In Vivo Analysis of Drosophila Cell Signaling Using Bimolecular Fluorescence Complementation (BiFC)
Date of Award
Campus Access Thesis
Master of Science (MS)
Cell signaling is a fundamental biological process that allows cells to respond to environmental stimuli and communicate with each other by transmitting information from the cell surface to the nucleus through a network of signaling molecules. Dysregulation of signaling pathways is often involved in human diseases including autoimmune diseases, neurodegenerative diseases, and cancer. Identifying protein-protein interactions can help uncover the mechanisms of pathway regulation and inform development of treatment therapies. Here, I studied protein-protein interactions in two different signaling pathways using bimolecular fluorescence complementation (BiFC), an in vivo assay involving complementary fragments of a fluorescent protein that are each tagged to the proteins of interest. If the proteins of interest interact, the fragments are drawn close enough to each other to reform the fluorescent protein that then emits the BiFC signal. I explored how BiFC can be used to study cell signaling pathways in Drosophila by focusing on the known interactions between Capicua (Cic) and extracellular signal regulated kinase (ERK) from the RTK/RAS/MAPK signaling pathway, as well as the interaction between Yorkie (Yki) and Scalloped (Sd) from the Hippo pathway. My results confirmed that BiFC based on the GAL4/UAS expression system was suitable for detecting both the Cic-ERK and Yki-Sd interactions in various fly tissues. To verify the specificity of the BiFC signal, I compared the wild type interactions with the appropriate negative controls that inhibited the interaction between each pair. I also used the BiFC assay to further investigate some unknown aspects about each of these interactions in vivo. I found that the Cic-ERK BiFC signal occurred mostly in the nucleus, suggesting that Cic is not exported to the cytoplasm once it binds to ERK. Additionally, increased ERK pathway activation resulted in a stronger Cic-ERK BiFC signal, showing how the interaction between Cic and ERK is affected by upstream signaling. In the Drosophila ovary, the Yki-Sd BiFC signal appeared in the cytoplasm initially, but strain-induced negative regulation of the Hippo pathway resulted in nuclear signal localization in stretch cells, likely due to Yki activation. Yet Yki was not activated when it was expressed alone; instead, Yki translocated to the nucleus in stretch cells only when co-expressed with Sd. This suggests that Sd may be required for Yki nuclear localization in the absence of Hippo signaling. Overall, my results uncover new properties of Cic-ERK and Yki-Sd interactions in vivo and further emphasize the advantages of using BiFC to study protein-protein interactions that regulate cell signaling pathways.
Jackan, Claire S., "In Vivo Analysis of Drosophila Cell Signaling Using Bimolecular Fluorescence Complementation (BiFC)" (2021). Graduate Masters Theses. 716.
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