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Title: High speed imaging of nanoscopic organisation and dynamics in the cell membrane
Author: de Wit, Gabrielle
ISNI:       0000 0004 6495 9771
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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The ability of the membrane to dynamically organise its lipid and protein constituents is vital to the function of the cell. How this organisation is controlled on the molecular level, however, is poorly understood, in large part due to the two-fold diffculty of observing entities that are both nanoscopic and in rapid motion. In this thesis, we use interferometric scattering microscopy (iSCAT) for its unique combination of sensitivity, spatial precision and temporal resolution in order to investigate longstanding questions about the physical origins of anomalous diffusion and dynamic phase separation in cell and model bilayer membranes. Chapters 1 introduces the state of current knowledge in the field of membrane organisation and describes the model frameworks used to interpret and understand experimental observations. Chapter 2 provides an outline of the development of optical microscopy techniques towards the goals of single molecule tracking and label-free imaging of nanoscopic objects in cell and model membranes, particularly focusing on scattering detection. Chapter 3 describes the design principles and construction of the iSCAT microscope and characterises the optomechanical stability and localisation accuracy achieved in this work. In Chapter 4, we demonstrate the unprecedented ability to image single nanoscopic domains in motion, by exploiting the sensitivity of iSCAT to intrinsic differences in refractive index between lateral bilayer phases. We directly prove the existence of lipid bilayer mixtures that dynamically form nanoscopic domains, and quantify the sizes and lifetimes of these lipid assemblies for the first time. In Chapter 5 we study how a single lipid, ganglioside GM1, diffuses within the bilayer and interacts with its environment, in order to understand the mechanistic origins of heterogeneous mobility in a simple model membrane system. With iSCAT microscopy we achieve 2 nm spatial precision, and 20 μs temporal resolution, allowing for the facile identification of transient trapping events within sub-10 nm domains. By varying properties of the substrate and the lipid head and tail groups, we identify the factors necessary for the induction of anomalous diffusion and conclude that transbilayer interactions with immobilised lipids in the opposite leaflet explain the observed trapping events. This chapter also examines the effect of the size of the gold nanoparticle label on the measured trajectories, addressing a major concern in single particle tracking studies. In Chapter 6 we investigate the effect of the actin cytoskeleton on the mobility and spatial distribution of membrane proteins in hippocampal neurons. The periodic arrangement of sub-membrane actin rings found uniquely in this particular cell type permits the trajectories of single molecules to be spatially correlated with the cytoskeleton for the first time. Using both high speed tracking with 40 nm gold labels and fluorescent single molecule tracking at lower speeds, we observe periodicity in the localisation probability that coincides with the arrangement of actin. Our results suggest that the actin cytoskeleton controls the diffusion of membrane proteins not via its assumed role as a highly impermeable barrier, but by locally slowing diffusion via protein crowding effects.
Supervisor: Kukura, Philipp Sponsor: Clarendon Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available