Using Light to Control Protein-Protein Interactions: Optogenetics in Drosophila melanogaster
Repository Usage Stats
Recent advancements in genetically encoded light-sensitive protein systems, also known as optogenetic systems, have stemmed from the many benefits of using blue light stimuli to selectively initiate protein-protein interactions. Such benefits include the non-invasive nature of light, the precision of the stimulus, and the reversibility of the protein-protein interactions in the dark. One specific optogenetic system from Arabidopsis thaliana, the CRY2/CIB module, offers a powerful genetically encoded mechanism by which to study the role of proteins in a tissue-specific manner during various stages of development. Using cloning techniques to generate CRY2 and CIB constructs in Drosophila specific vectors, we have attempted to adapt the CRY2/CIB system to Drosophila. We tested an oligomerizing version of the CRY2 component as a tool for the negative regulation of targeted proteins in Drosophila. Although we were unable to repeat the clustering results observed in yeast, we worked on modifying our light activation protocol and discovered the sensitivity of the system to inadvertent light stimulation during preparation for imaging. We also conducted cloning in order to perform a proof-of concept experiment utilizing both cytoplasmically diffuse CRY2 and membrane-anchored CIBN. Thus far, germline transformants of the CIBN component have been generated, and work will continue to generate the CRY2 germline transformants. Additionally, we are also working on cloning variants of the small G protein Rho to form a fusion protein with the CRY2 component. At the plasma membrane, Rho proteins catalyze signaling cascades to affect actin and myosin formation and cytoskeletal changes. If Drosophila Rho1 proteins are successfully adapted to CRY2 components, upon blue light stimulation the recruitment of CRY2 to a CIB component anchored in the membrane could be spatially and temporally controlled to affect subsequent downstream events. The ability to drive Rho1 to the membrane at specific stages of development will generate a better understanding of the effects of altering cytoskeletal function during Drosophila morphogenesis and thereby give insight into wound healing and tissue regeneration processes in vertebrates.
CitationLo, Alexis (2016). Using Light to Control Protein-Protein Interactions: Optogenetics in Drosophila melanogaster. Honors thesis, Duke University. Retrieved from https://hdl.handle.net/10161/11849.
More InfoShow full item record
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.
Rights for Collection: Undergraduate Honors Theses and Student papers