Establishing a common function for ferlin proteins in membrane fusion using novel genetic code expansion and single molecule techniques.

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Membrane trafficking is regarded as a crucial and distinctive characteristic of eukaryotic cells, with both intracellular transport and secretion events playing an essential role in homeostasis and signaling. While much of what is known about membrane trafficking comes from studies in yeast, there are important proteins involved in membrane trafficking in multicellular organisms not seen in yeast. One family of these proteins are the ferlins, an evolutionarily ancient family of trafficking proteins linked to an increasingly diverse list of physiological activities, including fertility, the encoding of sound, muscle development, and repair of damaged cell membranes. This diversity of physiological roles has made establishing a common underlying molecular function for this family challenging. This project will address this gap in our knowledge of the Ferlin family proteins, and in so doing add to our understanding of fertility, hearing, and muscle development. The proposed work will also develop undergraduate laboratory research training opportunities and lab classes focused on membrane biology and provide graduate students with state-of-the-art training in interdisciplinary research.The project tests the idea that ferlins share a common function as calcium sensitive scaffolds for regulated exocytosis and endocytosis. Challenges due to the large size of the proteins, their transmembrane domains, and the high number of endogenous cysteines typical of ferlins have precluded many of the traditional recombinant protein assays typically used to test this hypothetical function. To overcome these challenges a novel single molecule protein-protein interaction assay that allows for probing the contacts of large multivalent membrane proteins has been developed. In addition, genetic code expansion techniques will be exploited to incorporate environmentally sensitive fluorescent unnatural amino acids into the ferlins. These technologies will be exploited to determine the functions that underlie ferlin activity. The proposed studies will define a set of underlying molecular-level functions that unite the ferlins, despite their disparate physiological roles. This information will allow integrating ferlins into the larger picture of membrane trafficking and cell signaling. The development of a novel single molecule fluorescence technique will provide a method for the study of large multi-domain membrane proteins and thus have applications beyond the study of ferlins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.