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. 2017 Jun 15;28(12):1688–1700. doi: 10.1091/mbc.E17-02-0128

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© 2017 Chehab, Santos, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Deletion of C2A or C2B abolishes the stimulation-induced clustering of Munc13-4 at the plasma membrane. TIRF time-lapse movies of live HUVECs expressing YFP–Munc13-4, Munc13-4–mKate or the indicated mutant constructs and VWF-RFP as WPB marker were recorded, and the obtained images were analyzed by the object detection algorithm MorphoQuant. (a) Representative graphs showing the number of Munc13-4–positive objects detected in the TIRF field of HUVECs stimulated with 100 µM histamine at t = 15 s. Cells were transfected with YFP- or mKate-tagged full-length Munc13-4, Munc13-4 variants with truncated C2 domains, Munc13-4 variants with mutations in the Ca²⁺-binding sites of the C2 domains, or Munc13-4(Δ280-285). Munc13-4(543-1090) lacks the C2A domain, Munc13-4(1-899) lacks the C2B domain, and Munc13-4(285-917) lacks both C2A and C2B (Elstak et al., 2011). Munc13-4(C2A*) carries two point mutations in the Ca²⁺-binding site of the C2A domain, Munc13-4(C2B*) carries two analogous point mutations in the Ca²⁺-binding site of the C2B domain, and Munc13-4(C2A*B*) carries these mutations in both C2 domains (Boswell et al., 2012). (b) Change in the number of Munc13-4–positive objects/100 µm² after histamine stimulation. Cells were transfected and stimulated as in a. Means of n-fold changes over all cells from at least five independent experiments were tested for statistical significance by one-way ANOVA with Tukey’s test (ns, statistically not significant, ****p ≤ 0.0001, as compared with the wild-type samples). Numbers of cells analyzed: Munc13-4, 33; Munc13-4(543-1090), 27; Munc13-4(1-899), 42; Munc13-4(285-917), 14; Munc13-4 (mKate), 32; Munc13-4(C2A*), 52; Munc13-4(C2B*), 35; Munc13-4(C2A*B*), 35; Munc13-4(D280-285), 35. Means ± SEM: 3.5 ± 0.3, 1.3 ± 0.03, 1.2 ± 0.02, 1.2 ± 0.02, 2.8 ± 0.1, 1.8 ± 0.1, 1.5 ± 0.1, 1.2 ± 0.02, and 2.3 ± 0.2. (c) Munc13-4(C2A*) is recruited more slowly to WPBs. HUVECs expressing VWF-RFP and YFP-Munc13-4 or YFP-Munc13-4(C2A*) were stimulated as in a. Given is the time from stimulation to the peak of Munc13-4–positive objects in the TIRF field. Means over all cells analyzed were tested for statistical significance by unpaired t test (****p ≤ 0.0001). Means ± SEM: 6.3 ± 0.1 (Munc13-4), 79 ± 8.6 (Munc13-4(C2A*)).