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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02573

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Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

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

View larger version (98K): [in a new window]   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.

View larger version (294K): [in a new window]   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.

View larger version (75K): [in a new window]   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 IRDyeTM 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.

View larger version (83K): [in a new window]   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.

View larger version (53K): [in a new window]   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/Fi–F0. (C) Mf values for apical actin filaments in resting and stimulated acini analyzed after the indicated exposures to CCH. (D) Turnover times (t) 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; *P0.05.

View larger version (70K): [in a new window]   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 P0.05 from comparable value in control cells and n=5-6 preparations.

View larger version (69K): [in a new window]   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.

View larger version (64K): [in a new window]   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 P0.05. 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.

View larger version (63K): [in a new window]   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.

View larger version (88K): [in a new window]   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.

View larger version (81K): [in a new window]   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 P0.05 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.

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