![Figure 4. EB3 changes the autoinhibitory conformation of TTBK2. (A) Schematic diagram of TTBK2. The domain organization of TTBK2 and its fragments is presented with a summary of their ability to bind to TTBK2-cat. (B) COS-7 cells expressing mGFP–TTBK2-FL were lysed and incubated with the indicated GST-fused proteins. mGFP–TTBK2-FL specifically coprecipitated with GST–TTBK2-cat. CBB, Coomassie Brilliant blue staining. (C) The TTBK2 fragments were expressed in COS-7 cells, followed by pull-down. The TTBK2 fragments containing the C-terminal region were coprecipitated with GST–TTBK2-cat. Asterisks indicate the intact bands of the TTBK2 fragments. (D) HeLa cells expressing GFP–TTBK2-FL or -C were fixed and stained with the indicated antibodies. TTBK2-C end-tracked more efficiently than TTBK2-FL. The GFP intensity at the MT plus ends was measured by averaging the peak intensities of >20 GFP-TTBK2 comets for each cell and then plotting against the mean GFP intensity in the whole cell. Each data point represents an individual cell (≥25 cells for each condition). The boxes in the left panels are enlarged in the right panels. Bars, 10 µm. A.U., arbitrary unit. (E) mGFP–TTBK2-C was expressed in COS-7 cells together with control mCherry or mCherry–TTBK2-cat. mGFP–TTBK2-C failed to exhibit localized distribution at MT plus ends in ∼40% of cells expressing mCherry–TTBK2-cat. The graph shows the quantitative results. We counted the numbers of cells exhibiting the localized distribution of the indicated mGFP construct in the presence of mCherry or mCherry–TTBK2-cat. Error bars indicate the SEM. ***, P < 0.001 versus the respective control (Student’s t test). All of the results are representative of more than four independent experiments. The boxes in the left panels are enlarged in the rightmost panels. Bars: (left) 20 µm; (magnified images) 10 µm. (F) TTBK2-C or -C-m1/2 was expressed in COS-7 cells, and the lysates were subjected to pull-down using GST–TTBK2-cat in the presence of the control RhoGDI or EB3. Excess EB3 inhibited the association between TTBK2-cat and -C in a dose-dependent manner. The graph shows the relative amounts of bound TTBK2-C from more than three independent experiments as a box plot. ***, P < 0.001 (one-way analysis of variance [ANOVA], Tukey's test). IB, immunoblotting.](https://cdn.rupress.org/rup/content_public/journal/jcb/210/5/10.1083_jcb.201412075/7/jcb_201412075_fig4.jpeg?Expires=1771597699&Signature=KUUvD3SYmngz07-rc3Fsy1kUVW64ge3oSz7YCWS-rEMIQcs7GWLPiOb1-9Ln~yB8BRnPwlTGzNiX9H~maLvnhEWRef-wfB9Bka4L-DbpIgYlH6vPp0kPl1VeCUYMSSkhuCsETCSa9YmGmir~UYM5nt4alyHBph7h3PkGSq-979twOOSTA7pzSJAFP8gJh7OwGzkhz8U64-NVuwmaQo7rcoI0XBXiKL2jy4wZVJxvtlY6GuNq-dcDfKDH7VWtm6q9w7rZ1V1Pa-xE8VTg2UY4iP47aXQvIZYd0E5m-ft2xqJKFcVjjk-BPhVAALjL0z4mJI8u8-Zhp0cJFQNEu-c5Jw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
EB3 changes the autoinhibitory conformation of TTBK2. (A) Schematic diagram of TTBK2. The domain organization of TTBK2 and its fragments is presented with a summary of their ability to bind to TTBK2-cat. (B) COS-7 cells expressing mGFP–TTBK2-FL were lysed and incubated with the indicated GST-fused proteins. mGFP–TTBK2-FL specifically coprecipitated with GST–TTBK2-cat. CBB, Coomassie Brilliant blue staining. (C) The TTBK2 fragments were expressed in COS-7 cells, followed by pull-down. The TTBK2 fragments containing the C-terminal region were coprecipitated with GST–TTBK2-cat. Asterisks indicate the intact bands of the TTBK2 fragments. (D) HeLa cells expressing GFP–TTBK2-FL or -C were fixed and stained with the indicated antibodies. TTBK2-C end-tracked more efficiently than TTBK2-FL. The GFP intensity at the MT plus ends was measured by averaging the peak intensities of >20 GFP-TTBK2 comets for each cell and then plotting against the mean GFP intensity in the whole cell. Each data point represents an individual cell (≥25 cells for each condition). The boxes in the left panels are enlarged in the right panels. Bars, 10 µm. A.U., arbitrary unit. (E) mGFP–TTBK2-C was expressed in COS-7 cells together with control mCherry or mCherry–TTBK2-cat. mGFP–TTBK2-C failed to exhibit localized distribution at MT plus ends in ∼40% of cells expressing mCherry–TTBK2-cat. The graph shows the quantitative results. We counted the numbers of cells exhibiting the localized distribution of the indicated mGFP construct in the presence of mCherry or mCherry–TTBK2-cat. Error bars indicate the SEM. ***, P < 0.001 versus the respective control (Student’s t test). All of the results are representative of more than four independent experiments. The boxes in the left panels are enlarged in the rightmost panels. Bars: (left) 20 µm; (magnified images) 10 µm. (F) TTBK2-C or -C-m1/2 was expressed in COS-7 cells, and the lysates were subjected to pull-down using GST–TTBK2-cat in the presence of the control RhoGDI or EB3. Excess EB3 inhibited the association between TTBK2-cat and -C in a dose-dependent manner. The graph shows the relative amounts of bound TTBK2-C from more than three independent experiments as a box plot. ***, P < 0.001 (one-way analysis of variance [ANOVA], Tukey's test). IB, immunoblotting.
