dc.contributor.author Colussi, Adeline dc.date.accessioned 2018-01-08T09:57:13Z dc.date.available 2018-01-08T09:57:13Z dc.date.issued 2017-11-07 dc.identifier.uri https://www.repository.cam.ac.uk/handle/1810/270350 dc.description.abstract Eukaryotic cells are characterised by membranes with varied and dynamic compositions and shapes. Consequently, membrane-binding proteins are tuned to recognise and modify these membrane states to perform their functions. To study the curvature sensitivity of proteins, I have developed a single-particle assay using NanoSight technology that tracks the Brownian motion of particles to measure their size. I optimised this system to track fluorescently labelled lipid-binding domains bound to liposomes of different sizes moving freely in solution. The comparison of the size distribution of the total liposomes with the fluorescently labelled population allowed me to determine their curvature preferences. To validate the method I tested proteins from the Bin/Amphiphysin/Rvs (BAR) superfamily, which are inherently curved and have known curvature preferences. My method was capable of recapitulating the behaviour of BAR domains with different curvature preferences. I then expanded the range of targets and showed that this assay is also capable of detecting curvature preferences for a variety of other lipid-binding domain families. As such, I identified AKT PH domain as a new curvature-sensing domain. Finally, using the ENTH domain of Epsin1 that causes vesicle budding, I demonstrated that this method can also be used to study membrane remodelling. Trafficking involves generation and sensing of membrane curvature combined with recognition of specific cargo. Endophilin consists of a curvature-sensitive BAR domain followed by an SH3 (Src-homology 3) domain and has recently been identified in a clathrin-independent endocytosis pathway, FEME (fast endophilin-mediated endocytosis), involved in the uptake of cell surface receptors. Endophilin recognises ligands via its SH3 domain, binding G-protein coupled receptors (GPCRs) directly in their intracellular loop 3 and receptor tyrosine kinase (RTKs) via adaptor proteins. However, a specific recognition motif has not been identified yet. Here, using a combination of biophysical approaches and NMR spectroscopy, I characterised the Endophilin binding motif of ALIX (ALG-2-interacting protein X) adaptor protein and of the GPCR $\alpha$2A adrenergic receptor. Comparison of SH3-peptide models resulted in a putative Endophilin recognition site. dc.description.sponsorship MRC dc.language.iso en dc.subject membrane curvature dc.subject curvature sensing dc.subject Endophilin dc.subject endocytosis dc.subject clathrin-independent dc.subject BAR dc.title Understanding Membrane Curvature Sensing dc.type Thesis dc.type.qualificationlevel Doctoral dc.type.qualificationname Doctor of Philosophy (PhD) dc.publisher.institution University of Cambridge dc.publisher.department MRC Laboratory of Molecular Biology dc.date.updated 2018-01-07T10:32:33Z dc.rights.general Permission to use third-party copyrighted content has still not been granted from one copyright holder. Therefore, two versions of the thesis have been uploaded, one with the complete content, including for which permission is still lacking (Thesis_corr_unredacted.pdf) as well as a version where this content has been removed (Thesis_corr_redacted.pdf). Currently, only this redacted version does not infringe third-party intellectual property rights. dc.identifier.doi 10.17863/CAM.17212 dc.publisher.college Wolfson dc.type.qualificationtitle PhD in Biological Science cam.supervisor McMahon, Harvey T rioxxterms.freetoread.startdate 2100-01-01
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