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First published online 15 November 2005
doi: 10.1242/jcs.02639

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Actin at cell-cell junctions is composed of two dynamic and functional populations

Juankun Zhang^1,*, Martha Betson^1,*,, Jennifer Erasmus¹, Kostas Zeikos¹, Maryse Bailly², Louise P. Cramer³ and Vania M. M. Braga^1,

¹ Molecular and Cellular Medicine, Faculty of Life Sciences, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK
² Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK
³ Laboratory for Molecular Cell Biology and Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (70K): [in a new window]   Fig. 1. Spatial distribution of actin populations during induction of cell-cell contacts. (A) Keratinocytes grown in low calcium medium (Low Ca2+), which does not induce cell-cell contacts, were induced to form cadherin-dependent cell-cell adhesion (standard calcium medium, Std. Ca2+). After junction formation, two actin populations can be distinguished on the basis of their location: one is present as a wavy, punctate line at junctions (junctional actin, arrowheads); the second population is a tighter array of thin bundles in the cytoplasm, flanking the junctional-actin population (arrow). By 60 minutes, the two populations are less readily distinguished (mature junctional actin). Bar, 50 µm. (B) Percentage of keratinocytes classified into four different categories according to the spatial organization of thin bundles following junction formation. Categories observed: (1) a wide, loose band of actin filaments, and no F-actin at cell-cell borders; (2) F-actin at junctions is visible (junctional actin) and the band of filaments localizes closer to cell-cell contacts; (3) junctional actin is more intense; filaments are more tightly bundled in a narrower region proximal to junctions or indistinguishable from F-actin at cell-cell contacts; (4) other phenotypes (less than 5-10% of total). Results are the average of two independent experiments, in which 200 cells were scored per time point in each experiment. (C) Following induction of cell-cell contacts in sub-confluent keratinocytes, cytoskeletal changes show a delayed formation of junctional actin and reorganization of thin bundles. For example, thin bundles are not coincident with junctional actin after 120 minutes of junction formation. Additional actin structures are also seen before junctional actin is properly stabilized (i.e. kissing structures, inset 30 minutes). Presumably these structures participate in the migration of two neighbouring cells towards each other as they are not readily observed in confluent cultures.

View larger version (106K): [in a new window]   Fig. 2. Junctional actin and thin bundles have distinct dynamics. Keratinocytes grown in low calcium medium (Low Ca2+), which does not induce cell-cell contacts were microinjected with labelled actin (3 mg/ml pipette concentration), and transferred immediately to standard calcium medium to induce cell-cell contacts (Std. Ca2+). Control cells were maintained in low calcium medium. Arrowheads point to junctional actin; arrows show thin bundles. (A) Junctional actin has fast dynamics. After junction formation, images were collected at places where E-cadherin clustering was visible and at the corresponding incorporated actin. (B) Thin bundles have slower kinetics of actin incorporation. Images were collected where filamentous actin (total actin) was present and the corresponding new actin labelling at the bundles (incorporated actin). In the absence of cell-cell contacts (Low Ca2+), very little actin incorporation was detected into thin bundles. (C) Quantification of actin dynamics. Junctional-actin fluorescence intensity was measured only where cadherin recruitment at junctions was observed. Thin bundles were detected in phalloidin stained images and the fluorescence intensity of labelled actin was measured in the corresponding area. (D) Thin bundles are less sensitive than junctional actin to latrunculin treatment. Keratinocytes were induced to form contacts (5 and 15 minutes, Std. Ca2+) in the presence of 0.2 µM latrunculin B (Latr.) or vehicle (DMSO); cells were fixed and stained with phalloidin (total actin) and anti-E-cadherin antibodies. Although the amount and organization of thin bundles are affected by latrunculin, thin bundles are clearly seen after 5 minutes incubation, when the majority of junctional actin was removed. (E) Quantification of the proportion of cells showing thin bundles and junctional actin in controls (C) or after latrunculin treatment (Latr., at 5 and 15 minutes). Error bars represent error obtained from at least two independent experiments. Bar, 50 µm.

View larger version (68K): [in a new window]   Fig. 3. Actin assembly mechanisms. (A) G- and F-actin pool were isolated from keratinocytes treated with 0.2 µM latrunculin, or left untreated, for 5 minutes. After separation using SDS-PAGE and Coomassie staining, bands were quantified. After latrunculin treatment, there were increased levels of G-actin, with a concomitant reduction in the amount of F-actin. (B) Global levels of G-actin and F-actin pools were quantified by immunofluorescence during a time course after induction of cell-cell contacts. Keratinocytes were stained with DNAse1 (G-actin) and phalloidin (F-actin) and the whole optical field quantified for each fluorophore. No significant changes were observed in the relative concentration of either actin pool during the time course examined by staining or SDS-PAGE (data not shown). Values are expressed as percentage of total actin. (C) Quantification of immunofluorescence of G- and F-actin pools at the junctional-actin region. Only junctions where junctional actin was clearly separated from thin bundles were examined. At time zero, areas of close apposition of neighbouring membranes were quantified. Data in A-C are representative of two independent experiments. Error bars represent standard deviation. *P<0.05. (D) Polymerization of actin at cell-cell junctions occurs preferentially via the barbed end. After formation of new contacts for different amounts of time, cells were permeabilized for 2 minutes in cytoskeleton buffer containing labelled Alexa Fluor 568-G-actin (0.45 µM), in the presence or absence of 2 µM cytochalasin B (see text for details). After fixation, E-cadherin, total actin (phalloidin) and incorporated actin were visualized. Results are representative of at least three independent experiments. Arrows indicate labelled actin incorporation at junctions. Bar, 50 µm.

View larger version (89K): [in a new window]   Fig. 4. Actin filament disassembly does not play a major role in the formation of either thin bundles or junctional actin. (A) Recently disassembled filaments do not contribute monomers for incorporation at cell-cell junctions. Cell-cell contacts were induced (Std. Ca2+) in the presence of 0.5 µM jasplakinolide (Jasp.) or vehicle control (methanol, MeOH). Cells were stained with anti-E-cadherin and anti-actin antibodies, because jasplakinolide competes with phalloidin for F-actin interaction. (B) Actin disassembly is not required for bundle reorganization. Keratinocytes expressing GFP-actin were treated with 0.02 µM jasplakinolide (Jasp.) or methanol (MeOH) during junction assembly. After 60 minutes, thin bundles were visible either as flanking filaments to junctional actin (middle images Jasp.) or coincident to junctions (bottom images, Jasp.). Arrows indicate thin bundles; arrowheads point to junctional actin. (C) Keratinocytes were classified into four different categories according to the spatial localization of thin bundles as described in Fig. 1 legend. The same proportion of cells was found in category 2 and 3 for both the Jasplakinolide-treated (Jasp.) and untreated (control) cells. Bar, 50 µm.

View larger version (129K): [in a new window]   Fig. 5. Contribution of myosin contractility to junctional actin and thin bundles. Confluent keratinocytes grown in low calcium medium (Low Ca2+) or induced to form junctions (Std. Ca2+) for 1 hour were incubated in the presence or absence of blebbistatin (Bleb.). Controls were treated with DMSO. Enlargements of the boxed regions in each image are shown on the right. In the absence of cell-cell contacts (Low Ca2+) thin bundles are present in control (DMSO-treated) cells, but not in blebbistatin-treated cells (Bleb.) After cell-cell adhesion, junctional actin is formed in the absence of thin bundles (Bleb. Std. Ca2+). Asterisks indicate the absence of thin bundles in blebbistatin-treated keratinocytes; arrow indicates junctional actin and arrowheads, wavy junctions. Images are representative of at least three independent experiments. Bar, 50 µm.

View larger version (84K): [in a new window]   Fig. 6. Following cell-cell junction formation, phosphorylated myosin light chain (P-MLC) localizes at thin bundles but not junctional actin. Keratinocytes grown in low calcium medium (Low Ca2+) were induced to form junctions (Std. Ca2+) and stained with phalloidin and anti-P-MLC. Merged confocal images are shown on the right; Enlargements of the boxed regions are shown below each image. After induction of cell-cell contacts, P-MLC is concentrated at peripheral thin bundles, but absent from junctional actin (Std. Ca2+). Arrowheads show junctional actin, arrows point to thin bundles and localization of P-MLC. Asterisks show absence of P-MLC at junctional actin. Bar, 32 µm; 8 µm for enlargements.

View larger version (26K): [in a new window]   Fig. 7. Spatial and global quantification of P-MLC levels during keratinocyte polarization. (A) P-MLC levels were quantified at thin bundles and in the cell body from confocal images obtained after 30 minutes of induction of cell-cell contacts. Following junction formation (Std. Ca2+), an increase in fluorescence intensity is seen both at thin bundles (2.7 fold) and in the cell body (1.4 fold). Values are from two independent experiments, using at least 50 different cells per condition. (B) Western blots showing the overall increase in P-MLC levels in keratinocytes. Quantification reveals that a threefold increase in phosphorylation levels is seen after 15 minutes of new junction formation (P<0.001). Values are the mean of four independent experiments.

View larger version (94K): [in a new window]   Fig. 8. Inhibition of contractility during keratinocyte polarization perturbs the acquisition of maximum height at lateral domains. E-cadherin (red) and phalloidin (green) staining was performed after 1 hour of cell-cell contact formation in the absence (control) or presence of blebbistatin (blebbistatin). Confocal sections (0.3 µm) were collected at different levels through a keratinocyte monolayer and processed as follows. (A) Three dimensional reconstruction. After blebbistatin treatment, F-actin is diffusely localized and cadherin staining is found as a wavy line between neighbouring cells. Small ruffles/projections are seen at the cell surface and on top of junctions (arrows). (B) Confocal sections taken from basal to top of the monolayer at 0, 1.5, 3, 4.5 and 6 µm. Arrow points to small ruffles/projections sees at the top of junctions. Arrowhead shows cadherin staining in controls whereas no similar linear cadherin staining is observed in blebbistatin-treated cells. (C) Quantification of the maximum height of the lateral domains (basal to apical distance). Confocal sections showing linear cadherin staining were quantified and values expressed relative to control, untreated cells polarized for 1 hour (see Materials and Methods for details). Maximum height seen in lateral domain of control cells is 4.5 µm compared with 3 µm in blebbistatin-treated cells and 2 µm in cells maintained in low calcium (Braga et al., 1998). After blebbistatin treatment, lateral domain height is decreased by 35% (*P<0.001). Results represent the mean of seven different Z-series collected from two independent experiments.

View larger version (37K): [in a new window]   Fig. 9. Summary of changes to the microfilament network triggered by formation of cadherin-dependent cell-cell contact. (A) Mechanisms of formation and functions of junctional actin and thin bundles. After cadherin receptors cluster, two distinct processes are observed: new actin polymerization and filament reorganization. Actin assembly is a major contributor to junctional actin (thick brown arrow), but has a minor effect on thin bundles (dashed thin brown arrow). In contrast, myosin function is essential for bundle stability, but does not affect initial clustering of cadherin receptors or junctional-actin formation. All the above events do not occur in cells maintained in low calcium medium or in which cadherin function is blocked. Two separate functions are identified (red arrows): junctional actin stabilizes cadherin receptors at puncta and thin bundles are essential for development of maximum height of the lateral domains. At later time points, these two actin populations apparently colocalize at cell-cell contacts to form mature junctional actin (merged blue and yellow lines; see below). *Adams et al., 1996; Nelson, 2003. (B) Temporal and spatial events. After initiation of cell-cell adhesion, distinct cellular processes are detected at different time points (horizontal bars). Once initiated, each event increases until a plateau after 60 minutes of cell-cell adhesion. E-cadherin clustering at puncta is observed within a couple of minutes, and is followed shortly by new actin incorporation as junctional actin ( minutes, fast dynamics) and then by thin bundle labelling (approximately minutes; slower dynamics). Increased P-MLC localization at bundles and bundle reorganization occurs later (from 15 minutes). The end result is the formation of a cuboidal epithelial morphology, with the spatial organization of microfilaments as shown. A possible mechanism, supported by our results, is myosin-dependent contraction of thin bundles from a loose band of filaments at the periphery to filaments that are coincident with junctional actin. Please see text for functional implications and more details. Arrows indicate bundles; arrowheads indicate junctional actin, open arrow indicates mature junctional actin.