LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 6

Figure 6. LRRK2 dependent phosphorylation stalls Rab10 on macropinosomes.

A,B. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were pre-incubated with FITC-conjugated dextran (70 kDa, green signal) prior to washing, fixing, and immunostaining with total-Rab10 (shown as red signal), or pT73-Rab10 antibody (shown as red signal). Alternatively, cells were co-stained with antibodies to Rab5, Rab7 or Lamp1 (shown as green signal in separate images). Representative photomicrographs (from >30 images analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels.

C. Analysis of 20 cells across three independent experiments are given for each indicated co-localization, where % of Rab10 or pT73-Rab10 vesicles positive with indicated markers was calculated for each cell (with signal inside of vesicles analyzed for TRITC-dextran and DQ-ovalbumin). Each dot represents the mean value of vesicles analyzed inside one cell. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated. Error bars represent ± SEM. Significance was assessed by Mann-Whitney tests for each marker with **** representing p<0.0001 and N.S representing p>0.05.

D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were treated with 0.001% DMSO or 100 nM MLi2 (i.e., IC90 concentration) for two hours prior to immunostaining with total Rab10 or pT73-Rab10 antibody (shown as red signal), and anti-Rab5 or anti-Rab7antibody (shown as green signal). Representative photomicrographs (from 20 cells analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker.

E. Calculated percentage of double-positive vesicles (with >50% of the vesicle boundary demarcated by white signal in the colocalization analysis) from 20 cells counted from n=3 biologically independent experiments are shown. Each dot represents 1 cell in the dotted plot. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated and shown. Error bars represent± SEM. Significance was assessed by Mann-Whitney test for each marker with * representing p<0.05, ** P<0.01.

F. Raw 264.7 macrophage cells were transfected with eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from 25 images analyzed for each condition across n=3 biologically independent experiments) are shown with eGFP(N-term)-Rab10 epifluorescence (shown as green signal) and pT73-Rab10 (shown as red signal), with EEA1 (shown as blue signal). White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 Rab10 and the indicated marker.

G. Calculated percentage of colocalized vesicles, with each dot representing mean vesicle frequency in each cell analyzed. The group mean of % Rab10 vesicles positive with EEA1 were calculated, and error bars represent ± SEM. Significance was assessed by Mann-Whitney test for each marker with *** representing P<0.005.