Fig. 2
From: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation
Pathogenic LRRK2 causes deficits in centrosomal positioning critical for cell polarization and directed migration. a Example of reorientation of the centrosome 4 h after wounding in SH-SY5Y cells stably expressing flag-tagged wildtype or G2019S mutant LRRK2. The white lines indicate scratch orientation, and cells were stained with anti-pericentrin, anti-Golgin97, and DAPI. Angles are labeled as having oriented (+) or not oriented (−) centrosomes for the first row of cells facing the scratch wound. Scale bar, 10 μm. b Quantification of centrosome reorientation in cells stably expressing GFP, flag-tagged wildtype or G2019S-mutant LRRK2 immediately after (t = 0 h), or 4 h after generating the wound (t = 4 h). Random orientation is expected to be 25%. N > 100 cells were quantified for each condition in each experiment. Bars represent mean ± s.e.m. (n = 3 independent experiments); **, p < 0.01. c Wound-healing assays in either flag-tagged wildtype or G2019S-mutant LRRK2-expressing SH-SY5Y cells. Phase-contrast images at the indicated times are shown. Scale bar, 150 μm. d Quantitative analysis of wound-healing assays as described in Methods. Bars represent mean ± s.e.m. (n = 6 independent experiments); ****, p < 0.001. e Example of tracking of individual cells expressing flag-tagged wildtype or G2019S mutant LRRK2. Individual cells in the first row facing the wound were tracked until reaching the middle of the wound. Directionality index (D) and forward migration index in Y axis (FMI Y) were calculated for at least 30 cells per condition in three independent experiments, and are expressed as mean ± s.e.m. on top of each graph. *, p < 0.05. f Single cell wound healing migration speed was calculated for at least 30 cells per condition in 3 independent experiments