The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

Introduction

The ability of actin filaments to remodel rapidly in response to changes in intracellular signaling is essential for their participation in a number of functions including cytokinesis (Bi, 2001), cell motility (Krause et al., 2003; dos Remedios et al., 2003), endocytosis (Qualmann and Kessels, 2002; Engqvist-Goldstein and Drubin, 2003) and exocytosis (Eitzen, 2003). Here we explore the changes in apical actin that occur during apical exocytosis in the secretory epithelial cells responsible for the production and release of tear proteins into ocular fluid, the acinar cells of the lacrimal gland. Like other epithelial cells, actin filaments in acinar cells from lacrimal gland are detected primarily beneath cell membranes, with an abundant enrichment beneath the apical plasma membrane (APM) (da Costa et al., 1998).

Earlier attempts to evaluate the role of actin filaments in lacrimal acinar exocytosis using the actin-targeted agents, cytochalasin D and jasplakinolide (da Costa et al., 1998; da Costa et al., 2003), did not reveal major changes in acinar secretion nor affect resting or carbachol (CCH)-stimulated distributions of the mature secretory vesicle (SV) marker, rab3D. It was unclear from these studies whether the actin filament array beneath the APM was substantially affected by these treatments. Apical actin filaments in epithelial cells are more resistant to actin-targeted drugs than are basolateral actin filaments (Ammar et al., 2001). Spurred by recent confocal fluorescence microscopy analysis revealing evidence for actin filament organization in acutely stimulated lacrimal acini exposed to CCH, we have re-evaluated actin filament participation in exocytosis in live acini.

Green fluorescent protein (GFP)-tagged proteins have been extensively used to measure the dynamics of different proteins including actin in live cells. Choidas et al. (Choidas et al., 1998) found that GFP-actin co-assembled with endogenous actin into a variety of actin-based structures. GFP-actin has also been utilized to measure actin dynamics in microvilli (Tyska and Mooseker, 2002; Loomis et al., 2003) and stereocilia (Rzadzinska et al., 2004). Here we used high efficiency (80-90%) transduction with replication-defective adenovirus (Ad) encoding GFP-actin to label the actin filament array in live lacrimal acini and to obtain qualitative (time-lapse imaging) and quantitative (fluorescence recovery after photobleaching or FRAP) measures of its dynamics. This approach, combined with additional functional and morphological analyses of lacrimal acini exposed to the general myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM), and the more selective myosin light chain kinase inhibitor, ML-7, has enabled us to demonstrate that the filamentous actin array beneath the APM of stimulated lacrimal acini participates actively in exocytosis, in conjunction with non-muscle myosin II.

Materials and Methods

Reagents

CCH, rhodamine-phalloidin, BDM and goat anti-rabbit secondary antibody conjugated to FITC were obtained from Sigma Chemical Co (St Louis, MO). Latrunculin A (LAT A), latrunculin B (LAT B) and myosin light chain kinase inhibitor, ML-7 [1-(5-Iodonaphthalene-1-sulfonyl)homopiperazine, HCl] were purchased from EMD Biosciences, Inc. (San Diego, CA). Rabbit ProLong antifade mounting medium was from Molecular Probes (Eugene, OR). Cell culture reagents were from Life-Technologies. Rabbit polyclonal antibodies to actin and GFP were obtained from NOVUS (Littleton, CO) or Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Rabbit polyclonal antibody to non-muscle myosin II was obtained from Biomedical Technologies Inc. (Stoughton, MA). Goat anti-rabbit IRDye™800-conjugated secondary antibody was from Rockland (Gilbertsville, PA). Adeno-X™ virus purification and Adeno-X™ rapid titer kits were from BD Biosciences (Palo Alto, CA).

Cell isolation, culture and treatments

Isolation of lacrimal acini from female New Zealand white rabbits (1.8-2.2 kg) obtained from Irish Farms (Norco, CA) was in accordance with the Guiding Principles for Use of Animals in Research. Lacrimal acini were isolated as described (da Costa et al., 1998) and cultured for 2-3 days. Cells prepared in this way aggregate into acinus-like structures; individual cells within these structures display distinct apical and basolateral domains and maintain a robust secretory response (da Costa et al., 1998; da Costa et al., 2003; Wang et al., 2003). CCH was used at 100 μM for the indicated times, while BDM treatment was for 15 minutes at 10 mM and ML-7 was for 15 minutes at 40 μM.

Production and purification of recombinant Ad

Ad-Tc-GFP-actin contained full-length EGFP fused to the Dictostelium discoideum actin 15 gene (accession number, M14146) using a polylinker sequence in frame. This cDNA construct was inserted immediately downstream of the tetracycline repressor binding sequence (Tc) followed by the minimal human CMV immediate early promoter and a transcription start site and upstream of a pA sequence. Co-transduction with the Tet transcriptional activator (Ad-tTA) promoted GFP-actin expression in acinar cells. Preliminary studies showed that this protein co-assembled with endogenous actin. Given the availability of this construct and the high sequence homology with mammalian β-actin (94%), we felt it unnecessary to construct a new recombinant adenovirus with a mammalian actin. Ad-syncollin-GFP was generated as described previously (Ma et al., 2004). For amplification, QB1 cells, a derivative of HEK293 cells, were infected with Ad-Tc-GFP-Actin, Ad-tTA, Ad-GFP or Ad-syncollin-GFP and grown at 37°C and 5% CO2 in DMEM (high glucose) containing 10% fetal bovine serum for 66 hours until completely detached from the flask surface. The Adeno-X™ virus purification kit was used for virus purification and the Adeno-X™ rapid titer kit for viral titration.

Detection of GFP-actin in transduced acini

Lacrimal acinar cells cultured on Matrigel-coated coverslips in 12-well plates at a density of 2×106 cells per well were co-transduced with Ad-Tc-GFP-Actin and Ad-tTA at MOIs ranging from 1.5-6.0 at 37°C and 5% CO2 for 2 hours. Cells were rinsed with PBS before addition of fresh culture medium and incubation for 20 hours. Non-transduced cells and cells transduced with Ad-GFP served as controls. Cells were rinsed with PBS and lysed in RIPA buffer containing protease inhibitor cocktail (da Costa et al., 1998) on a rocker platform at 4°C for 1 hour. Lysates were clarified by centrifugation, and equal amounts of total proteins from each sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with Odyssey blocking buffer, followed by hybridization with appropriate primary and IRDye™800-conjugated secondary antibodies and quantified using an Odyssey Scanning Infrared Fluorescence Imaging System (Li-Cor, Lincoln Nebraska).

Confocal fluorescence microscopy

For analysis of actin filaments in fixed cells, reconstituted rabbit lacrimal acini cultured on Matrigel-coated coverslips were fixed and processed as described (Wang et al., 2003; da Costa et al., 2003) and incubated with rhodamine-phalloidin. For detection of myosin II, acini were fixed in 4% paraformaldehyde and permeabilized with ice cold acetone according to the manufacturer's instructions prior to blocking and primary and fluorescent secondary antibody addition. Confocal images were obtained with a Zeiss LSM 510 Meta NLO imaging system equipped with Argon, red HeNe and green HeNe lasers mounted on a vibration-free table. Panels were compiled in Adobe Photoshop 7.0 (Adobe Systems Inc, Mountain View, CA).

For live cell imaging of transduced acini expressing GFP-actin, rabbit lacrimal acini seeded on Matrigel-covered glass-bottomed round 35 mm dishes (MatTek, Ashland MA) at a density of 4×106 cells per dish for 2 days were co-transduced with Ad-Tc-GFP-actin and Ad-tTA at an MOI of 6 for each for 1-2 hours. Cells were then rinsed and cultured in fresh medium for 18-24 hours to allow protein expression. Dual transduction efficiency (as measured by GFP-actin expression) ranged from 80-90% in each experiment. On day 3, lacrimal acini were analyzed by time-lapse confocal fluorescence and DIC microscopy or FRAP analysis using Zeiss Multiple Time Series V3.2 and Physiology V3.2 software modules. Live cell analyses were performed at 37°C. For time-lapse analysis, acini of similar size (4-6 cells arranged around a central lumen) were chosen. DIC images and GFP fluorescence were acquired simultaneously using the 488 line of the Argon Laser.

For measurement of the diameter of actin-coated structures, z-stack images from lacrimal acini were combined into projections and the Ruler tool of the Zeiss LSM 510 software was used to measure the maximum diameter of the structures (including the fluorescence signal at the periphery) within the projection. Acini were selected at random and all actin-coated structures within a chosen acinus were scored.

FRAP analysis

A 30 mW Argon Laser (488 nm) set at 60% power with 100% transmission was used to photobleach a circular region of interest (ROI) ∼1-2 μm in diameter; image acquisition post-bleach was at 0.1% of transmission with the same laser power without frame averaging to avoid photobleaching of the ROI during imaging acquisition. The fluorescence associated with the entire acinus was simultaneously recorded to ensure that image acquisition did not significantly reduce the fluorescence associated with the cells under study. The loss in total cellular fluorescence did not exceed 10-20% during 90 seconds of observation. Also, since recording of the fluorescence of the whole cell area was available together with recording of the fluorescence of the circular bleached ROI, it was obvious whether the ROI moved out of focus. If this occurred during the experiment, the data were discarded. A region of comparable size within the cytosol (containing G-actin) was photobleached in parallel and shown to exhibit almost complete recovery (∼95%) over the time period of interest, demonstrating that the parameters chosen for photobleaching were appropriate. Additional controls were performed as recommended (Snapp et al., 2003, Lippincott-Schwartz et al., 2003).

Published rates of actin filament turnover suggest that complete filament exchange normally requires time scales of minutes. We limited our observation time to 90-100 seconds due to the extreme mobility of the apical actin filaments in stimulated acini; during this shorter time scale, problems associated with remodeling out of the plane of focus or away from the photobleached spot were minimized. This time frame of observation was sufficient to demonstrate dramatic differences in the mobile fraction (Mf) of apical GFP-actin under the different conditions in our study. Mf was calculated from the equation:
\[\ \mathrm{M}_{\mathrm{f}}=Y{\times}(F_{4}-F_{0}){/}(F_{\mathrm{i}}-F_{0}){\times}100\%.\ \]
Where Fi is initial fluorescence recorded right before the bleach, F0 is fluorescence recorded right after the bleach, F4 is fluorescence at the end of the observation time, and Y is correction factor for the bleach during observation.
For analysis of actin filament turnover time (t½), FRAP recovery curves (FF0/FiF0) were fitted to equation 12 published by Axelrod et al. (Axelrod et al., 1976) using the method of least squares. Turnover time t½ (or tDeff) is related to Deff, the effective diffusion coefficient, by the equation:
\[\ t_{\mathrm{Deff}}=w^{2}{\gamma}{/}(4D_{\mathrm{eff}}),\ \]
where w is half-width of the intensity at e–2 height, Deff is effective diffusion coefficient, and γ is correction factor for the amount of bleaching. For statistical analysis, paired t-tests were utilized to compare CCH-treated samples with and without BDM or ML-7 with their own internal dish controls. Mf and t½ were evaluated at four intervals after CCH stimulation: 1-4 minutes CCH, 5-10 minutes CCH, 10-12 minutes CCH, and 15-18 minutes CCH.

Electron microscopy (EM)

Acini were fixed and processed as previously described (Schechter et al., 2002) and analyzed using a JEOL 1200 EX transmission electron microscope. For analysis of SV diameter and morphology, images (15-17 fields) of the subapical region beneath a defined lumenal space were acquired at 7500× magnification under each experimental condition. The longest diameter of each detectable single, dual fused and multiple fused SV in the field, as previously defined (da Costa et al., 2005), was measured and grouped for calculation of average vesicle diameter in these subcategories.

Secretion assays

Measurement of protein secretion were conducted as described (Wang et al., 2003) in control, BDM-treated (10 mM, 15 minutes), ML-7-treated (40 μM, 15 minutes) and LAT B-treated (10 μM, 60 minutes) rabbit lacrimal acini seeded in Matrigel-coated 24-well plates. In each assay, protein release was calculated from 5-6 replicate wells per treatment and normalized to total cellular protein. Basal, total and stimulated (total minus basal) releases were plotted. Differences in experimental groups were determined using a paired t-test with P≤0.05. For analysis of syncollin-GFP release into culture medium from acini transduced with Ad-syncollin-GFP, medium was collected, concentrated on Centricon 10 filters, equal volumes resolved by SDS-PAGE, and syncollin-GFP detected by western blotting using a polyclonal antibody to GFP. Blots were quantified using an Odyssey Scanning Infrared Fluorescence Imaging System (Li-Cor, Lincoln Nebraska). Signal intensity was normalized to pellet protein in each sample and expressed as fluorescence intensity/mg protein before normalization to control and comparison across treatments.

Results

Actin filaments are reorganized following acute exposure to CCH

Fig. 1 shows representative confocal fluorescence micrographs of actin filaments in resting (CON) acini and acini exposed to CCH for 5 or 15 minutes. The lumenal regions in reconstituted acinar cells were distinguished by intense actin filament labeling detected in circular regions (*) attributable to actin filament enrichment beneath the APM. Fainter actin filament labeling was detected beneath basolateral membranes. The schematic diagram in Fig. 1 depicts the cellular boundaries and organization of the acini in the control image. Actin filaments at the apical membrane exhibited two changes after CCH stimulation (100 μM) for 5 minutes: (1) decreased intensity and increased irregularity in the continuity of apical actin filaments and (2) formation of actin-coated structures beneath the APM (arrows). Increased intensity of actin filament labeling beneath the basolateral membrane was also detected in acini exposed to CCH (arrowheads, 5 and 15 minutes). Formation of actin-coated structures was not as evident at 15 minutes of exposure to CCH, although the apparent lumenal extension and discontinuity of labeling was still noticeable. We and others have previously established that CCH evokes exocytosis in cultured rabbit or rat lacrimal acinar cells with a response curve exhibiting substantial amounts of secretory protein release by 5 minutes (40-50% of the total release) (Hodges et al., 1992; Zoukhri et al., 1994; da Costa et al., 1998; Ota et al., 2003) with a subsequent slowing of secretory product release. This release profile is consistent with the dramatic changes in the shape of the lumen at 5 minutes of stimulation, the time course associated with maximal rates of exocytosis, and also with the restoration of lumenal dimensions to the resting state by 15 minutes when rates of secretion have diminished.

Fig. 1.

CCH alters actin organization in lacrimal acini. Confocal fluorescence micrographs of control acini (CON) and acini exposed to 100 μM CCH for 5 minutes (CCH 5) or 15 minutes (CCH 15) and fixed and processed as described in Materials and methods to label actin filaments. The top right panel shows a schematic outline of the actin filaments in CON, with thick lines representing apical actin filaments and thinner lines representing basolateral actin filaments. *, Lumenal regions; arrows, actin-coated structures; arrowheads, increased basolateral actin; bar, 5 μm.

Fig. 1.

CCH alters actin organization in lacrimal acini. Confocal fluorescence micrographs of control acini (CON) and acini exposed to 100 μM CCH for 5 minutes (CCH 5) or 15 minutes (CCH 15) and fixed and processed as described in Materials and methods to label actin filaments. The top right panel shows a schematic outline of the actin filaments in CON, with thick lines representing apical actin filaments and thinner lines representing basolateral actin filaments. *, Lumenal regions; arrows, actin-coated structures; arrowheads, increased basolateral actin; bar, 5 μm.

Fig. 2.

EM images of resting and CCH-stimulated lacrimal acini. (A) Filament distribution in resting (CON) and CCH-stimulated (5 minutes, 100 μM) lacrimal acini. (B) Filament distribution in BDM-treated (10 mM, 15 minutes) and ML-7-treated (40 μM, 15 minutes) lacrimal acini exposed to CCH (5 minutes, 100 μM). Boxed regions in the left column are magnified in the right column. L, lumen; SV, secretory vesicle; arrowheads, swaths of filaments beneath the APM or, in CCH-stimulated acini, assembled beneath multiple SVs and arrows, filaments associated with individual SVs.

Fig. 2.

EM images of resting and CCH-stimulated lacrimal acini. (A) Filament distribution in resting (CON) and CCH-stimulated (5 minutes, 100 μM) lacrimal acini. (B) Filament distribution in BDM-treated (10 mM, 15 minutes) and ML-7-treated (40 μM, 15 minutes) lacrimal acini exposed to CCH (5 minutes, 100 μM). Boxed regions in the left column are magnified in the right column. L, lumen; SV, secretory vesicle; arrowheads, swaths of filaments beneath the APM or, in CCH-stimulated acini, assembled beneath multiple SVs and arrows, filaments associated with individual SVs.

As shown in Fig. 2A, EM analysis of the subapical cytoplasm of resting rabbit lacrimal acini periodically revealed areas of abundant filament enrichment (arrows) which were often localized between subapical SVs and the APM, suggesting that they might restrict access of SVs to the APM. As shown in Fig. 2A, we could periodically detect larger swaths of filaments that assembled beneath groups of SVs in acini acutely exposed to CCH (arrowheads); we also occasionally detected filament bundles associated closely with fusing SVs (arrows). Sampling of individual filament diameters in these bundles revealed values between 4-7 nm (average ±s.d. was 5±0.28 nm, n=20 fields).

GFP-actin co-assembles with endogenous actin in transduced lacrimal acini

Co-transduction of lacrimal acini with Ad-Tc-GFP-actin and Ad-tTA resulted in a dual transduction efficiency of 80-90%, similar to previous reports for other Ad constructs in lacrimal acini (Wang et al., 2003). Western blot analysis of lysates from co-transduced lacrimal acini confirmed that GFP-actin was expressed at the expected MW of ∼66 kDa (Fig. 3A). This label was co-localized with actin filaments labeled with rhodamine-phalloidin, indicating co-assembly with endogenous actin (Fig. 3B).

Analysis of GFP-actin fluorescence in live acini revealed intense labeling beneath the APM surrounding the lumenal region (Fig. 3C), similar to non-transduced acini. GFP-actin was also detectable at basolateral surfaces while fainter fluorescence could be detected throughout the cytoplasm, which was excluded from large circular regions identified as SVs from the paired DIC image. Occasionally, some SVs were ringed with apparent actin filaments (arrowheads).

Time-lapse confocal fluorescence microscopy reveals substantial apical actin filament reorganization in CCH-stimulated acini

Images obtained at chosen intervals from time-lapse confocal fluorescence microscopy sequences are shown for resting (Fig. 3C) and CCH-stimulated acini (Fig. 4), while the entire sequences are available online (supplementary material Movies 1-3). The same resting and CCH-stimulated acinus is shown in Fig. 3C and Fig. 4A, respectively, to illustrate the remarkable increase in actin remodeling associated with CCH while Fig. 4B shows a second CCH-stimulated acinus for comparison. Image acquisition of CCH-treated acini was initiated 30-60 seconds after CCH addition, due to the time required to refocus on the appropriate focal plane. Therefore, the acinus in Fig. 4A at 0 seconds reflects the rapid appearance of GFP-actin labeled invaginations relative to the same unstimulated acinus in Fig. 3C. In the absence of CCH, there was little global remodeling of apical or basolateral actin filaments, although subtle changes suggestive of basal release of a few SVs at the APM were detected after 469 seconds (Fig. 3C, arrow).

After CCH addition, the intensity of the apical actin filament array was noticeably diminished in places (barbed arrows, Fig. 4), suggestive of CCH-induced disassembly. Invaginated regions underlaid with actin filaments (arrowheads) also formed rapidly upon stimulation. These actin-coated structures appeared to represent transient fusion intermediates, since they encircled SVs detectable by DIC microscopy (Fig. 4A, bottom row, arrowheads). In many cases, the actin associated with these structures was retracted steadily toward the apical actin network in successively acquired images until the underlying actin coat could no longer be distinguished from the apical filaments. Although most of the actin-coated intermediates formed close to the APM, some could be detected deep within the cytoplasm.

In addition to the profound changes in actin filament organization detected at the APM, changes in basolateral actin filaments were also seen. Within a few minutes of CCH stimulation, GFP fluorescence associated with basolateral actin filaments increased, followed by the appearance of small actin-coated vesicular structures that appeared to `bubble' and extend from the surface (Fig. 4, arrows).

FRAP analysis reveals increased apical actin filament turnover

The relative rates of apical actin filament turnover in resting and CCH-stimulated acini expressing GFP-actin were measured by FRAP. Observation time following bleaching were limited to 90-100 seconds due to the extreme mobility of the apical actin filaments in stimulated acini; during this shorter time scale, problems associated with remodeling out of the plane of focus or away from the photobleached spot were minimized. Representative images clearly delineated the more complete recovery of fluorescence post-bleaching in the ROI of CCH-stimulated samples (Fig. 5A). We did not observe complete recovery of filaments at 100 seconds, possibly indicative of the presence of some capped actin filaments. This observation was consistent with previous findings that cytochalasin D did not significantly disassembly the apical actin array (da Costa et al., 1998). However, CCH-stimulated acini always exhibited more recovery than unstimulated acini. Fig. 5C shows composite data from multiple experiments comparing Mf values (% of Fi) detected under each condition at defined time intervals after addition of CCH. The Mf is significantly (P≤0.05) increased by CCH stimulation when FRAP is conducted immediately (1-4 minutes) or up to 15-18 minutes after stimulation.

Fig. 3.

GFP-actin expression and distribution in lacrimal acini. (A) Western blots of lysates (100 μg protein/lane) from non-transduced and transduced lacrimal acinar cells developed in parallel using a polyclonal anti-actin antibody (left) and a polyclonal anti-GFP antibody (right) and goat anti-rabbit IRDye™ 800-conjugated secondary antibody. Lane 1, rabbit lacrimal acinar cells without transduction; lane 2, rabbit lacrimal acinar cells transduced with Ad-GFP, MOI of 6; lanes 3-5, rabbit lacrimal acinar cells co-transduced with Ad-Tc-GFP-Actin and Ad-tTA at MOIs of 1.5, 3 and 6, respectively. (B) Co-transduced lacrimal acini fixed and processed as in Materials and methods to label actin filaments with rhodamine-phalloidin (Rho-Phall, red). GFP-actin fluorescence is shown in green while blue indicates nuclei labeled with DAPI. Most soluble GFP-actin is quenched by fixation/permeabilization. (C) Confocal fluorescence and DIC images of rabbit lacrimal acini co-transduced to express GFP-actin were acquired at 10.5 seconds intervals over 16 minutes. The top row shows GFP-actin fluorescence, the DIC image, and an overlay. GFP-actin fluorescence at intervals throughout the time-lapse sequence is shown in the second row. In images: bars, 5 μm; *, lumena; arrow, region of actin invagination; arrowheads, SVs.

Fig. 3.

GFP-actin expression and distribution in lacrimal acini. (A) Western blots of lysates (100 μg protein/lane) from non-transduced and transduced lacrimal acinar cells developed in parallel using a polyclonal anti-actin antibody (left) and a polyclonal anti-GFP antibody (right) and goat anti-rabbit IRDye™ 800-conjugated secondary antibody. Lane 1, rabbit lacrimal acinar cells without transduction; lane 2, rabbit lacrimal acinar cells transduced with Ad-GFP, MOI of 6; lanes 3-5, rabbit lacrimal acinar cells co-transduced with Ad-Tc-GFP-Actin and Ad-tTA at MOIs of 1.5, 3 and 6, respectively. (B) Co-transduced lacrimal acini fixed and processed as in Materials and methods to label actin filaments with rhodamine-phalloidin (Rho-Phall, red). GFP-actin fluorescence is shown in green while blue indicates nuclei labeled with DAPI. Most soluble GFP-actin is quenched by fixation/permeabilization. (C) Confocal fluorescence and DIC images of rabbit lacrimal acini co-transduced to express GFP-actin were acquired at 10.5 seconds intervals over 16 minutes. The top row shows GFP-actin fluorescence, the DIC image, and an overlay. GFP-actin fluorescence at intervals throughout the time-lapse sequence is shown in the second row. In images: bars, 5 μm; *, lumena; arrow, region of actin invagination; arrowheads, SVs.

Fig. 5B shows two representative plots of fluorescence recovery under each experimental condition, plotted as F/Fi. These recovery curves were biphasic, with the second phase exhibiting a more pronounced CCH effect including an increased slope. Fig. 5D shows composite data obtained from measurement of individual turnover times (t½) calculated from individual recovery plots such as those in Fig. 5B as described in Materials and Methods. Actin filament t½ was significantly decreased in CCH-treated acini by ∼2-fold compared to unstimulated controls, confirming a reduced lifetime for these apical filaments relative to those in resting acini.

BDM and ML-7 stabilize actin-coated structures and suppress some apical actin dynamics

In resting lacrimal acini, BDM (10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes) did not significantly alter actin filament organization in resting acini; however, CCH stimulation of BDM- or ML-7-treated acini caused the formation of large actin-coated structures at and beneath the APM by 5 minutes (Fig. 6A, arrows). These actin-coated structures persisted stably in the cytoplasm for up to 60 minutes (data not shown) in contrast to their rapid turnover in untreated acini. The remarkable trapping of apparent actin-coated structures by BDM and ML-7 was accompanied by a significant inhibition of CCH-stimulated protein secretion that was detectable by 5 minutes of CCH exposure (Fig. 6B).

Since these agents are commonly employed as inhibitors of non-muscle myosin II, we investigated its distribution in lacrimal acini. By western blotting, the antibody to non-muscle myosin II recognized a band at 220 kDa in concentrated supernatant fractions from lacrimal acini, which was also present in crude cell lysates (data not shown). Myosin II immunofluorescence in resting lacrimal acini displayed a diffuse labeling pattern, while stimulation of acini with CCH for 5 minutes resulted in detection of filamentous immunofluorescence, some of which was co-localized with actin assembled into actin-coated structures (arrows, Fig. 6A). Myosin II distribution in acini exposed to BDM or ML-7 prior to stimulation with CCH also exhibited filamentous immunofluorescence localized in roughly circular structures beneath the apical pole of the cells, some of which was co-localized with the actin-coated structures increased in these acini (arrows, Fig. 6A).

Fig. 4.

Time-lapse confocal fluorescence microscopy of GFP-actin in CCH-stimulated lacrimal acini reveals considerable actin remodeling. Confocal fluorescence microscopy images of rabbit lacrimal acini transduced to express GFP-actin were exposed to 100 μM CCH at the onset of the time-lapse sequence. Selected images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown. (A) The same acinus shown in Fig. 3C immediately after CCH. Boxed image at 298 seconds is magnified and shown on the bottom row as GFP-actin, DIC and as an overlaid image. (B) Another CCH-stimulated acinus. Arrowheads, actin-coated structures; arrows, basolateral actin filament `bubbling'; barbed arrows, regions of apical actin filament thinning. Bars, 5 μm.

Fig. 4.

Time-lapse confocal fluorescence microscopy of GFP-actin in CCH-stimulated lacrimal acini reveals considerable actin remodeling. Confocal fluorescence microscopy images of rabbit lacrimal acini transduced to express GFP-actin were exposed to 100 μM CCH at the onset of the time-lapse sequence. Selected images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown. (A) The same acinus shown in Fig. 3C immediately after CCH. Boxed image at 298 seconds is magnified and shown on the bottom row as GFP-actin, DIC and as an overlaid image. (B) Another CCH-stimulated acinus. Arrowheads, actin-coated structures; arrows, basolateral actin filament `bubbling'; barbed arrows, regions of apical actin filament thinning. Bars, 5 μm.

Analysis of live lacrimal acini expressing GFP-actin and exposed to BDM or ML-7, then CCH by time-lapse confocal fluorescence microscopy revealed that these actin-coated structures formed sequentially adjacent to the APM after CCH stimulation (Fig. 7A,B, arrowheads). The actin filaments associated with actin-coated structures in ML-7-treated acini exposed to CCH appeared to first accumulate and then to condense; actin-coated structures in BDM-treated acini appeared, in contrast, to retain their vesicular shape.

In addition to the remarkable stabilization of actin-coated structures, analysis of live lacrimal acini expressing GFP-actin and exposed to BDM or ML-7, then CCH by time-lapse confocal fluorescence microscopy shown in Fig. 7 revealed subtle changes in the apical actin array. The intensity of the apical actin filament network in these treated acini was not diminished by CCH, unlike acini in the absence of these agents (Fig. 4), suggesting possible suppression of CCH-stimulated apical actin filament turnover. Consistent with this, EM analysis of apical actin filaments revealed bundles of filaments located between SVs and the lumenal surface in acini treated with BDM or ML-7 prior to CCH stimulation (Fig. 2B). Measurements of Mf for apical actin filaments in acini exposed to BDM +CCH confirmed that this agent blunted the significant CCH-induced increases in Mf elicited by CCH (Table 1), while eliciting an increased t½ that was significant relative to CCH-stimulated acini alone at most time points evaluated. ML-7 elicited a similar trend towards suppression of the increased Mf elicited by CCH stimulation (Table 1), although this suppression was significant at only one time point evaluated. ML-7 significantly increased t½ only at one time point relative to CCH-stimulated acini alone. An additional feature of BDM- and ML-7-treated acini seen in acini expressing GFP-actin included stabilization of basolateral actin blebbing. The complete time-lapse sequences for lacrimal acini exposed to BDM or ML-7 and stimulated with CCH are available online (supplementary material Movies 4,5).

Table 1.

Mf and t½ values for apical actin in BDM- and ML-7-treated acini expressing GFP-actin without and with CCH

Treatment Mf (%) t½ (minutes)
Control (BDM series)   39±3 (n=8)   11±1 (n=8)  
CCH (1-4 minutes)   49±2 (n=6)   7±2 (n=6)  
CCH (5-7 minutes)   69±9 (n=9)   8±0 (n=9)  
CCH (10-12 minutes)   58±4 (n=4)   6±1 (n=4)  
CCH (15-18 minutes)   49±4 (n=4)   7±2 (n=4)  
BDM   40±5 (n=8)   18±2 (n=8) 
BDM±CCH (1-4 minutes)   39±3 (n=6)*  13±4 (n=6)  
BDM±CCH (5-7 minutes)   41±5 (n=7)*  15±4 (n=7) 
BDM±CCH (10-12 minutes)   42±5 (n=4)*  15±3 (n=4) 
BDM±CCH (15-18 minutes)   29 (n=2)   16 (n=2)  
Control (ML-7 series)   41±5 (n=4)   14±2 (n=4)  
CCH (1-4 minutes)   70±7 (n=4)   4±0 (n=4)  
CCH (5-7 minutes)   55±6 (n=4)   7±1 (n=4)  
CCH (10-12 minutes)   54±5 (n=4)   10±2 (n=4)  
CCH (15-18 minutes)   48±10 (n=4)   8±3 (n=4)  
ML-7   45±3 (n=4)   12±3 (n=4)  
ML-7±CCH (1-4 minutes)   52±4 (n=4)*  5±1 (n=4)  
ML-7±CCH (5-7 minutes)   51±5 (n=4)   10±1 (n=4) 
ML-7±CCH (10-12 minutes)   45±5 (n=4)*  7±1 (n=4)  
ML-7±CCH (15-18 minutes)   37±4 (n=4)   15±1 (n=4) 
Treatment Mf (%) t½ (minutes)
Control (BDM series)   39±3 (n=8)   11±1 (n=8)  
CCH (1-4 minutes)   49±2 (n=6)   7±2 (n=6)  
CCH (5-7 minutes)   69±9 (n=9)   8±0 (n=9)  
CCH (10-12 minutes)   58±4 (n=4)   6±1 (n=4)  
CCH (15-18 minutes)   49±4 (n=4)   7±2 (n=4)  
BDM   40±5 (n=8)   18±2 (n=8) 
BDM±CCH (1-4 minutes)   39±3 (n=6)*  13±4 (n=6)  
BDM±CCH (5-7 minutes)   41±5 (n=7)*  15±4 (n=7) 
BDM±CCH (10-12 minutes)   42±5 (n=4)*  15±3 (n=4) 
BDM±CCH (15-18 minutes)   29 (n=2)   16 (n=2)  
Control (ML-7 series)   41±5 (n=4)   14±2 (n=4)  
CCH (1-4 minutes)   70±7 (n=4)   4±0 (n=4)  
CCH (5-7 minutes)   55±6 (n=4)   7±1 (n=4)  
CCH (10-12 minutes)   54±5 (n=4)   10±2 (n=4)  
CCH (15-18 minutes)   48±10 (n=4)   8±3 (n=4)  
ML-7   45±3 (n=4)   12±3 (n=4)  
ML-7±CCH (1-4 minutes)   52±4 (n=4)*  5±1 (n=4)  
ML-7±CCH (5-7 minutes)   51±5 (n=4)   10±1 (n=4) 
ML-7±CCH (10-12 minutes)   45±5 (n=4)*  7±1 (n=4)  
ML-7±CCH (15-18 minutes)   37±4 (n=4)   15±1 (n=4) 
*

Significant (P≤0.05) decrease in Mf relative to the paired treatment without or with CCH; significant increase in t½ relative to the paired treatment without or with CCH; P≤0.07.

Fig. 5.

FRAP analysis of GFP-labeled apical actin filaments in lacrimal acini. (A) Representative scans of apical actin intensity before [Pre-(–1s)], during [Bleaching (0 seconds)] and after [Post-(100 seconds)] photobleaching in unstimulated (CON) and CCH-stimulated (100 μM, 10 minutes) lacrimal acini. The circular region is the ROI and bar, 5 μm. (B) Typical plots of fluorescence over initial fluorescence (F/Fi) for resting and CCH-stimulated (100 μM, 10 minutes) acini. Fractional fluorescence was calculated by F–F0/FiF0. (C) Mf values for apical actin filaments in resting and stimulated acini analyzed after the indicated exposures to CCH. (D) Turnover times (t½) for apical actin filaments in resting and stimulated acini analyzed after the indicated ranges of exposure to CCH as described in Materials and Methods. Results from (C) and (D) were obtained from 3-7 dishes in each preparation from n=5 preparations; *P≤0.05.

Fig. 5.

FRAP analysis of GFP-labeled apical actin filaments in lacrimal acini. (A) Representative scans of apical actin intensity before [Pre-(–1s)], during [Bleaching (0 seconds)] and after [Post-(100 seconds)] photobleaching in unstimulated (CON) and CCH-stimulated (100 μM, 10 minutes) lacrimal acini. The circular region is the ROI and bar, 5 μm. (B) Typical plots of fluorescence over initial fluorescence (F/Fi) for resting and CCH-stimulated (100 μM, 10 minutes) acini. Fractional fluorescence was calculated by F–F0/FiF0. (C) Mf values for apical actin filaments in resting and stimulated acini analyzed after the indicated exposures to CCH. (D) Turnover times (t½) for apical actin filaments in resting and stimulated acini analyzed after the indicated ranges of exposure to CCH as described in Materials and Methods. Results from (C) and (D) were obtained from 3-7 dishes in each preparation from n=5 preparations; *P≤0.05.

BDM and ML-7 promote accumulation of syncollin-GFP in actin-coated structures

Studies with a syncollin-GFP fusion protein have shown labeling of large protein-enriched SVs in diverse systems including zymogen granules in pancreatic acini (Hodel and Edwardson, 2000), insulin granules in pancreatic β-cells (Ma et al., 2004) and SVs in lacrimal acini (Jerdeva et al., 2005). Syncollin-GFP in transduced, unstimulated acini was detected in a series of large SVs, discernable by DIC microscopy, that were enriched around a lumenal region (Fig. 8A). Exposure of acini to CCH resulted in rapid loss of this punctate fluorescence that increased up to 10 minutes, as shown in the 2.5 D-graphical reconstruction of syncollin-GFP intensity at 0 and 600 seconds. This loss in vesicular syncollin-GFP fluorescence was accompanied by a 3.5-fold increase in the recovery of syncollin-GFP in the culture medium (Fig. 8B). Although syncollin-GFP labeled SVs with the same intensity in acini treated with ML-7 or BDM, stimulation with CCH resulted in discharge of only a fraction of the syncollin-GFP stores (Fig. 8A), consistent with findings that these agents significantly decreased syncollin-GFP recovery in culture medium by ∼50-60% (Fig. 8B).

Confocal fluorescence microscopy revealed that actin-coated structures trapped by BDM and ML-7 were frequently rich in syncollin-GFP as shown in Fig. 9A (arrowheads), confirming that these actin-coated structures were fusion intermediates. This effect was even more apparent when z-series from acini were combined into projections (Fig. 9B).

Fig. 6.

BDM and ML-7 alter CCH-stimulated actin remodeling while inhibiting protein secretion in lacrimal acini. (A) Confocal fluorescence micrographs of control (CON), BDM-pretreated (10 mM, 15 minutes) or ML-7-pretreated (40 μM, 15 minutes) lacrimal acini exposed to 100 μM CCH for 5 minutes, and then fixed and processed to detect myosin II (green, also shown as monochrome image) and actin filaments (red, also shown as monochrome image). Arrows, recruitment of myosin II immunoreactivity to areas enriched in actin-coated fusion intermediates; *, lumena; bar, 5 μm. (B) Bulk protein secretion in acini treated with BDM and ML-7 as described above and stimulated with CCH for 5, 10 or 30 minutes. Basal release, total release and the stimulated component (total minus basal) are each plotted. Values were normalized to cell protein before comparison across samples. Bars, s.e.m.; *significant at P≤0.05 from comparable value in control cells and n=5-6 preparations.

Fig. 6.

BDM and ML-7 alter CCH-stimulated actin remodeling while inhibiting protein secretion in lacrimal acini. (A) Confocal fluorescence micrographs of control (CON), BDM-pretreated (10 mM, 15 minutes) or ML-7-pretreated (40 μM, 15 minutes) lacrimal acini exposed to 100 μM CCH for 5 minutes, and then fixed and processed to detect myosin II (green, also shown as monochrome image) and actin filaments (red, also shown as monochrome image). Arrows, recruitment of myosin II immunoreactivity to areas enriched in actin-coated fusion intermediates; *, lumena; bar, 5 μm. (B) Bulk protein secretion in acini treated with BDM and ML-7 as described above and stimulated with CCH for 5, 10 or 30 minutes. Basal release, total release and the stimulated component (total minus basal) are each plotted. Values were normalized to cell protein before comparison across samples. Bars, s.e.m.; *significant at P≤0.05 from comparable value in control cells and n=5-6 preparations.

Actin-coated structures encompass multiple SVs

Accumulation of syncollin-GFP in actin-coated structures in acini treated with BDM or ML-7 suggested that they were fusion intermediates. To gain additional information regarding the role of these actin-coated structures in the secretory process, we quantified SV diameter and fusion status as well as actin-coated structure diameter under each experimental condition. In EM micrographs, all SVs were categorized into one of three categories: single vesicles, dual fused vesicles and multiple fused vesicles. As shown in Fig. 10 and Table 2, in control acini, the majority of SVs (>85% of total) were single vesicles with an average diameter of ∼1 μm while a smaller number were categorized as dual and multiple fused SVs. CCH stimulation did not alter the percentage of total vesicles within each category, although it significantly increased the average diameter of the SVs in the single and dual categories by 20-25%. This increase in diameter may reflect CCH-stimulated fusion of smaller single SVs in the cytoplasm prior to release of their contents at the APM. Both BDM and ML-7 treatments altered the percentage of SVs recovered in each category, causing a trend towards formation and/or stabilization of multiple fused SVs. The increased vesicle diameter detected in single and dual fused SVs from CCH-stimulated acini was suppressed by both agents.

Table 2.

Analysis of SV diameter and morphology in lacrimal acini

Vesicle diameter (μm) Vesicle diameter range (μm) Proportion of total (%) Vesicle count
CON      
Single   1.01±0.02   0.26-2.46   87.35   449  
Dual   1.42±0.06   0.42-2.36   10.70   55  
Multiple   1.97±0.15   1.47-2.98   1.95   10  
CCH      
Single   1.18±0.03  0.37-4.19   87.82   339  
Dual   1.90±0.09  0.94-3.09   10.36   40  
Multiple   2.51±0.39   0.79-3.56   1.81   7  
BDM±CCH      
Single   0.92±0.02*   0.31-3.25   81.13   288  
Dual   1.42±0.07*   0.73-2.41   9.86   35  
Multiple   2.67±0.19   1.20-5.00   9.01   32  
ML-7±CCH      
Single   1.00±0.03*   0.26-2.72   74.36   261  
Dual   1.46±0.06*   0.58-3.04   17.09   60  
Multiple   2.58±0.16   1.20-4.71   8.55   30  
Vesicle diameter (μm) Vesicle diameter range (μm) Proportion of total (%) Vesicle count
CON      
Single   1.01±0.02   0.26-2.46   87.35   449  
Dual   1.42±0.06   0.42-2.36   10.70   55  
Multiple   1.97±0.15   1.47-2.98   1.95   10  
CCH      
Single   1.18±0.03  0.37-4.19   87.82   339  
Dual   1.90±0.09  0.94-3.09   10.36   40  
Multiple   2.51±0.39   0.79-3.56   1.81   7  
BDM±CCH      
Single   0.92±0.02*   0.31-3.25   81.13   288  
Dual   1.42±0.07*   0.73-2.41   9.86   35  
Multiple   2.67±0.19   1.20-5.00   9.01   32  
ML-7±CCH      
Single   1.00±0.03*   0.26-2.72   74.36   261  
Dual   1.46±0.06*   0.58-3.04   17.09   60  
Multiple   2.58±0.16   1.20-4.71   8.55   30  

Lacrimal acini were pretreated without or with BDM (10 mM) or ML-7 (40 μM) for 15 and stimulated with CCH (100 μM) for 5 minutes, collected by centrifugation and fixed as described in Materials and Methods for EM analysis. 15-17 images of equivalent subapical areas under each treatment condition were evaluated for vesicle diameter and number within each category. Diameter is mean ± s.e.m.; *significant decrease in diameter relative to matched CCH-stimulated samples at P≤0.05; significant increase in diameter relative to matched unstimulated control at P≤0.05.

The diameter of the actin-coated structures formed transiently in CCH-stimulated acini and stabilized by BDM and ML-7 were likewise quantified. Because detection of these actin coats was infrequent in EM micrographs, we conducted this analysis in projections from z-series acquired from acini under each experimental condition where actin-coated structures were readily identified. The average diameters of the actin-coated structure in acini stimulated with CCH (100 μM, 5 minutes) was 1.80±0.01 μm (70 vesicles, n=3) while actin-coated structures in acini exposed to either BDM or ML-7 prior to CCH for 5 minutes were significantly increased to 2.11±0.02 μm (156 vesicles, n=3) and 2.15±0.03 μm (n=98 vesicles, n=3), respectively. These values are larger than the average diameter of a single SV, corresponding with the average diameters of dual or multiple fused SVs (Table 2) and suggesting assembly around multiple SVs. The significant increase in size of the actin-coated structure associated with BDM and ML-7 is also consistent with measurements of larger aggregated SVs by EM (Table 2), and may reflect the inhibition of non-muscle myosin II in contraction of the actin coat around these aggregates involved in compound fusion. These findings suggest that actin-coated structures form around clusters of fusing vesicles. This model is also consistent with observations that syncollin-GFP-enriched individual SVs do not completely `fill' the actin-coated structures.

Fig. 7.

Time-lapse confocal fluorescence microscopy of GFP-actin in lacrimal acini exposed to BDM or ML-7, then CCH reveals stabilization of actin-coated structures. Lacrimal acini transduced to express GFP-actin and exposed to BDM (10 mM, 15 minutes, A) or ML-7 (40 μM, 15 minutes, B) were imaged immediately upon CCH addition (100 μM). Selected images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown. Arrowheads, actin-coated structures; arrow, actin bubble at the basolateral membrane; bar, 5 μm.

Fig. 7.

Time-lapse confocal fluorescence microscopy of GFP-actin in lacrimal acini exposed to BDM or ML-7, then CCH reveals stabilization of actin-coated structures. Lacrimal acini transduced to express GFP-actin and exposed to BDM (10 mM, 15 minutes, A) or ML-7 (40 μM, 15 minutes, B) were imaged immediately upon CCH addition (100 μM). Selected images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown. Arrowheads, actin-coated structures; arrow, actin bubble at the basolateral membrane; bar, 5 μm.

LAT B decreases apical actin filaments while enhancing secretory response

The results described above suggested that actin-coated fusion intermediates formed in response to inhibition of non-muscle myosin II were participants in exocytosis, since their stabilization was associated with decreased secretory capacity as well as accumulation of multivesicular aggregates within the cytoplasm. Although we detected evidence for enhanced turnover of apical actin filaments with CCH stimulation, we wanted to investigate further the barrier role of apical actin filaments in exocytosis. To do this, we used LAT A and LAT B, which destabilize actin filaments through binding and sequestration of actin monomers (Spector et al., 1983). Fig. 11A shows that LAT B decreased the intensity of labeling of apical and basolateral actin, an effect increased by CCH stimulation. LAT B also elicited a modest but significant release of bulk protein in the absence or presence of CCH (Fig. 11B, left), and a modest and significant enhancement of CCH-stimulated exocytosis of syncollin-GFP release in Ad-syncollin-GFP transduced acini (Fig. 11B, right).

Preliminary investigations comparing LAT B and the related agent, LAT A, in lacrimal acini showed comparable effects on secretion and actin filaments (data not shown). Although LAT A was not utilized extensively in biochemical assays due to its higher cost, we utilized it in EM analysis of acinar morphology (Fig. 11C,D). The image on the left depicts a representative lumenal region enriched in SVs in a LAT A-treated acinus, while a higher magnification image of the boxed region to the right shows SVs poised at the APM (arrowheads) without the intervening actin filament barrier normally restricting SVs from APM that is evident in the control acinus in Fig. 2A. The images in Fig. 11D are also of LAT-A treated, unstimulated acini, and depict secretagogue-independent fusion of an unusually large SV at the APM (left image, arrowhead) and apparent CCH-independent compound fusion at the APM (right image, arrows). These data are consistent with biochemical findings that disassembly of apical actin filaments modestly enhances exocytosis.

Discussion

Although a considerable amount of work has considered the role of actin filaments in neuronal and neuroendocrine secretion, much less is known about the mechanisms of acinar SV exocytosis. Lacrimal, parotid and pancreatic acini are different in many aspects from neuronal and neuroendocrine cells, including: (1) the absence of a readily releasable, pre-docked SV pool; (2) maintenance of SVs of significantly larger diameter; (3) slower SV release kinetics; (4) release at only one domain within the polarized epithelial cells and (5) significantly restricted surface area available for fusion at the APM. Here we demonstrate for the first time using time-lapse confocal fluorescence microscopy and FRAP analysis of lacrimal acini expressing GFP-actin that these acini maintain an actin filament structure beneath the APM that is actively remodeled during exocytosis, and that non-muscle myosin II participates in some aspects of this remodeling.

Fig. 8.

BDM and ML-7 reduce CCH-stimulated exocytosis of syncollin-GFP in lacrimal acini. (A) Confocal fluorescence microscopy images acquired at the indicated times after CCH addition (100 μM) without inhibitor treatments (CCH) or with ML-7 treatment (40 μM, 15 minutes) or BDM treatment (10 mM, 15 minutes) prior to CCH addition. DIC and overlay images are also shown at 0 seconds. *, Lumen; bars, 5 μm. Plots to the right of each treatment group depict 2.5 D graphical reconstructions of the overall intensity profile of the imaged areas at 0 and 600 seconds of stimulation with CCH, illustrating individual intensities per pixel utilizing the rainbow scale. The resolution is ∼10 pixels per μm. (B) Syncollin-GFP release (plotted as percentage of control) in resting and CCH-stimulated (100 μM, 30 minutes) acini without or with BDM and ML-7 pretreatment as described above. *Significant from paired control at P≤0.05. Although the time-lapse sequences indicate rapid release of syncollin-GFP, the signal in the culture medium reached appropriate intensity for analysis by western blotting after 30 minutes, likely due to the time taken for exocytosed material in the lumen to diffuse into culture medium.

Fig. 8.

BDM and ML-7 reduce CCH-stimulated exocytosis of syncollin-GFP in lacrimal acini. (A) Confocal fluorescence microscopy images acquired at the indicated times after CCH addition (100 μM) without inhibitor treatments (CCH) or with ML-7 treatment (40 μM, 15 minutes) or BDM treatment (10 mM, 15 minutes) prior to CCH addition. DIC and overlay images are also shown at 0 seconds. *, Lumen; bars, 5 μm. Plots to the right of each treatment group depict 2.5 D graphical reconstructions of the overall intensity profile of the imaged areas at 0 and 600 seconds of stimulation with CCH, illustrating individual intensities per pixel utilizing the rainbow scale. The resolution is ∼10 pixels per μm. (B) Syncollin-GFP release (plotted as percentage of control) in resting and CCH-stimulated (100 μM, 30 minutes) acini without or with BDM and ML-7 pretreatment as described above. *Significant from paired control at P≤0.05. Although the time-lapse sequences indicate rapid release of syncollin-GFP, the signal in the culture medium reached appropriate intensity for analysis by western blotting after 30 minutes, likely due to the time taken for exocytosed material in the lumen to diffuse into culture medium.

The GFP-labeled apical actin filament array in lacrimal acini showed rapid CCH-induced remodeling of the subapical actin network: specifically, CCH elicited a significant increase in Mf and a significant decrease in t½ for these filaments. These parameters suggest that the overall dynamics of apical actin filaments are increased by secretagogue stimulation. These changes could occur through modulation of actin filament assembly and disassembly, increased diffusional mobility or even by active transport of filaments into the region. In addition to the changes elicited specifically in the apical actin filaments network immediately beneath the APM, we noted another striking phenomenon – transient actin assembly into actin-coated structures in the subapical cytoplasm, which appeared to contract towards the APM and to merge with apical actin. Non-muscle myosin II showed some co-localization with actin-coated structures in CCH-stimulated acini, suggesting that myosin-dependent contractile force might participate in their migration towards the APM. The average diameter of these actin-coated structures in CCH-stimulated acini was greater than that of a single SV, suggesting that they encompassed and possibly compressed dual or multiple SVs.

Fig. 9.

Syncollin-GFP is enriched in actin-coated structures in lacrimal acini exposed to BDM or ML-7. (A) Confocal micrographs of lacrimal acini transduced with Ad-Syncollin-GFP on day 2 of culture. On day 3 of culture, acini were stimulated with CCH (100 μM) for 5 minutes after pretreatment without (CON) or with BDM (10 mM) or ML-7 (40 μM) for 15 minutes. Cells were fixed with 4% paraformaldehyde to preserve Syncollin-GFP fluorescence (green) and labeled in parallel with rhodamine-phalloidin to visualize actin filaments (red). (B) Cells were treated and fixed as in A.; serial Z-stacks of images were obtained at 0.25 μm intervals and compiled into projection images in transparency mode using the 3D function of the Zeiss LSM software. Acinar thickness was ∼8-10 μm. Arrows, syncollin-GFP-enriched SVs beneath the actin-enriched lumen; arrowheads, syncollin-GFP-enriched SVs enveloped in actin coats; *, lumena; bars, 5 μm.

Fig. 9.

Syncollin-GFP is enriched in actin-coated structures in lacrimal acini exposed to BDM or ML-7. (A) Confocal micrographs of lacrimal acini transduced with Ad-Syncollin-GFP on day 2 of culture. On day 3 of culture, acini were stimulated with CCH (100 μM) for 5 minutes after pretreatment without (CON) or with BDM (10 mM) or ML-7 (40 μM) for 15 minutes. Cells were fixed with 4% paraformaldehyde to preserve Syncollin-GFP fluorescence (green) and labeled in parallel with rhodamine-phalloidin to visualize actin filaments (red). (B) Cells were treated and fixed as in A.; serial Z-stacks of images were obtained at 0.25 μm intervals and compiled into projection images in transparency mode using the 3D function of the Zeiss LSM software. Acinar thickness was ∼8-10 μm. Arrows, syncollin-GFP-enriched SVs beneath the actin-enriched lumen; arrowheads, syncollin-GFP-enriched SVs enveloped in actin coats; *, lumena; bars, 5 μm.

Our work made use of two different myosin inhibitors: BDM and ML-7. BDM has been utilized as an uncompetitive inhibitor of myosin ATPase activity (Higuchi and Takemori, 1989; Herrmann et al., 1992). Exposure of cells to BDM dissociates myosin from actin filaments, impairing myosin involvement in events as varied as muscle contraction (Herrman et al., 1992) and myosin-based vesicle transport (Bennett et al., 2001; Duran et al., 2003). The ability of BDM to inhibit myosins other than myosin II family members has recently been questioned (Ostap, 2002), and other work suggests that it may affect actin dynamics through myosin-independent mechanisms (Yarrow et al., 2003). ML-7 and the related inhibitor, ML-9, have been utilized extensively as selective inhibitors of myosin light chain kinase (Saitoh et al., 1987).

The use of BDM and ML-7 enabled us to resolve myosin-dependent and myosin-independent events associated with actin remodeling in exocytosis. The most prominent effect of BDM and ML-7 was their stabilization of actin-coated structures. These actin-coated structures appeared to represent fusion intermediates since (1) they encompassed the secretory protein, syncollin-GFP and (2) their accumulation was associated with inhibition of secretory product release. In addition to trapping of actin-coated fusion intermediates, BDM and ML-7 also significantly increased the average diameter of actin-coated intermediates while concomitantly eliciting a shift in the percentage of total SVs from single SVs towards dual and multiple SV aggregates. Although the magnitude of this change was small (e.g., from ∼10% to 25% of total vesicles), EM analysis of resting and stimulated acini has consistently shown that only a portion of the SVs are released in response to CCH. Comparison of the total numbers of SVs in resting and CCH-stimulated acini in Table 2 suggests release of about ∼25% of the total SVs within a 5 minutes interval. A shift to 20-25% of total vesicles incorporated in multivesicular aggregates suggests that essentially all fusing vesicles may be incorporated into these aggregates.

We propose that CCH-stimulated release of SVs at the APM of lacrimal acini involves at least two actin-facilitated processes: (1) formation of actin-coated fusion intermediates and their subsequent movement toward the APM and (2) rapid turnover of the apical actin filament network to increase accessibility of these fusion intermediates to the APM. We suggest that the formation of actin-coated fusion intermediates is triggered by the initiation of compound fusion of individual SVs in the cytosol immediately upon CCH stimulation. Evidence for compound fusion of SVs has previously been obtained in lacrimal acini (Satoh et al., 1997) and related models (Cochilla et al., 2000; Ishihara et al., 2000; Campos-Toimil et al., 2002). This model is also supported by our findings of a significant increase in the individual SV diameter in CCH-stimulated acini. We propose that compound fusion is accompanied by assembly of actin and non-muscle myosin II filaments beneath the fusing SVs, generating contractile forces which aid in compound fusion while also pushing the contents towards the APM for extrusion into the lumen. Evidence for the contractile role of non-muscle myosin II in compound vesicle fusion and compression is provided by the finding that the formation of multivesicular aggregates is increased by BDM and ML-7, while the increase in the diameter of individual SVs evoked by CCH is suppressed. Also consistent with this model, the diameter of the actin-coated structures formed in BDM- and ML-7-treated acini is significantly increased. The comparable stabilization of actin-coated structures elicited by both agents reinforces a common inhibition of non-muscle myosin II.

Fig. 10.

EM analysis of SV diameter in lacrimal acini exposed to BDM or ML-7, then CCH. The maximal SV diameter of vesicles categorized as single, dual fused or multiple fused was measured as described in Materials and methods in untreated lacrimal acini (CON) or acini treated without or with BDM (10 mM, 15 minutes) or ML-7 (40 μM, 15 minutes) prior to CCH (100 μM, 5 minutes). Vesicle diameter per category under each experimental condition is shown in the histogram while a sample of the vesicle type is outlined in blue in the EM image to the right. Arrows, filaments associated with a SV cluster; bars, 1 μm.

Fig. 10.

EM analysis of SV diameter in lacrimal acini exposed to BDM or ML-7, then CCH. The maximal SV diameter of vesicles categorized as single, dual fused or multiple fused was measured as described in Materials and methods in untreated lacrimal acini (CON) or acini treated without or with BDM (10 mM, 15 minutes) or ML-7 (40 μM, 15 minutes) prior to CCH (100 μM, 5 minutes). Vesicle diameter per category under each experimental condition is shown in the histogram while a sample of the vesicle type is outlined in blue in the EM image to the right. Arrows, filaments associated with a SV cluster; bars, 1 μm.

CCH stimulation caused filamentous myosin II immunofluorescence to co-localize with actin-coated structures, an effect which was not blunted by BDM or ML-7. This observation suggests that non-muscle myosin II assembly can occur in stimulated acini even in the absence of regulatory light chain phosphorylation (ML-7) or myosin ATPase activity (BDM). Non-muscle myosin II assembly is regulated at multiple levels by phosphorylation of its light and heavy chains (Bresnick, 1999). Although many studies with BDM and ML-7 have shown loss of non-muscle myosin II filaments, some studies reveal retention of myosin II filaments with these treatments. Recent studies investigating the role of myosin light chain phosphorylation in HeLa cells have shown that its activity is necessary for cell migration but not for recruitment and localization of myosin II with actin cytoskeleton at the leading edge (Fumoto et al., 2003). Assembly of non-muscle myosin II into filaments associated with fusing secretory vesicles in CCH-stimulated lacrimal acini appears to be a complex process that can occur even if the assembled motor is inactive.

We also propose that the turnover of the apical actin filament network triggered by CCH stimulation occurs as a means of enhancing the accessibility of actin-coated fusion intermediates enveloping fusing SVs to the APM. This additional role for actin filament dynamics in exocytosis is consistent with our findings that CCH stimulation significantly increased Mf and decreased t½ for this filament population. It is also consistent with our findings that LAT-induced apical actin disassembly significantly enhanced CCH-stimulated secretion of protein and syncollin-GFP. Apical actin filament turnover is likely to involve other factors directly, besides myosin motors, which can facilitate filament turnover by binding to actin filaments and monomers, and influencing their dynamics. Although BDM and ML-7 were able partially to suppress the CCH-stimulated changes in Mf and t½ of apical actin, it is unlikely that these effects are due to a direct role for myosin II in apical actin turnover. In stimulated acini, BDM and ML-7 sequester actin in actin-coated fusion intermediates that form a dense meshwork beneath the APM, which may indirectly influence apical actin filament turnover by altering cytoplasmic viscosity and/or reducing the amount of GFP-actin available for exchange. Moreover, inhibition of myosin's contractile function may impair the transport of filaments associated with actin-coated fusion intermediates towards the APM region, which may contribute to the total actin filament pool beneath the APM; this effect may also influence Mf and t½. The more potent effects of BDM on Mf and t½ relative to ML-7 may reflect its ability to influence actin filaments through myosin-independent processes (Yarrow et al., 2003).

Fig. 11.

LAT treatment of lacrimal acini enhances CCH-stimulated secretion in parallel with depletion of apical actin filaments. (A) Confocal fluorescence micrographs of lacrimal acini fixed and processed for labeling of actin filaments as described in Materials and Methods. Treatments included untreated lacrimal acini (CON) and acini exposed to LAT B (10 μM, 15 minutes) in the absence (LAT) or presence (LAT + CCH) of CCH (100 μM, 15 minutes). *, Lumenal regions; bar, 5 μm. (B) Effects of LAT B on bulk protein secretion (left) and syncollin-GFP release (right) without or with CCH stimulation. Values were normalized to cell protein before comparison across samples. *Significant at P≤0.05 from paired control value; n=6 for protein release and n=3 for syncollin-GFP release. (C) Left, EM image at lower magnification from rabbit lacrimal acini exposed to LAT A (1 μM, 15 minutes) and right, boxed region at higher magnification. Arrowheads depict SVs at the APM that are beginning to fuse. (D) Images showing premature fusion of mSVs in LAT A-treated, unstimulated acini. L, lumenal region; SV, secretory vesicle; bars (C,D), 500 nm.

Fig. 11.

LAT treatment of lacrimal acini enhances CCH-stimulated secretion in parallel with depletion of apical actin filaments. (A) Confocal fluorescence micrographs of lacrimal acini fixed and processed for labeling of actin filaments as described in Materials and Methods. Treatments included untreated lacrimal acini (CON) and acini exposed to LAT B (10 μM, 15 minutes) in the absence (LAT) or presence (LAT + CCH) of CCH (100 μM, 15 minutes). *, Lumenal regions; bar, 5 μm. (B) Effects of LAT B on bulk protein secretion (left) and syncollin-GFP release (right) without or with CCH stimulation. Values were normalized to cell protein before comparison across samples. *Significant at P≤0.05 from paired control value; n=6 for protein release and n=3 for syncollin-GFP release. (C) Left, EM image at lower magnification from rabbit lacrimal acini exposed to LAT A (1 μM, 15 minutes) and right, boxed region at higher magnification. Arrowheads depict SVs at the APM that are beginning to fuse. (D) Images showing premature fusion of mSVs in LAT A-treated, unstimulated acini. L, lumenal region; SV, secretory vesicle; bars (C,D), 500 nm.

Several groups have reported that the apical actin network beneath the APM of acinar epithelial cells from pancreas and parotid gland undergoes reorganization in stimulated acini (Perrin et al., 1992; Valentijn et al., 1999), leading to the `barrier' hypothesis proposing that apical actin filaments restrict access of SVs to the APM in resting acini while permitting access in stimulated acini. Other studies in pancreatic acini have shown that some actin filaments are necessary for exocytosis to proceed (Muallem et al., 1995), possibly for force generation. This study furthers our understanding of acinar exocytosis, supporting roles for apical actin filament turnover and non-muscle myosin II-mediated actin filament remodeling around actin-coated fusion intermediates as integral components of the exocytotic process.

Acknowledgements

This work was supported by NIH grant EY-11386 to S.F.H.-A. and EY-10550 to J.E.S. Additional salary support to S.F.H.-A. was from NIH grants EY-13949, EY-05081, NS-38246, DK-56040 and GM-59297. We thank Darren Michaels, Judy Garner and Austin Mircheff for helpful comments.

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