Characterisation of LAMP1- and LAMP2A-positive organelles in neurons

LAMP1 and LAMP2A are abundant proteins of late endosomal/lysosomal compartments, which are often used interchangeably to label what is thought to be the same pool of organelles, potentially obscuring their unique physiological roles. Here, we characterised the transport dynamics of LAMP1- and LAMP2A-positive compartments in human iPSC-derived cortical neurons. We found that axonal LAMP1-positive organelles move more slowly in the retrograde direction, pause more frequently, and show a broader velocity distribution in the anterograde direction than LAMP2A-positive vesicles, suggesting they are distinct compartments with differential trafficking behaviour. To explore the molecular mechanism underlying these differences, we characterised with high spatiotemporal precision, the protein interactomes of LAMP1 and LAMP2A-positive compartments through proximity labelling, using full-length LAMP1 or LAMP2A fused to the light-activated biotin ligase LOV-Turbo. We identified and validated the endosomal protein, ZFYVE16, as a novel member of LAMP1 and LAMP2A interactomes. We suggest that LAMP2A-positive organelles represent a subset of LAMP1-positive compartments, which are surprisingly enriched in synaptic vesicle proteins.