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. 2016 Feb 1;129(3):592–603. doi: 10.1242/jcs.178285

Fig. 2.

Fig. 2.

Actin remodelling is not required for Ca2+-driven WPB exocytosis. (A) Effect of jasplakinolide on actin cytoskeleton and cell morphology. Images show the effect of a 20-min incubation with vehicle (i) or 0.35 µM jasplakinolide (Jas, ii) on the actin cytoskeleton and cell morphology visualised by expression of YFP–actin (green in colour merged images). Competitive binding of jasplakinolide to actin is manifested by the displacement of Rhodamine–phalloidin staining (red in colour merged images). Scale bars: 10 µm. (B) Effect of a 20-min pre-treatment of HUVECs with jasplakinolide (at the indicated concentrations) on ionomycin-evoked (1 µM) VWF (i) or VWFpp (ii) secretion. Stimulation was for 10 min in the continued presence of vehicle or jasplakinolide. Experiments shown are representative of thre independent experiments performed in triplicate (mean±s.e.m.). (C) Images of single HUVECs 24 h after nucleofection with mCherry–actin WT (red; anti-RFP antibody) (i), or mCherry–actin V159N (red; anti-RFP antibody) (ii), and counterstained with Alexa-Fluor-488–phalloidin (green). Regions indicated by white boxes are shown as greyscale inserts. Arrowheads indicate colocalisation of RFP fluorescence with actin filaments. Scale bars: 10 µm. (Di) Cumulative plots of the total number of WPB fusion events detected during ionomycin stimulation (as in Fig. 1Bi) of HUVECs co-expressing VWFpp–EGFP and mCherry–actin WT (black, n=1038), or mCherry–actin V159N (grey, n=999). Data are scaled to the mean fraction of fluorescent WPBs that underwent fusion in each case (actin WT, 62.8±2.9%, n=21 cells; actin V159N, 60.4±3.0%, n=21 cells; mean±s.e.m.). (Dii) Summary of the delay, maximum rate and percentage of fluorescent WPBs exocytosed for cells expressing mCherry–actin WT (black; mean±s.e.m., n=21) or mCherry-actin V159N (grey; mean±s.e.m., n=21). (E) Ionomycin-stimulated secretion of VWFpp–EGFP in mCherry–actin WT (black) or mCherry–actin V159N (grey) co-expressing cells. Plots show data pooled from three independent experiments performed in triplicate (mean±s.e.m.).