Figure 3

Expression of VPS4^EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. (a) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4^EQ. Cells were then processed for electron microscopy as described in material and methods. Cells selected by fluorescence microscopy were located in the TEM and serial sections were collected to locate the centrosome (supplementary Fig. S3). Scale, 0.2 μm. (b–d) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4^EQ, immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). (b) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4^EQ n = 20. (c) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4^EQ n = 15. (d) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4^EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4^EQ expression, γ tubulin staining is severely reduced.