The role of canonical and non-canonical regulators of heterotrimeric G protein signaling during Drosophila melanogaster morphogenesis

Peters, Kimberly Ann

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March 22, 2019

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Peters, Kimberly Ann
- Affiliation: College of Arts and Sciences, Department of Biology

Abstract

Morphogenesis of multicellular organisms requires precise regulation of cell movements and cell shape changes. The first morphogenetic movement to occur in Drosophila melanogaster embryogenesis is ventral furrow formation during gastrulation; wherein a subset of presumptive mesodermal cells undergoes cytoskeletal rearrangements to invaginate into the embryo. This process is regulated by a secreted ligand, Folded gastrulation (Fog), that binds a G-protein-coupled receptor (GPCR), which activates Concertina (Cta), a Gɑ12/13 protein, and triggers a signaling cascade resulting in contraction of the apical actomyosin network to drive cell shape changes leading to internalization of the ventral furrow. We used an RNAi screen in Drosophila tissue culture cells targeting all known and putative GPCRs to identify a receptor required for Fog signaling which we named Mist (Mesoderm Invagination Signal Transducer). We determined that Mist is an essential component of the Fog signaling pathway, and is sufficient to mediate Fog susceptibility in otherwise Fog unresponsive cells. I further identified specific domains within the receptor involved in signal transduction. Mist loss-of-function in the Drosophila melanogaster embryo revealed a role for Mist in gastrulation, and exhibited defects similar to Fog mutants. Ultimately our examination of Mist function within cellular morphogenesis has defined a role for Mist from its transcriptional regulation, to its function within developmental processes. I also investigated the role for Ric-8, a conserved cytoplasmic protein, in Cta function. In Drosophila tissue culture I used RNAi to show that Ric-8 is necessary for Cta-induced cellular constriction triggered by ectopic Fog application. Biochemical analyses and molecular mis-targeting demonstrated that Ric-8 directly binds to- and localizes Cta, with a much higher affinity for constitutively inactive Cta. Further, I found that Ric-8 modulation directly impacts productive Fog signaling. Finally, by mutagenizing amino acids conserved across species I identified specific residues within Ric-8 required for Cta function and/or establishing a binding interface between the two molecules. These two projects converge to further our understanding of the regulation of Gα signaling during gastrulation events by two distinct mechanisms, canonical GPCR activation via Mist signaling and non-canonical modulation of Cta, by the highly conserved cytosolic protein Ric-8.

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