LAMP1 and LAMP2A localise to axonal organelles with distinct motility dynamics and partially overlapping molecular signatures in human neurons

LAMP1 and LAMP2A (an isoform of LAMP2) are abundant proteins of late endosomal/lysosomal compartments that are often used interchangeably to label what is assumed to be the same organelle population, potentially obscuring distinct physiological roles. Here, we characterised the axonal transport dynamics of LAMP1- and LAMP2A-positive compartments in human induced pluripotent stem cell (hiPSC)-derived cortical neurons. We found that LAMP1-positive organelles move slower in the retrograde direction, pause more frequently, and display a broader anterograde velocity distribution than LAMP2A-positive vesicles, indicating distinct trafficking behaviours. Co-transport analysis revealed that ∼65% of motile LAMP1-positive organelles carry LAMP2A, and vice versa, with higher co-transport in the retrograde direction. To explore molecular differences underlying these behaviours, we performed proximity labelling using full-length LAMP1 or LAMP2A fused to the light-activated biotin ligase LOV-Turbo. This approach revealed largely overlapping interactomes, with LAMP2A-associated proteins forming a subset of the LAMP1 interactome and showing an enrichment for synaptic vesicle-related proteins. We further validated ZFYVE16 as a novel interactor of both compartments. Together, our findings indicate that LAMP1- and LAMP2A-positive organelles share overlapping molecular identities but represent functionally distinct axonal populations with divergent transport dynamics.