TTBK2 tracks MT plus ends in an EB-dependent manner. (A) Schematic diagram of TTBK1 and TTBK2. Kinase, protein kinase domain; SxIP, EB-binding motif. The red X indicates the mutation site in the SxIP motif. (B) Immunoprecipitation (IP) between TTBK2 and EB3 using COS-7 cells. R IgG indicates rabbit IgG. (C) COS-7 cells expressing mGFP-TTBK2 were lysed and incubated with GST-fused proteins. mGFP-TTBK2 coprecipitated with EB3-FL and EB3 lacking 3 aa from the C terminus but not with the further deletion mutant EB3 1–251 aa. Note that GST–EB3 1–251 aa and GST–EB3 Δ3 aa contain a 13-aa linker after the GST tag (see Protein purification and biochemistry in Materials and methods). IB, immunoblotting. CBB, Coomassie Brilliant blue staining. (D) Mutation of both SxIP motifs diminished the association between TTBK2 and EB3. (E) COS-7 cells expressing TTBK2 were imaged using epifluorescence microscopy. mGFP–TTBK2-WT accumulated at EB3-positive MT plus ends, but the mutant TTBK2-m1/2 did not. Bar, 10 µm. (F) mGFP–TTBK2-C colocalized with EB3 at the MT ends. Bar, 5 µm. (G) In vitro reconstitution of TTBK2 tracking. Purified mGFP–TTBK2-FL tracked the MT plus ends only in the presence of EB3. Protein concentrations are as follows: 15-µM tubulin (containing 3.2% rhodamine-labeled tubulin), 25-nM FLAG–mGFP-TTBK2, and 400-nM EB3. Kymograph images are presented. Horizontal and vertical bars, 5 µm and 50 s, respectively. See Fig. S1 for identification of the TTBKs as EB-binding proteins.