Figure 4. LRRK2 phosphorylation of AP2M1 increases AP2M1 membrane association.

(A and B) Cellular fractionation assays of AP2M1 intensity on membrane and cytosol fractions in LRRK2 KO SH-SY5Y cells transfected with HA-AP2M1-WT or T156A (TA) with or without MYC-LRRK2-GS. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Data are mean ± SEM from n=5 independent experiments. *P < 0.05 by Student’s t tests. (C and D) Cellular fractionation assays of AP2M1 intensity on membrane and cytosol fractions upon LRRK2 inhibitor MLi-2 treatment. Data are mean ± SEM, n=3 independent experiments. *P < 0.05 by Student’s t tests. (E) Liquid nitrogen coverslip freeze–thaw methods assessing AP2M1 membrane association. LRRK2 KO SH-SY5Y cells were transfected with DsRed-LRRK2 WT, GS, or DA and transduced with lentiviruses carrying eGFP-AP2M1 WT or TA. After 48 hours, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol and then fixed and visualized by confocal microscopy. Scale bars, 20 μm. (F) TIRF microscopy assessing AP2M1 membrane association. LRRK2 KO SH-SY5Y cells were cotransfected with DsRed-LRRK2 WT, GS, or DA and eGFP-AP2M1 WT or TA at a plasmid ratio of 5:1. After 48 hours, cells were fixed and imaged by TIRF microscopy. Scale bars, 20 μm. (G and H) Quantification of AP2M1 intensity on the membrane by the methods described and results represented in panels (E) and (F), respectively. Images were quantified by ImageJ software. Data are mean ± SEM from three independent experiments, with the means from the n ≥ 10 cells quantified in each experiment (data file S1) shown in the triangles. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by one-way ANOVA followed by a Tukey’s post hoc test.