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. 2016 Jul 1;27(13):1990–1999. doi: 10.1091/mbc.E15-12-0848

FIGURE 4:

FIGURE 4:

CENP-F is required for establishment of MT array asymmetry, and loss of CENP-F leads to an abundance of posttranslational modifications and resistance to depolymerization by NOC. Immunostaining of MTs with DM1A antibody (green) and DAPI (blue) in CENP-F+/+ (A) and CENP-F−/− (C) mouse embryonic fibroblasts. Scale bar, 15 μm. Schematic for fluorescence intensity quantification is shown on the right in A and C. Representative fluorescence intensity plots (30, each) of CENP-F+/+ (B) and CENP-F−/− (D) for each sample. Immunostaining of MTs (green) in wild-type MEFs (E–E′′) shows minimal amounts of acetylated (E′) and Glu (E′′) tubulin in relation to the typical staining of the complete MT network (E). In contrast, the MT network of CENP-F−/− MEFs (F) contains an abundance of posttranslational modifications, as indicated by immense acetylated (F′) and Glu (F′′) tubulin staining. Scale bar, 10 μm. (G) Western blot demonstrating the abundance of acetylated tubulin and tubulin in CENP-F+/+ and CENP-F−/− MEFs. Immunofluorescence analysis of the MT network (green) in CENP-F+/+ (H–H′′) and CENP-F−/− (I–I′′) MEFs treated for 2 h with no NOC (H, I), 1 μg/ml NOC (H′, I′), or 2.5 μg/ml NOC (H′′, I′′). Numerous short MTs persist in CENP-F−/- MEFs (I′, I′′), as compared with full depolymerization of MTs in CENP-F+/+ MEFs (H′, H′′). n = 25. Scale bar, 10 μm. Time-lapse imaging of CENP-F+/+ (J–J′′) and CENP-F−/- (L–L′′) MEFs expressing 3xEGFP-EMTB (black) to highlight the MT network. Images showing dynamic pixel differences were created by projecting pixel differences (purple) between sequential movie frames. CENP-F−/− MTs are very stable and move as an entire unit, as opposed to dynamic instability, exclusively at the tips of CENP-F+/+ MTs. Life history plots of MT dynamic instability can be observed in CENP-F+/+ MEFs (K) with periods of growth and shrinkage at the MT tips. In contrast, CENP-F−/- MEFs (M) experience very few periods of growth and catastrophe with long periods of pause.