Quantification of actin nanoarchitecture at the T cell immunological synapse using single molecule localisation microscopy

Student thesis: Doctoral ThesisDoctor of Philosophy


Much of our knowledge regarding cellular structure and function has derived from our ability to visualise distinct processes through fluorescence microscopy. The unparalleled combination of specificity and compatibility with live samples renders the fluorescent microscope an invaluable imaging modality across the life sciences. Owing to the diffraction of light, the resolution of an optical system is inherently limited. For centuries, a fundamental, immutable resolution was thus imposed on the fluorescence microscope and the study of cellular features existing beyond this limit was unfathomable. Circumvention of the diffraction barrier however was achieved experimentally in 2006, and the advent of super resolution microscopy, for whose discovery was awarded the 2014 Nobel Prize in Chemistry, represented a paradigm shift in our apperception of the resolution issue in microscopy. A repertoire of super resolution microscopy methods have since emerged, allowing unprecedented access to the cellular architecture on the nanoscale. The focus of this Thesis is the implementation of single molecule localisation microscopy (SMLM) for the study of sub-cellular fibrous organisation, which routinely achieves ~ 30 nm spatial resolution.
Concurrent with the remarkable technological advances in the field of SMLM is the need for complimentary data analysis methods, to help tune SMLM into a quantitative imaging tool. Despite variations in the working principles employed to achieve SMLM images, all modalities share a common data output: a spatial point pattern (SPP). One must therefore embrace new strategies for the study of SMLM images, compared to conventional fluorescence microscopy methods; a technical challenge. This challenge is exacerbated when studying filamentous structures, whose topologies and cytoarchitectures are typically complex. This Thesis aims to deliver analysis methods for the study of fibrous SPPs generated by SMLM, to compliment the varied tools currently available for clustered SPPs.
The presented analysis methods will be validated by use of simulated data and applied to the study of the actin cytoskeleton at the T cell synapse. The actin cytoskeleton is responsible for maintaining a wide range of cellular behaviours, and its malfunction has been implicated in various human diseases. Filamentous actin is subject to extensive remodelling to execute diverse cellular tasks, mediated by a catalogue of accessory proteins. Besides its structural role in maintaining cell morphology, the functional role of actin in regulating cell signals is a current topic of great interest. This Thesis primarily investigates the nanoarchitecture of the actin cytoskeleton at the T cell immunological synapse via SMLM. The effect of the actin crosslinking protein α-actinin on the nanoscale organisation of actin at the mature immunological synapse, and its role in the clustering of the linker for activation of T cells (LAT), a prominent signalling molecule involved in T cell activation, will be investigated.
Date of Award2019
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorDylan Owen (Supervisor) & Chris Lorenz (Supervisor)

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