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. 2018 Jul 2;46(1):40–58.e8. doi: 10.1016/j.devcel.2018.05.032

Figure 2.

Figure 2

The Second TOG-like Domain of CLASP2α Is Necessary and Sufficient for Catastrophe Suppression

(A) A scheme of different CLASP2 constructs used. Processive MT growth is the condition in which no catastrophes were observed within 10 min in the assay with 20 nM mCherry-EB3.

(B) Representative kymographs showing MT plus end growth in the presence of 20 nM mCherry-EB3 and GFP fusions of the indicated fusion proteins. EB3-CH domain fusion was used at 100 nM, all the other proteins at 30 nM. Scale bars, 2 μm (horizontal) and 60 s (vertical).

(C) Parameters of MT plus end dynamics in the presence of 20 nM mCherry-EB3 alone or together with the indicated GFP-fusion proteins. Protein concentrations were as in (B). Number of growth events analyzed: for mCherry-EB3 alone, n = 207, together with GFP-CLASP2α, n = 110, with TOG12-S, n = 110, with S-TOG3-CLIP-ID, n = 117, with S-TOG3, n = 70, with S-CLIP-ID, n = 136, with L-TOG2-S, n = 110, with ΔTOG2, n = 154, with L-TOG2-S W339E, n = 118, with chTOG-TOG1-S, n = 47, with chTOG-TOG2-S, n = 78, and for TOG2-EB3CH alone, n = 110. Error bars represent SEM. For catastrophe frequency plots, ∗p < 0.05, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001, for rescue frequency plots, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.0001, and for growth rate plots, ∗p < 0.05, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001, and ns, no significant difference with control, Mann-Whitney U test.

(D) Representative kymographs showing MT plus end dynamics in the presence of 20 nM mCherry-EB3 and 5 μM concentration of the indicated TOG domains from CLASP2α or Stu2. Scale bars, 2 μm (horizontal) and 60 s (vertical).

(E and F) MT plus end rescue and catastrophe frequencies in the presence of 20 nM mCherry-EB3 alone (n = 207) or together with 5 μM of CLASP2α TOG1 (n = 61) or TOG2 (n = 100), or with Stu2-TOG1 (n = 146). Error bars represent SEM. For all plots, ∗p < 0.05, ∗∗∗∗p < 0.0001 and ns, no significant difference with control, Mann-Whitney U test.

(G) Plot of the fraction of the total GMPCPP seeds that show MT outgrowth at increasing tubulin concentrations with tubulin alone (black curve) or GFP-EB3 (200 nM) together with either GFP-TOG3-S (100 nM) (blue) or GFP-TOG2-S (100 nM) (purple). For increasing tubulin concentrations in case of tubulin alone, n = 92, 96, 105, 82, 97, 87, 161, and 127 GMPCPP seeds, respectively, for GFP-TOG3-S, n = 61, 52, 53, 56, 71, 59, 61, and 88 GMPCPP seeds, respectively, and for GFP-TOG2-S, n = 70, 64, 50, 50, 55, 66, 63, and 63 GMPCPP seeds, respectively. Data are from two experiments. Error bars represent SD. Solid lines indicate the sigmoidal equation fit to the data. Tubulin concentration for half-maximal MT outgrowth for tubulin alone = 7.28 ± 0.08, for GFP-TOG2-S with GFP-EB3 = 3.29 ± 0.07, and for GFP-TOG3-S with GFP-EB3 = 5.54 ± 0.32. Hill slope for the fits with tubulin alone = 5.99 ± 0.34, for GFP-TOG2-S with GFP-EB3 = 3.56 ± 0.21, and for GFP-TOG3-S with GFP-EB3 = 4.59 ± 1.11.

See also Figure S2.