Myosin 2 maintains an open exocytic fusion pore in secretory epithelial cells

Abstract

Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (-)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (-)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening.

Figure 1.

Immunofluorescence localizes myosin 2A to the apical domain of pancreatic acinar cells. Paraformaldehyde-fixed and permeabilized pancreatic acinar clusters retain the polarized enrichment of the F-actin cytoskeleton in the subapical domain as shown with phalloidin staining. Extracellular lysine-fixable FITC dye is retained in the acinar lumens after fixation and is surrounded by the phalloidin staining as expected for the lumenal structure. Myosin 2B immunostaining (MY2B) is not found close to the luminal markers instead it is found in the basal part of the cell. Myosin 2A (MY2A) in contrast is located in a narrow band along the lumen (see enlarged images at bottom of the figure).

Figure 2.

Phospho-myosin levels increase rapidly after stimulation and are maintained for a protracted time even after stimulus removal. In these experiments, cell clusters were stimulated with ACh (10 μM) and atropine (100 μM) applied 1 min later to stop stimulation. Then, at the indicated times after the start of stimulation, the cell cluster suspension was rapidly frozen and the tissue processed for Western blotting. Phospho-myosin band densities (A), normalized to the β-actin loading controls, showed that phospho-myosin levels increased after stimulation, reaching maximal levels by 4 min, and maintaining a high expression level even at 11 min post-ACh stimulation (B).

Figure 3.

Drug actions on phospho-myosin levels. Cell clusters, treated with drugs for 20 min, at room temperatures were then ACh stimulated (10 μM) for 1 min before atropine (100 μM) was added. At 4 min after stimulation, cells were rapidly frozen and lysed. Quantitation of bands (shown in A) normalized for β-actin loading controls are expressed as a percentage of control (B). ML-9 (30 μM) significantly decreased phospho-myosin levels in ACh stimulated cell clusters (p < 0.05), whereas blebbistatin (50 μM) and Y27632 (50 μM) had no significant effect (data from 5 replicate experiments).

Figure 4.

The numbers of agonist-stimulated granule fusion events are not affected by myosin 2 inhibitors. The histogram shows that cell stimulation (with 10 pM CCK) increases granule fusion events measured per cell in the 5-min period after stimulation. Neither 50 μM blebbistatin nor 30 μM ML-9 pretreatment 20 min before CCK addition had any significant effect on the numbers of fusion events (n = 15 in each group; p = 0.9, p = 0.7, respectively, compared with no drug controls).

Figure 5.

Blebbistatin increases the numbers of closed fusion pores. (A) Representative images of CCK stimulated clusters showing the lumens and fused granules labeled with extracellular dyes (FITC, green; TMRE, red). The FITC was present throughout the experiment and the TMRE added 5 min after stimulation with CCK (10 pM). FITC-only labeling of a granule indicates a closed fusion pore. Pretreatment with (−)-blebbistatin (50 μM) resulted in increased number of closed fusion pores. (B) The ratio of TMRE/FITC in the granules was calculated. As a value of 1.00 is consistent with the fusion pore being open and the two dyes equilibrating, lower ratios (arbitrarily set as <0.4) indicate a closed fusion pore preventing complete intragranular equilibration with extracellular TMRE. The number of these granules is significantly higher after blebbistatin treatment (n = 201) compared with untreated (n = 251) resulting in a left-shift of the frequency graph (p < 0.0001). The (+)-blebbistatin enantomer was used as a negative control and showed no significant difference from no drug (n = 183).

Figure 6.

ML-9 dose-dependently increases the numbers of closed fusion pores. As in Figure 5, at two-dye technique was used to identify granules with closed fusion pores. ML-9 pretreatment (n = 264) resulted in a dose-dependent increase in the numbers of granules that predominantly are single-labeled with FITC (green). (A) Highlights of a single green granule, whereas the majority of granules are yellow in control. In contrast, after ML-9 (30 μM) treatment the majority of granules are green. (B) Quantification of these data showed that the graph of the normalized dye ratios within each granule shifted to the left indicative of an increased prevalence of closed granules (p < 0.001; n = 85–100 granules in each dose).

Figure 7.

Myosin 2 inhibition decreases fusion pore lifetimes. Live cells were imaged during entry of extracellular dye into fused granules, and local photobleaching was applied to a region drawn around an individual granule. (A) Shows a typical record. The fluorescence signal increases as it enters the granule, and is bleached down in the first cycle. The granule recovers as fresh unbleached dye from the lumen enters the granule through an open fusion pore. In the controls, sequential cycles of photobleaching showed ongoing granule recovery. The short straight lines imposed on the fluorescence trace show the lines fitted by linear regression to the recovery slopes. (B) After pretreatment with 50 μM (−)-blebbistatin we observed failure of fluorescence recovery sooner (on the fifth cycle in the example shown, shown by the asterisk [*]), indicating earlier closure of the fusion pore. (C) Frequency histogram of data from multiple experiments, indicating the times where fluorescence recovery first failed (i.e., the time of no fluorescence recovery when the fusion pore closed). The average time to fusion pore closure in control cells (n = 33, black bars) was 10.44 min (SEM 0.41), compared with blebbistatin-treated cells (n = 16, gray bars) 4.86 min (SEM 0.43), indicating significantly earlier granule closing with inhibition of myosin 2 (p < 0.0001).

Figure 8.

Myosin 2 inhibitors do not affect F-actin coating of postfusion secretory granules. The images (A) show cells stimulated for 5 min with CCK (10 pM) in the presence of an extracellular FITC dye (green) and labeled (after fixation and permeabilization) with phalloidin staining F-actin (red). Inhibition of myosin 2 or its upstream regulatory elements did not affect the pattern of coating of membrane-fused granules with F-actin, which occur as rings surrounding the granule. (B) Data from multiple separate experiments examining >200 granules in each treatment group shows that the number of F-actin–coated granules in stimulated acinar cells did not change with treatment with myosin 2 inhibitors (50 μM (−)-blebbistatin, 50 μM Y27632, and 30 μM ML-9; p = ns compared with controls). Bar, 5 μm.