Molecular mechanisms underlying the establishment of cell polarity.

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Project Summary (parent grant): R35GM145340 A fundamental feature of eukaryotic life is the establishment and maintenance of cellular polarity. Molecular motors help to establish polarity by transporting mRNAs, proteins, vesicles, and organelles to specific sites within the cell. A variety of organisms from the single-celled yeast to humans use mRNA localization coupled with translational regulation as a way to asymmetrically sort proteins. The prevalence of this phenomenon is best illustrated in developing embryos, neurons and epithelial, in which thousands of mRNAs are spatially localized. When this process is compromised, it can result in developmental and neurological disorders. Despite the importance of this topic and the prevalence of this phenomenon, we lack a mechanistic understanding of mRNA localization. A critical gap in our understanding pertains to how mRNAs destined for localization are recognized by the cell and distinguished from non- localizing mRNAs. In addition to sequences present within localizing mRNAs, the proteins that bind these mRNAs are key to their cellular fate. This complex of proteins is responsible for linking localizing mRNAs with molecular motors and for regulating their translation. However, identifying these critical proteins has proven to be extremely challenging. By its very nature, the process of mRNA localization is highly dynamic. Consequently, the protein-protein and protein- RNA interactions required for assembling localizing messenger ribonucleoprotein (mRNP) particles are weak and transient. This has made their identification using classical biochemical approaches almost impossible; the complex falls apart during the purification step. In this application, we propose novel strategies to address this critical knowledge gap. The model we propose to use for these studies is the well characterized Drosophila melanogaster egg chamber. In Objective 1 of this application, we propose to use proximity biotin ligation to define the core components of transport particles and to examine the conservation of these factors between flies and mammals. In Objective 2, we propose to test the hypothesis that granular structures referred to as P bodies coordinate the localization of mRNAs with their translational regulation.