Fig. 4: The rescue activity of TOGARAM1 depends on the TOG3 and TOG4 domains.
a, Schematic representation of different TOGARAM1 constructs and summary table highlighting their effects on MT dynamics. Vertical lines indicate point substitutions predicted to ablate tubulin-binding activity. b, Fields of view (top) and histogram plot (bottom) of fluorescence intensities of single GFP molecules, EB3 dimers and TOGARAM1 molecules immobilized in separate chambers of the same coverslip. Number of molecules analyzed: GFP, n = 57,865; EB3, n = 73,074; TOGARAM1, n = 74,306. Scale bar: 2 µm. c, Parameters of MT plus-end dynamics in the presence of 20 nM EB3 in combination with indicated concentrations of TOGARAM1 constructs (from kymographs shown in d–h,k and Fig. 1c,p). For growth rates, bars represent the pooled data from three independent experiments. Total number of growth events: EB3 alone, n = 938; EB3 with TOGARAM1, n = 861; EB3 with TOMOGRAM1 1′2′34, n = 350; EB3 with TOMOGRAM1 123′4′, n = 337; EB3 with TOMOGRAM1 123′4, n = 560; EB3 with TOMOGRAM1 1234′, n = 253; EB3 with L-TOG-34, n = 131; EB3 with 200 nM TOG3–TOG4, n = 150. For transition frequencies, bars represent the averaged means from three independent experiments. Error bars represent the s.e.m., ****P < 0.0001 (Kruskal–Wallis test followed by Dunn’s post hoc test). d–k, Fields of view (left; scale bar: 2 µm) and kymographs (right; scale bars: 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seeds with 20 nM GFP–EB3 or mCherry–EB3 and indicated concentrations and colors of TOGARAM1 constructs. Assays were repeated three independent times. Blue arrowheads, rescues.
