Fluctuation imaging of LRRK2 reveals that the G2019S mutation alters spatial andmembrane dynamics

Abstract

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are themost common genetic cause of autosomal and sporadic Parkinson's disease (PD). LRRK2 is a largemultidomain kinase that has reported interactionswith severalmembrane proteins, includingRab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclearwhether or howthe spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties andmembrane recruitment ofWT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasmamembrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasmamembrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation-all of which could lead to neurodegeneration associated with PD.