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Fig. 6. GFP-Kif5c preferentially associates with the detyrosinated-MT subpopulation in COS-7 cells. (A) Triple immunostaining shows that COS-7 cells contain detyrosinated, acetylated and tyrosinated tubulin. Microtubules can contain all three types of tubulin (see the MT in the boxed region). Other MTs stain predominantly for either tyrosinated (blue), acetylated (green) or detyrosinated (red) tubulin (merged image). The double arrow is shown parallel to a MT that is both detyrosinated and acetylated, but has very little tyrosinated tubulin. Arrows show an MT that is predominantly detyrosinated. Scale bar, 5 µm. (B) COS-7 cells transfected with GFP-Kif5c, and fixed and co-stained with antibodies specific for Kif5c (green) and tyrosinated, detyrosinated or acetylated tubulin (red). Box 1: MT decorated with GFP-Kif5c, showing very little staining for tyrosinated tubulin or acetylated tubulin. Boxes 2 and 6: MTs decorated with GFP-Kif5c and tyrosinated or acetylated tubulin, respectively. Boxes 3 and 4: MTs decorated with GFP-Kif5c and are stained for detyrosinated tubulin. Box 5: acetylated MT showing low levels of decoration by GFP-Kif5c. Scale bar, 5 µm. (C) 30 cells were used for the analysis. Ten were co-stained for GFP-Kif5c and tyrosinated tubulin, ten for GFP-Kif5c and detyrosinated tubulin, and ten for GFP-Kif5c and acetylated tubulin. Ten MTs were then randomly selected from each cell and scored for the level of GFP-Kif5c decoration along the length of MT visible at <10% (light grey), 10-50% (mid grey) or >50% decoration (dark grey) and are shown in the graph. The data shows that detyrosinated MTs are most likely to be decorated by GFP-Kif5c at high densities (greater than 50%).
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