Figure 6. EB1 and ch-TOG do not affect the binding of each other to microtubules.

The effects of overexpression of mEB1-GFP or ch-TOG-GFP on the distributions of endogenous ch-TOG or EB1, respectively, were examined. (A, B) mEB1-GFP (green) was transiently expressed in HeLa cells, and the cells were fixed and stained for endogenous ch-TOG using a RhoX-conjugated secondary antibody (red). To analyse the effect of mEB1-GFP overexpression on the distribution of ch-TOG, cells expressing a large amount of mEB1-GFP that distributed throughout the entire microtubule lattice, in which microtubule ends were expected to be saturated with EB1, were selected and the images were acquired on a SIM microscope. The boxed areas in (A) are enlarged in (B). Microtubule ends positive for ch-TOG clusters are indicated by arrowheads. Scale bars, 10 µm (A), 2 µm (B). (C) Average FI profiles of overexpressed mEB1-GFP and ch-TOG were obtained by analysing multiple SIM images (n = 71, 4 cells in 4 images) and plotted. The data were normalised and the peak intensities were set to 1. The error bars are SEM. (D) ch-TOG-GFP was exogenously expressed in HeLa cells, and the cells were fixed and stained for ch-TOG and EB1. The images were acquired on a confocal microscope. The expression level of exogenous ch-TOG-GFP was determined by comparing the fluorescence intensity of ch-TOG staining in GFP-negative and positive cells. The cell indicated by the asterisks has an ∼8-fold higher expression level of ch-TOG than untransfected cells. The inset shows the boxed areas at 2.5× magnification. Scale bar: 20 µm. In (E), average FI profiles of EB1 in untransfected control cells and ch-TOG-GFP expressing cells (> 6-fold overexpression) were obtained by analysing multiple confocal images (n = 132, 7 cells in 3 images for control; n = 135, 5 cells in 3 images for ch-TOG-GFP expressing cells) and plotted without normalization. The comet-like distribution of EB1, as well as the fluorescence intensity of the EB1 comet, was not altered by ch-TOG-GFP overexpression. (F) A control experiment showing competitive binding of EB1 to microtubule ends. mEB1-RFP was transiently overexpressed at a level at which it distributes throughout entire microtubule lattices in HeLa/mEB1-GFP clones (2F10) stably expressing mEB1-GFP at ∼40% the level of endogenous EB1 (Figure S2D, S6E). In RFP-negative cells, mEB1-GFP distributes in a typical comet shape. Overexpression of mEB1-RFP inhibited the accumulation of mEB1-GFP to microtubule ends and redistributed it throughout the entire microtubule lattice. The insets show the boxed areas at 2.5× magnification. Scale bar: 20 µm.