucleated by formins and running parallel to the axis of cell division, provide the framework for polarized growth and organelle segregation. We and others have also shown that an essential myosin-V, whose heavy chain is encoded by MYO2, uses these cables to transport secretory vesicles for polarized growth, as well as many organelles, including microtubules for nuclear orientation, the TGN, the vacuole, peroxisomes, etc. for segregation during the cell cycle. In this renewal, we propose to extend our studies on yeast formins, as well as undertake a new project investigating the breadth of functions that formins perform in a simple multicellular organism, the nematode Caenorhabditis elegans. We shall also continue with our studies on Myo2p, the best-understood member of the highly conserved myosin-V family, to investigate how it selects, delivers and releases it cargos. Specifically, we propose to address the following questions: (1) How is the nucleating activity of yeast formins controlled in time and space? We propose to use genetic and biochemical approaches to identify factors that cooperate with the yeast formin Bnilp, and understand in biochemical terms how these function; (2) What is the range of structures that formins perform? The nematode has single representative formins for almost all the classes of formins common to metazoans. We shall initiate a project to study the location, biochemical properties and functions of these different formins; (3) What proteins function with Myo2p, and how does it select between cargos? Genetic and cell biological studies will be expanded to identify factors (including specific phosphoinositides) that function with Myo2p in cargo selection, in its regulation, and in cargo delivery. These studies will contribute to a fundamental understanding of how cellular order is generated. Defects in related proteins in vertebrates are associated with diseases, including mutations in formin DNFA1, resulting in non-syndromic deafness, as well as in myosin-Vs, for example in Griscelli's syndrome. Thus, the proposed studies will provide insight into a basic cell biology question as well as have clinical relevance. ? ? ?
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