ORP1L regulates dynein clustering on endolysosmal membranes in response to cholesterol levels

Abstract

The sub-cellular positioning of endolysosomes is crucial for regulating their function. Particularly, the positioning of endolysosomes between the cell periphery versus the peri-nuclear region impacts autophagy, mTOR (mechanistic target of rapamycin) signaling and other processes. The mechanisms that regulate the positioning of endolysosomes at these two locations are still being uncovered. Here, using super-resolution microscopy, we show that the retrograde motor dynein forms nano-clusters on endolysosomal membranes containing 1-2 dyneins, with an average of ~3 nanoclusters per endolysosome. These data suggest that a very small number of dynein motors (1-6) drive endolysosome motility. Surprisingly, dynein nano-clusters are slightly larger on peripheral endolysosomes having higher cholesterol levels compared to peri-nuclear ones. By perturbing endolysosomal membrane cholesterol levels, we show that dynein copy number within nano-clusters is influenced by the amount of endolysosomal cholesterol while the total number of nano-clusters per endolysosome is independent of cholesterol. Finally, we show that the dynein adapter protein ORP1L (Oxysterol Binding Protein Homologue) regulates the number of dynein motors within nano-clusters in response to cholesterol levels. We propose a new model by which endolysosomal transport and positioning is influenced by the cholesterol sensing adapter protein ORP1L, which influences dyneins copy number within nano-clusters.