Endoplasmic reticulum junctions serve as a platform for endosome-lysosome interactions through their stop-and-go motion switching.

Endosomes and lysosomes (collectively termed "endolysosomes") traverse the cytoplasm in a stop-and-go manner, but the mechanisms underlying this motion remain poorly understood. Using deep learning-based image analyses, including particle tracking, spatial distribution, and endoplasmic reticulum (ER) morphology analysis, we found that ER junctions facilitate stop-and-go motion switching and serve as platforms for endolysosome interactions. Within the ER network, endolysosomes exhibit three dynamic states: fast movement, local slow movement, and pausing. Pauses occur mainly at ER junctions, where transient endosome-lysosome interactions often coincide with organelle fission and are followed by departure. Disruption of ER junctions impairs lysosomal motility and maturation. We further show that actin condensation around endolysosomes mediates motion switching, involving VAP-STARD3 interaction and the actin regulator YWHAH. Other organelles, such as lipid droplets and peroxisomes, also pause near ER junctions. These findings highlight ER junctions as regulatory hubs that orchestrate organelle dynamics, contributing to the spatial coordination of organelle distribution and interactions within the cytoplasm.