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dc.contributor.authorWard, Devin
dc.date.accessioned2019-09-12T18:35:14Z
dc.date.available2019-09-12T18:35:14Z
dc.identifier.urihttp://hdl.handle.net/10464/14504
dc.description.abstractThe regulated transport of materials in cells is an essential function of all living organisms. In eukaryotes, one main family of transport regulators is the Rab GTPases. Rab GTPases utilize GTP to move materials throughout the cell by binding to the membrane of vesicles or endosomes, and trafficking distinct, membrane-associated components throughout the cell. One member of this large family of proteins is Rab11. Rab11 is responsible for endosome recycling: returning membrane proteins and receptors from intracellular recycling endosomes to the cell membrane, where these membrane proteins and receptors may be reused. Although the exact mechanism of Rab11 trafficking is not known, Rab11 appears to be critical for the development and survival of many organisms. Drosophila mutants for the Rab11 gene are not viable, where lethality manifests during embryonic development. This early lethality has imposed significant limitations on elucidating the immediate effects of Rab11 inhibition. Thus, the goal was to engineer a novel method of inhibiting Rab11 in vivo in Drosophila melanogaster. Specifically, the goal was to generate a genomically non-invasive construct (Opto-Nanobody) utilizing an optogenetic, light-sensitive Cryptochrome 2 (Cry2) fused to YFP-targeting nanobodies to bind functional, endogenous, YFP-tagged Rab11. This system promises to provide precise light-responsive spatio-temporal control of Rab11 function in response to blue-light exposure through homo-oligomeric clustering, which has been shown to inhibit Rab-dependent trafficking. Using the Drosophila embryo as a model system, these tools were applied to directly determine the effects of Rab11 inhibition on Notch signaling, and to determine the mechanisms that govern Rab11 trafficking. The Opto-Nanobody was tested in vitro in S2 cells, and was shown to form homo-oligomeric clusters in the presence of blue light and demonstrated the ability to bind to YFP-Rab11. This Opto-Nanobody construct has been inserted into a D. melanogaster injection vector, so that the Opto-Nanobody may be inserted into the D. melanogaster genome, and used to control YFP::Rab11 activity in vivo to elucidate the role of Rab11 in Notch signalling.en_US
dc.language.isoengen_US
dc.publisherBrock Universityen_US
dc.subjectRab11en_US
dc.subjectNotchen_US
dc.subjectOptogeneticsen_US
dc.subjectNanobodiesen_US
dc.titleEngineering Optogenetic Control of Endocytic Recycling: Controlling Rab11 Function in Drosophila melanogaster using Engineered Light-Responsive Nanobodiesen_US
dc.typeElectronic Thesis or Dissertationen_US
dc.degree.nameM.Sc. Biological Sciencesen_US
dc.degree.levelMastersen_US
dc.contributor.departmentDepartment of Biological Sciencesen_US
dc.degree.disciplineFaculty of Mathematics and Scienceen_US
refterms.dateFOA2021-08-18T01:44:09Z


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