Figure 3
The SxLP/SxIP motifs of DDA3 are essential for its EB1-dependent MT plus-end accumulation and tracking.
(a) Shown is the sequence alignment of the basic Pro/Ser-rich regions in DDA3 from human (Homo), mouse (Mus) and rat (Rattus). Dark and light shading indicate the identical and conserved residues, respectively. The residues in the black squares indicate the potential EB1-binding motifs. Numbers indicate the amino acid positions. (b, c) SxLP/SxIP motifs are essential for DDA3-EB1 binding in vitro. (b) GST-EB1 was used as an affinity matrix to isolate various GFP-DDA3 mutants from 293T cell extracts, followed by Western blotting analysis with GFP antibody to indicate the specific interaction. (c) Quantitative analysis of the relative binding activity in b. The ratio of GFP-DDA3/GST-EB1 was normalized to 1 in each experiment. Data are presented as means ± SD from three independent experiments. ***, P < 0.001 by t-test. (d) SxLP/SxIP motifs are essential for the plus-end accumulation of DDA3 in cells. Shown is live imaging of HeLa cells transiently co-transfected with EB1-RFP (red) and the indicated GFP-DDA3 mutants (green). (e, f) TIRF experiments indicated that SxIP/SxLP motifs are essential for the EB1-dependent MT plus-end tracking of DDA3122–363 in vitro. The corresponding kymograph at the bottom shows the same MT over a period of 2 min. (g) Statistical analysis of the relative intensity of DDA3122–363 at MT plus ends in e and f, as described in Fig. 2j. **, P < 0.01 by t-test. Scale bars, 5 μm (all image panels).
