Late Endosomal Membrane-Lipid Composition Imparts Change in Oxysterol-Binding Protein-Related Protein 1L's (ORP1L) Nano-Scale Organization Which Effects Organelle Motility

Abstract

Intracellular organelle transport along microtubules (MTs) is highly regulated spatiotemporally and is orchestrated by motor proteins dynein and kinesins. The directionality and uninterrupted long-range motion of the organelles are achieved by multilevel regulatory mechanisms including motor recruitment and motor activation by adapter and accessory proteins. Recently, the motor organization on organelle membranes, in particular motor clustering, has been suggested to play an important role in transport regulation and processes including organelle biogenesis and maturation. While the lipid composition of the organelle membrane, specifically the cholesterol content, has been suggested to play a role in regulating the molecular arrangement or clustered organization of motors, the exact mechanisms are not clear. We hypothesized that clustering/molecular organization of the motor protein dynein is mediated by the nanoscale organization of Oxysterol-Binding Protein-Related Protein 1L (ORP1L), which is the main cholesterol sensing protein present on late endosomal, lysosomal and phagosomal compartments. To determine the dependence of motor and ORP1L nano-organization on cholesterol levels, we have been using Stochastic Optical Reconstruction Microscopy (STORM). We found that motor proteins like dynein and ORP1L indeed form nano-clusters on late endosomal membranes. By coupling super-resolution imaging with drug treatments that modulate the cholesterol content of organelle membranes, we observed ORP1L nano-organization is sensitive to cholesterol content of the late endosomal membrane. Using quantitative analysis, we showed that ORP1L is more clustered on late endosomal membranes under high cholesterol conditions compared to low cholesterol conditions. Further, high cholesterol led to a redistribution and repositioning of late endosomes at the peri-nuclear region, suggesting that dynein activity and dynein mediated motility are sensitive to cholesterol levels. Overall using high resolution microscopy we are dissecting the link between organelle membrane nano-organization and organelle motility/positioning.