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Title: The optimisation of correlative light electron microscopy techniques and their application in the study of endocytic sorting events
Author: Brown , Edward Lloyd
ISNI:       0000 0004 2739 8119
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
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The endosomal network comprises a series of compartments which co- ordinate sorting and trafficking of internalised cargo. The early endosome sorts internalised cargo by two distinct trafficking pathways, depending the ultimate fate of the cargo. Cargo is either recycled back to the plasma membrane or degraded by lysosomes. Given the ability of the light microscope (LM) to observe living cells, much of our understanding of the dynamic nature of cargo sorting in the endosomal network has come from observations from the LM. However, in order to fully appreciate the localisation of cargo in the context of its membranous environment requires the resolving power of the electron microscope (EM). The examination of the same area of a biological specimen by both LM and EM can provide more information than the application of either technique alone. In particular correlative light electron microscopy (CLEM) combined with high pressure freezing (HPF) allows observation of a live cellular event to be correlated with a high-resolution snapshot of said event, preserved in a near native state. The current system for performing CLEM with HPF as the method of fixation limits the capabilities of live cell imaging. In this thesis I describe modifications to the system that have increased the achievable resolution and sensitivity of fluorescent live cell imaging and also improved the method of cellular relocation. To observe endocytic sorting using CLEM required two novel functionalised ligand probes to represent the differentially sorted cargos epidermal growth factor receptor and transferrin receptor. The validity of these ligand- fluorophore-gold conjugates, with respect to detection by both fluorescence and electron microscopy and their ability to represent the native ligands has been shown here. With the validity of the probes established, the new system was then employed to illustrate how CL EM combined with electron tomography can provide new insights into the sorting events occurring in early endocytic compartments. As a result of this investigation however, it was found that disruption of cells during HPF is a major issue with this system of CLEM that still needs to be resolved. As an additional investigation into endocytic sorting, I have studied the potential role of sorting nexin 15 (SNXI5) in receptor trafficking. This research has contributed to the characterisation of the SNXI5, whilst also providing mechanistic details of SNX15s role in regulation of receptor degradation. SNX 15 associates with clathrin during clathrin-mediated endocytosis and remains bound to the fully formed vesicles. The characteristic phosphoinositide-binding domain possessed by all SNX proteins means that SNX15 is also present on early endosomes. The association of SNX15 with early endocytic compartments potentially facilitates their directional movement via microtubules away from the periphery of the cell allowing compartment maturation and lysosomal fusion to occur.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available