Abouward, Reem Ahmed Elsayed; (2024) Investigating the interactomes of LAMP proteins using optogenetic proximity labelling in neurons. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lysosome-associated membrane glycoproteins (LAMPs) are a family of glycosylated proteins distributed not only to the lysosomal membrane but also across a heterogeneous population of endocytic organelles. Due to a lack of in-depth characterisation, it remains unclear whether different LAMPs, specifically the two most abundant LAMP1 and LAMP2, label the same type of organelle. They however continue to be used interchangeably in the field despite reported functional differences, potentially obscuring the interpretation of findings related to the role of lysosome-like organelles in neurodegenerative diseases. In my PhD project, I aimed to characterise and compare the transport dynamics and interactomes of LAMP1- and LAMP2A-positive organelles, using human induced pluripotent stem cell-derived cortical neurons. I found that in axons, retrogradely-moving LAMP1-positive organelles move slower, cover a wider range of motion types in the anterograde direction and pause more frequently in either direction, compared to LAMP2A-positive organelles. Additionally, I captured the interactomes of LAMP1- and LAMP2A-positive organelles using a blue-light activated biotin ligase known as LOV-Turbo. When LOV-Turbo is genetically coupled to a protein-of-interest and expressed in cells, it biotinylates nearby proteins in the presence of blue-light and biotin, capturing endogenous protein interactions. This new proximity labelling method offers increased spatiotemporal control of biotinylation, needed for the unique demands of in vitro neuronal cultures. Using a custom-built blue-light box, I optimised the use of LOV-Turbo in neurons targeting it to LAMP1- and LAMP2A-positive organelles. The comprehensive interactome datasets generated suggest that LAMP2A labels a subset of LAMP1-positive organelles with an early endosome-like protein signature. I also found that both organelles have several synaptic protein interactors, bolstering evidence for a synaptic role for LAMP-positive organelles. Finally, I described the use of proximity labelling in special devices which allow the isolation of large quantities of somatodendritic and axonal compartments, thus permitting the study of compartment-specific interactomes.

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