Determining binding specificities of cell adhesion molecules from Drosophila and other related Dipterans
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Date
2018-05
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The Ohio State University
Abstract
Neurons of both vertebrates and invertebrates exhibit a complex set of cell-to-cell interactions during successful development of the nervous system. Cell adhesion molecules (CAMs) play an important role in mediating many of these specific and stereotyped cell-cell interactions. I am investigating the binding specificities of two CAMs from Dipteran insects: Lachesin (Lac) and Amalgam (Ama). Ama arose as a duplication of Lac early in Dipteran evolution, and both proteins still share extensive sequence similarity. In the fruit fly Drosohila melanogaster, Lac is membrane-linked and homophilically binds itself. Ama, which is secreted into the extracellular matrix, has both a homophilic binding property as well as the ability to heterophilically bind another CAM: the transmembrane protein Neurotactin (Nrt). Despite the high level of amino acid sequence similarity between Ama and Lac, the two proteins are unable to bind each other, and Lac does not display any interaction with Nrt. This project is an attempt to identify the precise domain(s) of Lac and Ama that produce these differences in binding specificity. To accomplish this, chimeric constructs of the three immunoglobulin-like domains of Ama and Lac from D. melanogaster have been created and cloned into a vector for regulated expression in Schneider 2 (S2) cell lines. The S2 cells are then to be used for aggregation assays, which will reveal binding patterns of the chimeric proteins. Using this approach, a thorough model can be devised for the specific interactions of Lac, Ama, and Nrt in D. melanogaster. Additionally, I am utilizing bioinformatic databases to locate and subsequently clone out orthologs of Ama and Lac in other Dipteran species. Testing these clones in further aggregation assays will help develop a better understanding of how the unique binding properties of Ama and Lac have changed over evolutionary time.
Description
This research was presented and awarded at the Denman Undergraduate Research Forum and the 2018 Annual Drosophila Research Conference
Keywords
molecular genetics, Drosophila, molecular evolution, neurogenesis