Figure 5 | Scientific Reports

Figure 5

From: Interaction of microtubules and actin during the post-fusion phase of exocytosis

Figure 5

Cell treatment with colchicine and nocodazole influences the extent of actin polymerisation on fused secretory vesicles. (A) ATII cells transfected with actin-GFP were treated with colchicine or nocodazole and stimulated for secretion under the microscope. Exocytosis was detected with LTR. Arrows and inserts show actin coats on fused vesicles. Scale bar: 10 µm (above), 2 µm (below). (B) Actin-GFP fluorescence was measured in a ring-shaped region of interest on the actin coat (insert) and expressed as percent increased fluorescence intensity compared to cell cytoplasm fluorescence. Mean +/− SEM is shown and the numbers indicate the number of actin coats. Actin coats were from 5 (control) or 6 independent experiments (colchicine and nocodazole) and 3 cell isolations (***P < 0.001, *P < 0.05, one-way ANOVA with Tukey’s multiple comparison test). (C) ATII cells were transfected with lifeact-GFP, treated with colchicine or nocodazole and stimulated for secretion under the microscope to image actin coats (arrows and inserts). Exocytosis was detected with LTR. Scale bar: 10 µm (above), 2 µm (below). (D) Lifeact-GFP fluorescence was measured in a ring-shaped region of interest on the actin coat (as shown on 4B) and expressed as percent increased fluorescence intensity compared to cell cytoplasm fluorescence. Mean +/− SEM is shown and the numbers indicate the number of actin coats. Actin coats were from 4 independent experiments and 4 cell isolations (control), 8 independent experiments and 5 cell isolations (colchicine), and 9 independent experiments and 4 cell isolations (nocodazole). P = 0.07; one-way ANOVA with Tukey’s multiple comparison test. (E) ATII cells were treated with colchicine or nocodazole, stimulated for secretion, fixed and stained with alexa fluor 568 phalloidin. Actin coats on fused secretory vesicles (arrows) are enlarged below. Secretory vesicles were recognised by anti-ABCa3 staining (Supplementary Fig. 6). Scale bar: 10 µm (above), 2 µm (below). (F) Alexa fluor 568 phalloidin fluorescence was measured in a ring-shaped region of interest on the actin coat (as shown on 4B) and expressed as percent increased fluorescence intensity compared to cell cytoplasm fluorescence. Mean +/− SEM is shown and the numbers indicate the number of actin coats. The actin coats were from 5 independent experiments and 5 cell isolations (***P < 0.001, *P < 0.05, one-way ANOVA with Kruskal-Wallis test and Dunn’s multiple comparison test).

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