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An actin barrier to resealing

Katsuya Miyake1,*,{ddagger}, Paul L. McNeil2,{ddagger}, Kazunori Suzuki1, Rikiya Tsunoda1 and Naonori Sugai1

1 Second Department of Anatomy, School of Medicine, Fukushima Medical University, Fukushima 960-1295, Japan
2 Institute of Molecular Medicine and Genetics, Department of Cellular Biology and Anatomy, The Medical College of Georgia, Augusta, GA 30912-2000, USA
* Present address: Institute of Molecular Medicine and Genetics, Department of Cellular Biology and Anatomy, The Medical College of Georgia, Augusta, GA 30912-2000, USA



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  		Fig. 1. Confocal imaging of phalloidin staining of wounded RGM1 cells. At 15 seconds (a,b), 1 minute (c,d) or 30 minutes (e,f) after scratching a monolayer in medium containing FDx, cultures were fixed and stained with TRITC-phalloidin. Shown are paired, confocal microscope images of fluorescein fluorescence indicating cell permeation with FDx as a result of a plasma membrane disruption event (a,c,e) and rhodamine fluorescence indicating F-actin distribution (b,d,f). At 15 seconds post-disruption-injury, FDx-labeled zones of cytoplasm bordering on the scratch site were (a) often apparently depleted of F-actin (b, arrowhead). By contrast, at 1 or 30 minutes post disruption-injury, FDx labeled cells that had survived a disruption (c,e) characteristically displayed very strong phalloidin staining in cortex bordering on the scratch site (d,f, arrowheads). Bar, 10 µm. W, wounded; NW, not wounded.

 


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  		Fig. 2. Flow cytofluorometric analysis of phalloidin staining of RGM1 cells as a function of the presence or absence of Ca2+ during wounding. (a) Cells were wounded by scraping them from the substratum or incubated undisturbed in FDx for an equivalent interval. Flow cytometry was then used to measure the fluorescence of 10,000 cells in the undisturbed (gray field) and scraped (bold trace) populations. Greater than 92% of the scraped (W, demarcated by right arrow) population fell above a fluorescence threshold set to contain 95% of the undisturbed population (NW, demarcated by left arrow), indicating that this percentage incurred plasma membrane disruptions as a result of scraping. (b) Cells were scraped from the substratum in PBS containing 1.5 mM Ca2+ (Ca2+; bold trace) or containing no added Ca2+ and 1.0 mM EGTA (EGTA; gray filled). Flow cytometry was used to measure the fluorescence of 10,000 cells in each population. (c) The mean (and s.d.) of the FITC-phalloidin fluorescence measured by flow cytofluorometry from 4 populations scraped separately from four dishes, in EGTA (EGTA)- or Ca2+ (Ca2+)-containing medium. Applying the Kruskal-Wallis test to these two data sets gave P<0.05.

 


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  		Fig. 3. Electron microscope comparison of native RGM1 cytoplasm with cytoplasm surrounding a disruption site. (a) A section through the cortex of a RGM1 cell not subject to shear stress but incubated in HRP. Note the lack of HRP staining of cytoplasm typical of such an incubation carried out in the absence of concurrent plasma membrane disruption. (b) A section through the cortex of a cell syringed in HRP. Cytosolic labeling with HRP indicates that this cell experienced a plasma membrane disruption. Typical of such cells is the striking accumulation of abnormally large vesicles in cortical cytoplasm. Bars, 0.5 µm.

 


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  		Fig. 4. Cytochalasin B treatment of undisturbed RGM1 cells does not effect resealing. (a) Control RGM1 cells stained with TRITC-phalloidin. (b) Cytochalasin B (5 minutes, 3 µg/ml)-treated cells stained with TRITC-phalloidin. Note that, by comparison with the untreated cells in (a), there is a marked disruption of cortical and stress fiber F-actin staining in these cells. (c) Nearly complete closure of monolayer injury sites was observed 2 hours after scratching a cover slip in PBS. (d) Closure failure was observed when cytochalasin B (3 µg/ml, added 5 minutes before scratching) was added to the PBS. (e) Fixable FDx (Mr 10,000)-positive cells, which suffered and survived a plasma membrane disruption, line a control wound site created in the presence of this marker (10 mg/ml) added to PBS. (f) FDx-positive cells, in apparently equivalent density, line a wound site in a culture treated with cytochalasin. (g) Quantitation of the density cell survivors of PMD along scratch sites. Scratch-loading was performed in PBS containing FDx and no additive (Cont), 5 mM-EGTA (EGTA) or 5 µg/ml cytochalasin B added 5 minutes before monolayer injury (CyB), and the number of FDx-positive cells lining a 100 mm distance along scratch edges counted for each condition. The mean and s.d. of three separate experiments are shown. Comparison of the EGTA treated and control samples yielded P<0.05 (*) (Kruskal-Wallis test), and for cytochalasin and control samples P=0.51269 (**). Bars, 30 µm.

 


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  		Fig. 5. DNase1-mediated actin depolymerization can enhance, and phalloidin and jasplakinolide stabilization can inhibit, resealing of RGM1 cells. (a) The total amount of ATP released by cells upon syringe injury was measured in a chemiluminesence assay as a function of phalloidin (Pha-2; 2 µg/ml; or Pha-20; 20 µg/ml) or DNase1 (DN-2; 2 µg/ml; or DN-20; 20 µg/ml) loading by a first syringing event. Control (Cont) cells were subject to the loading procedure (syringing) carried out in PBS with no additives. Total cell population content of ATP was measured after lysis initiated by the addition of a kit-supplied cell lysis reagent (All). Values plotted represent mean and s.d. of three experiments, and differed significantly from the control (Cont) condition (P<0.05, Kruskal-Wallis test). (b) The total amount of ATP released upon no injury (NW), and upon syringe injury of cells pre-incubated for 20 minutes in plain PBS (Cont), jasplakinolide (Jasp; 3 µg/ml) or cytochalasin B (CyB; 3 µg/ml). Values represent the mean and s.d. of three experiments. Comparison of the control and jasplakinolide samples yields P<0.05 (Kruskal-Wallis test). Plasma membrane disruptions were induced in an automated syringe-loading device designed to inflict a reproducible level of mechanical stress on cells (Clarke and McNeil, 1992).

 


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  		Fig. 6. Inhibition of resealing by biologically mediated actin polymerization is reversible with cytochalasin or DNase1. (a) RGM1 cells lining a scratch site 30 minutes after its creation were heavily stained in their cortices with TRITC-phalloidin. (b) When this scratch maneuver was carried out in FDx, so that those cells along the scratch site incurring plasma membrane disruptions could be identified, those cells most heavily labeled with TRITC-phalloidin were co-labeled with the FDx marker of a plasma membrane disruption event (arrowheads). (c) A monolayer was scratched first in PBS containing cytochalasin B (3 µg/ml) and FDx (10 mg/ml), and then rinsed with fresh medium minus cytochalasin. Two hours later it was scratched a second time (scratch lines approximately at right angle to first set) in the presence of PBS containing fixable Texas Red-labeled dextran (TRDx; Mr 70,000; 10 mg/ml). Double-labeled (yellow) cells (arrows) are clearly present in the population and represent those that survived two PMD events. (d) A monolayer was treated identically to that above, except that it was scratched first in PBS minus cytochalasin. No double-labeled (yellow) cells are observed. (e) The percentage of yellow (double-labeled) relative to green cells (single label from first scratch only) observed as a function of scratching the first time plus (CyB+) or minus (CyB-) cytochalasin B (3 µg/ml). The number of yellow cells relative to green were scored along a total of 200 mm of scratch site length in five separate experiments. The mean and s.d. of these measurements are plotted. Comparison of the two data sets yielded P=0.00882 (Kruskal-Wallis test). (f) Analysis of cells surviving two disruptions made by glass beads instead of scratching the monolayer. During the first plasma membrane disruption event induced by beads, the PBS medium contained no additives (CyB-), or cytochalasin B at 3 µg/ml (CyB+) or DNase1 at 100 µg/ml (DN1). The data represents the mean and s.d. of five experiments. The CyB- (*) sample differs significantly from the other two samples (P< 0.01, Kruskal-Wallis test). Bars, 20 µm.

 


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  		Fig. 7. Proposed role of actin depolymerization in resealing. (a) A cortical barrier to vesicle movement consisting, in part, of filamentous actin is present in undisturbed cells. (b) A plasma membrane disruption allows entry of Ca2+ that promotes consequent Ca2+-induced actin depolymerization locally in peri-disruption cortex. (c) This facilitates Ca2+-induced membrane fusion events required for resealing by removing an obstacle to vesicle-vesicle and vesicle-membrane interaction. (d) Actin polymerization occurring in wounded and other cells lining injury sites many minutes after PMD events may be required for successful restitution or repair of monolayer injury.

 

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© The Company of Biologists Ltd 2001