Noncanonical regulatory mechanisms in cell biology

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Project summary: This project expands on decades of success elucidating the genetic and molecular underpinnings of intercellular communication and cytoskeletal remodeling in gamete development in females and males. We propose research in two interesting areas: 1) the inhibition of cellular abscission during cytokinesis of dividing germline cells, and 2) the molecular regulation and functional significance of highly efficient tissue-specific stop codon readthrough during protein translation. Proliferating germline cells in animal ovaries and testes characteristically fail to complete abscission, leading to cell clusters that remain connected by intercellular bridges, called ring canals. Incomplete cytokinesis also occurs in several lymphoproliferative disorders, highlighting the importance of understanding how this noncanonical endpoint to mitosis is controlled. Using live imaging of germline mitosis in the Drosophila testis, we discovered a previously unknown intermediate step in ring canal formation involving a midbody-like structure that remodels into a channel between daughter cells. To learn how this maturation step occurs and how the molecular machinery that drives abscission is inhibited from localizing to midbodies, we will use localized biotinylation to identify midbody components and probe the genetic requirement for proteins known to function during cytokinesis. We will also use localized biotinlylation to identify ring canal proteins at the plasma membrane-cytoskeleton interface. Our interest in stop codon readthrough stems from our extensive analysis of the kelch gene and its function during oogenesis. The kelch mRNA encodes a large open reading frame (ORF) punctuated by a single stop codon. In ovaries, translation terminates at the stop to produce a ring canal protein and there is no apparent function for the second ORF. In contrast, we have observed remarkably high efficiency stop codon readthrough in nerves of the central nervous system (CNS) of larvae and adults, producing abundant ORF1+ORF2 protein. Furthermore, kelch and several other genes in Drosophila display efficient stop codon readthrough specifically in the CNS, suggesting the presence of many proteins with carboxy-terminal extensions of unknown function. We will systematically analyze the scope and scale of stop codon readthrough using ribosome profiling of neuronal tissue and mass spectrometry of total protein lysates to identify readthrough peptides. To define the mechanism of readthrough, we will use genetic screens to investigate both stimulatory cis-acting sequences flanking stop codons in readthrough genes and trans-acting factors. Finally, to understand the function of neuronal stop codon readthrough, we will use gene editing to ablate ORF2 from kelch and several other genes and carefully analyze phenotypes using cell biological and behavioral assays. This research program will lead to discoveries concerning the fundamental cell biological and genetic mechanisms that regulate important noncanonical biological phenomena – an alternate ending to cytokinesis that produces syncytia of animal germline cells and ribosomal readthrough of stop codons that expands the neuronal proteome.