Journal: Molecular Biology of the Cell
Article Title: A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel–Palade body exocytosis in endothelial cells
doi: 10.1091/mbc.E17-02-0128
Figure Lengend Snippet: 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 Ca 2+ -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 Ca 2+ -binding site of the C2A domain, Munc13-4(C2B*) carries two analogous point mutations in the Ca 2+ -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 µm 2 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*)).
Article Snippet: Munc13-4–positive objects were detected in TIRF live recordings before and after stimulation using the Matlab-based image processing algorithm MorphoQuant, which was originally developed to detect Golgi structures ( Schuberth et al. , 2015 ).
Techniques: Clinical Proteomics, Membrane, Expressing, Mutagenesis, Construct, Marker, Transfection, Binding Assay
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