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Exploring Polarized Membrane Protein Distribution in Drosophila Follicle Cells using Proximity Labelling https://doi.org/10.17863/CAM.125189 Sen, Samarpita St Johnston, Daniel The establishment and maintenance of apical–basal polarity in epithelial cells depend on the faithful sorting and delivery of proteins to distinct plasma membrane domains. Using the Drosophila follicular epithelium as my model, this thesis applies peroxidase-mediated proximity labelling and mass spectrometry to systematically characterize the polarized membrane proteome. I first demonstrate that peroxidase-catalyzed labelling with biotin-phenol derivatives is a diffusion-controlled process: excess phenoxy radicals diffuse from their site of production to places where they can find more reactive sites, such as for example the electron-rich ECM at the basal side of follicle cells, generating artefactual basal enrichment. By titrating biotin-label concentration and minimizing labelling duration, I optimized labelling conditions to restore the expected proximal biotinylation signals. Using HRP-tagged Cadherin99c, Fasciclin 3, and Nidogen as apical, lateral, and basal baits respectively, I identified several membrane-domain specific transmembrane hits through mass spectrometry. Toll7 and Arrow were two interesting candidates that showed a basal staining in the follicle cells. To investigate the uncharacterized basal trafficking route in follicle cells, I used the RUSH system to track real-time trafficking of these two basal transmembrane cargoes. Interestingly, even though endogenous Arrow and Toll7 exhibited basal/basolateral localization, overexpression of these two cargoes resulted in mis-routing of the majority of the protein to the apical/apicolateral route. The basal surface view however showed some basal staining for both proteins, indicating that overexpression of endogenous basal cargoes might saturate the basal trafficking route and consequently shift the excess to the default apical/apicolateral route in the follicular epithelium. GP64, a viral envelope protein localizes basally/basolaterally when ectopically expressed in the follicle cells. However, tagging it for RUSH result in its mislocalization as well as misfolding of the cargo causing it to become trapped in the ER. Subsequent work will involve optimizing ER hook-binding tags and fluorescent labels for GP64 to enable successful RUSH, in order to finally elucidate the basal trafficking pathway in the Drosophila follicle cells. Collectively, this work advances proximity labelling as a tool for in vivo proteomic mapping in epithelial tissues, resolves key technical limitations of the method, and provides indications of a possibly new membrane trafficking route in the Drosophila follicular epithelium. University of Cambridge 2025-09-27 Thesis Doctoral Doctor of Philosophy (PhD) eng Gates Cambridge Scholarship https://www.repository.cam.ac.uk/handle/1810/395792 https://www.repository.cam.ac.uk/bitstreams/ff153fe5-6ee2-44af-814e-396b485d9549/download b8a58da5495bcd9986180b678944f36d https://www.repository.cam.ac.uk/bitstreams/f6514c0b-52eb-44ee-afd3-a0826b78976c/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved follicle cells live-cell imaging proteome mapping proximity labelling trafficking