1 Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, PSC 406A, Los Angeles, CA 90033, USA
2 Department of Cell and Neurobiology, University of Southern California, 1985 Zonal Avenue, PSC 406A, Los Angeles, CA 90033, USA
3 Department of Physiology and Biophysics, University of Southern California, 1985 Zonal Avenue, PSC 406A, Los Angeles, CA 90033, USA
4 Department of Ophthalmology, University of Southern California, 1985 Zonal Avenue, PSC 406A, Los Angeles, CA 90033, USA
5 Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA
6 Department of Pathology, Emory University, 201 Dowman Drive, Atlanta, GA 30322, USA
* Author for correspondence (e-mail: shalvar{at}usc.edu)
Accepted 6 July 2005
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Summary |
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Key words: Secretion, Fluorescence recovery after photobleaching, Confocal microscopy, Actin, Myosin
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Introduction |
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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.
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Materials and Methods |
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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 ß-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-XTM virus purification kit was used for virus purification and the Adeno-XTM 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 2x106 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 IRDyeTM800-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 4x106 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:
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For analysis of actin filament turnover time (t), 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 t
(or tDeff) is related to Deff, the effective diffusion coefficient, by the equation:
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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 7500x 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.
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Results |
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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).
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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).
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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.
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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 (t) calculated from individual recovery plots such as those in Fig. 5B as described in Materials and Methods. Actin filament t
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).
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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.
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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 ß-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).
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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.
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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.
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).
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Discussion |
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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 t 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.
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.
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 t 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 t
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 t
. The more potent effects of BDM on Mf and t
relative to ML-7 may reflect its ability to influence actin filaments through myosin-independent processes (Yarrow et al., 2003
).
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.
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Acknowledgments |
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Footnotes |
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References |
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