Journal: Scientific Reports
Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis
Figure Lengend Snippet: Ciliogenesis is defective in cells with altered VPS4 activity. ( a ) NIH3T3 transfected with the indicated plasmids or siRNA constructs were fixed, immunostained with anti-acetylated antibodies and imaged. The percentage of ciliated cells is displayed in the graph (short cilia ≤ 2 µm, cilia ≥ 2 µm). Statistical analysis was calculated using a one-way ANOVA (***p- value ≤ 0.0001). Maximum intensity projection images of representative cells are shown in the right panel. Arrows indicate cilia (scale, 10 μm). scRNA n = 113, siVPS4A/B n = 103, siCHMP2A n = 64, siCHMP2B n = 173, siCHMP4B n = 68, GFP-VPS4 EQΔMIT n = 185. Data was obtained from at least two independent experiments. GFP and GFP-VPS4 EQ transfections were repeated in each experiment for reference. For these conditions data was obtained from more than 10 experiments, n ≥ 700. ( b ) Zebrafish embryos were injected with mRNA encoding either GFP or GFP-VPS4 EQ . 32 h post injection embryos were analyzed for survival (GFP n = 112, GFP-VPS4 EQ n = 132). Live embryos were analyzed for developmental defects (GFP n = 71, GFP-VPS4 EQ n = 21). Representative images are shown. Scale, 100 μm. ( c ) Embryos (15 h) were fixed and immunostained with acetylated-tubulin antibodies. The number of cilia in the Kupffer’s vesicle of each animal was counted and embryos were categorized as control (not injected or injected with GFP), GFP-VPS4 EQ injected developmentally normal, or GFP-VPS4 EQ injected with developmental defects. Control n = 41, GFP-VPS4 EQ normal n = 17, GFP-VPS4 EQ defective n = 31. Statistical analysis was calculated using t-test. *p- value ≤ 0.1, **p- value ≤ 0.05. Scale, 10 μm. ( d ). Cells were then treated and serially sectioned as described in material and methods and imaged by TEM. Left panel: representative image of a GFP expressing cells. Second to fourth panels: representative images of cells expressing GFP-VPS4 EQ . GFP n = 16, GFP-VPS4 EQ n = 19. Scale, 0.2 μm. Right panel: percentage of ciliated cells and cells with a docked ciliary vesicle. ( e ) NIH3T3 cells, expressing PACT-TagBFP (blue) and GFP-VPS4 EQ (green) and immunostained for the cilia marker Arl13b (red) were imaged using Airyscan confocal microscopy. Scale, 0.5 μm. ( f ) NIH cells transfected with mCherry-VPS4 EQ (green) and PACT tagBFP (blue) and Smo GFP (green) were imaged live using Airyscan microscopy. Scale, 0.5 μm.
Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).
Techniques: Activity Assay, Transfection, Construct, Injection, Transmission Electron Microscopy, Expressing, Marker, Confocal Microscopy, Microscopy