Journal: PLoS Genetics
Article Title: Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics
doi: 10.1371/journal.pgen.1005063
Figure Lengend Snippet: (A) GBA2 expression in dermal fibroblasts from adult mice. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Heterologously expressed HA-tagged GBA2 was used as a positive control, calnexin (Clnx) as a loading control. (B) Accumulation of GlcCer in GBA2 knockout-fibroblasts. Thin layer chromatography (TLC) analyzing glycosphingolipids from wild-type (+/+) and GBA2 knockout-fibroblasts (-/-). Representative TLC analysis for neutral sphingolipids. GlcCer: glucosylceramide, LacCer: lactosylceramide, Spm: sphingomyelin. GlcCer levels were quantified by densitometry and are presented as mean ± S.D. The fold change in GlcCer levels in GBA2 knockout-fibroblasts was calculated. (C) Fluorescent labeling of the cytoskeleton in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were transfected with lifeact (green) to visualize F-actin and with EB3-cherry to visualize microtubules. Scale bars are indicated. (D) Fluorescent labeling of F-actin in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were seeded on CYTOO chips with micropatterns that are coated with fluorescently-labeled fibronectin (purple). F-actin was stained using Alexa Fluor Phalloidin 488 (green) and the DNA was stained with DAPI (blue). Scale bars are indicated. (E) Analysis of cytoskeletal structures. Cells were seeded on the crossbow shape. The number of cells containing filopodia or lamellipodia (left) and the average number of filopodia or lamellipodia per cell (right) were determined. (F) Gene expression-analysis. The mRNA expression level of Cdc42 , Rac1 , and Rho was analyzed by qRT-PCR. (G) Protein expression-analysis. Total protein lysates were probed with a GBA2- (2F8), a Cdc42-, and a Rac1-specific antibody on a Western blot. Calnexin (Clnx) was used as a loading control. (H) Quantification of protein expression based on (G). (I) Quantification of actin turnover in dermal fibroblasts. Expression levels of G- and F-actin in wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were determined using Western blot-analysis. Ratio of F-actin/G-actin for wild-type and GBA2 knockout-fibroblasts is expressed relative to the control. (J) See (I) for testis. (K-M) Analysis of microtubule dynamics in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). (K) Expression of EB3-cherry in dermal fibroblasts. Cells were transfected with EB3-cherry and microtubule dynamics were analyzed. Representative tracks of growing microtubule plus-ends are indicated with white lines. (L) Microtubule growth rate. Wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were transfected with EB3-cherry and the growth rate of growing plus-ends was analyzed. Per genotype, n = 3 animals with a minimum of 7 cells and 10 tracks per cell were analyzed. Data are presented as mean ± S.D. (M) see (L) for microtubule persistence. For all bar graphs, data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.
Article Snippet: For the analysis of cytoskeletal structures in dermal fibroblasts, cells were seeded on multi-pattern fibronectin-coated CYTOO chips (#10–900–13–06, CYTOO Cell Architects).
Techniques: Expressing, Western Blot, Positive Control, Knock-Out, Thin Layer Chromatography, Labeling, Transfection, Staining, Quantitative RT-PCR
