Fig. 7
From: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation
Expression of wildtype but not phosphorylation-deficient Rab8a causes centrosomal accumulation of phospho-Rab8a and centrosome cohesion deficits in wildtype LRRK2-expressing SH-SY5Y cells. a Example of SH-SY5Y cells stably expressing flag-tagged wildtype LRRK2, and transfected with mRFP-tagged wildtype or T72A-mutant Rab8a as indicated, stained with an anti-phospho-T72-Rab8a antibody preabsorbed with dephosphopeptide, for pericentrin and DAPI. Where indicated, cells were treated with 100 nM MLi2 for 2 h before immunocytochemistry. Note that phospho-Rab8 can be detected both in cells with one centrosome (first panel) as well as in cells with duplicated centrosomes (second panel). Scale bar, 10 μm. b Same as in a, but SH-SY5Y cells stably expressing flag-tagged G2019S mutant LRRK2. Scale bar, 10 μm. c Quantification of mean fluorescence intensity of phospho-Rab8a signal as described in Methods in cells either stably expressing wildtype (wt) or G2019S mutant LRRK2, transfected with RFP-tagged Rab8a or T72-mutant Rab8a, and treated with 100 nM MLi2 for 2 h before immunocytochemistry as indicated. Bars represent mean ± s.e.m., (n = 3 independent experiments); ****, p < 0.001; ***, p < 0.005. d Example of non-differentiated SH-SY5Y cells stably expressing GFP, flag-tagged wildtype or G2019S-mutant LRRK2 as indicated, and transfected with Rab8a or phosphorylation-deficient Rab8a (Rab8a-T72A) as indicated, and stained for pericentrin and TOPRO. Scare bar, 10 μm. e Quantification of the split centrosome phenotype in SH-SY5Y cells from the type of experiments depicted in a. Bars represent mean ± s.e.m. (n = 3 experiments); *, p < 0.05. f SH-SY5Y cells stably expressing GFP, flag-tagged wildtype or G2019S-mutant LRRK2 as indicated were transfected with mRFP-tagged wildtype or phosphorylation-deficient Rab8a (Rab8a-T72A) as indicated, and extracts blotted for Rab8a levels and GAPDH as loading control