Show simple item record

dc.contributor.authorColussi, Adeline
dc.date.accessioned2018-01-08T09:57:13Z
dc.date.available2018-01-08T09:57:13Z
dc.date.issued2017-11-07
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/270350
dc.description.abstractEukaryotic 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.sponsorshipMRC
dc.language.isoen
dc.subjectmembrane curvature
dc.subjectcurvature sensing
dc.subjectEndophilin
dc.subjectendocytosis
dc.subjectclathrin-independent
dc.subjectBAR
dc.titleUnderstanding Membrane Curvature Sensing
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentMRC Laboratory of Molecular Biology
dc.date.updated2018-01-07T10:32:33Z
dc.rights.generalPermission 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.doi10.17863/CAM.17212
dc.publisher.collegeWolfson
dc.type.qualificationtitlePhD in Biological Science
cam.supervisorMcMahon, Harvey T
rioxxterms.freetoread.startdate2100-01-01


Files in this item

Thumbnail
Thumbnail

This item appears in the following Collection(s)

Show simple item record