Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall A
Paola M. Miranda - University of Puerto Rico-Cayey
Co-Author(s): José Cruz-Arzón, University of Wisconsin-Madison, Madison, WI; Alex Murphy, University of Wisconsin-Madison, Madison, WI; Seth Blair, University of Wisconsin-Madison, Madison, WI.
In Drosophila melanogaster, the protocadherins Fat (Ft) and Dachsous (Ds) interact to regulate organ growth. The absence of either Ft or Ds causes overgrowth in wing imaginal discs, precursors of the adult wing, however, the ft- phenotype is substantially stronger than the ds- phenotype. Because of the interplay between these proteins and the subtle nature of the ds- phenotype, most studies have focused on Ft, with the assumption that the highly similar Ds works in an analogous fashion. Ft’s ability to control growth lies in its negative regulation of the growth-promoting myosin, Dachs (D). Phosphorylation of Ft’s intracellular domain (ICD) by the kinase, Discs overgrown (Dco), antagonizes D accumulation in the subapical cortex, suppressing its ability to enhance growth. Conversely, the palmitoyltransferase, Approximated (App), promotes growth by inhibiting Ft’s ability to interact with D and by working through the D binding partner, Dlish, to tether the protein complex to the subapical membrane. Similarly, D and Dlish have been shown to interact with the Ds-ICD, and we predict that App and Dco are able to interact with Ds, given Ds’s structural and functional similarity to Ft. To gain insight into Ds’s role in the growth control pathway and the biochemical mechanisms through which it operates, we have taken an in vitro approach to identifying Ds binding partners and investigating the sites at which they bind, reducing the factors that complicate such studies in vivo. We infer that these binding sites are specifically for D and Dlish interaction with Ds, and the importance is how the results will bring knowledge on the growth control pathway. We performed a co-immunoprecipitation (Co-IP) to identify which proteins of interest interact with the Ds-ICD and conducted a structure-function analysis to identify the sites at which these proteins bind Ds. We generated targeted deletions within regions of the Ds-ICD likely to be important for protein binding, based on secondary structure predictions and amino acid conservation, and assessed the ability of our proteins of interest to Co-IP with these mutant constructs. We found that neither App nor Dco interact with the Ds-ICD and that, while Dlish and D can interact with Ds, we have yet been unable to definitively identify the regions at which they bind. These data suggest that, contrary to previous assumptions, Ds works through different effectors than Ft to control growth, as it interacts with neither Dco, nor App, and implicates the presence of other effectors of the growth control pathway. Once we successfully identify regions of the Ds-ICD that are necessary for binding our proteins of interest in vitro, we would like to validate their importance by generating the deletions in vivo. Looking at the localization of Ds binding partners in imaginal wing discs containing our deletions will allow us to draw further conclusions about how their biochemical mechanisms function in the growth control pathway.
Funder Acknowledgement(s): WISCIENCE and the NSF REU Biological SIGNALS Summer Research Opportunity Program (NSF Award 1659159). Blair Lab, Department of Integrative Biology, UW-Madison. NIH Grant: AAC-5696.
Faculty Advisor: José Cruz-Arzón, email@example.com
Role: I conducted most of this research. Once I was told about and introduced to my project, with the help and guidance of my mentors, I planned and designed the experiments I was going to conduct. For example, I designed primers, chose the deletions to be made, and decided which proteins to test in addition to making decisions to solve slight complications that would arise. Additionally, I realized all the experiments that needed to be done and analyzed the results with minimal help from my mentors.