Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell-cell contacts in cultured epitheliocytes - PubMed (original) (raw)

Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell-cell contacts in cultured epitheliocytes

M Krendel et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The spatial organization of cell-cell adherens junctions is distinct in cultured cells from two different tissue types, specifically, epitheliocytes and fibroblasts. In epitheliocytes, contacts are localized tangentially, along contacting cell edges and in association with circumferential actin bundles. Contacts between fibroblasts are radially oriented; that is, they are perpendicular to the overlapping edges of the cells and are associated with straight bundles of actin filaments. In the present study, we establish that the spatial organization of cell-cell contacts in the epithelial cell line IAR-2 can be converted from the typical tangential pattern to the radial pattern observed in fibroblasts. This transition can be induced by treatment with two agents, phorbol 12-myristate 13-acetate and nocodazole, which have different modes of action. Inhibition of myosin contractility reverses tangential-to-radial conversion of cell-cell contacts. These data suggest that formation of radially aligned contacts depends on modulation of contractility within the actin cytoskeleton through the myosin motor protein. The results open the possibility that modulation of the spatial organization of cell-cell contacts may play important roles in regulating organization and physiological functions of epithelial tissues.

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Figures

Figure 1

Organization of the actin cytoskeleton and cell–cell contacts in control IAR-2 cells. Monolayers of IAR-2 cells were fixed and either double-labeled for F-actin (A and B) and β-catenin (C), F-actin (D), and myosin-II (E) or single-labeled for E-cadherin (F). Confocal images in A_–_C are optical sections of the same cells at the level of the apical cell surface (A) or 0.6 μm below the cell surface (B and C). Confocal images of the apical cell surface (A and D) revealed the presence of thin parallel bundles of actin filaments that exhibited punctate localization of myosin-II along the bundles (E). Apical actin bundles appeared to be connected to circumferential actin bundles (B), which colocalized with β-catenin (C). Adhesion belts in IAR-2 cells contained both both β-catenin (C) and E-cadherin (F). (Bars = 10 μm.)

Figure 2

Effect of PMA on contact formation during wound healing. Contact formation by control (A) or PMA-treated cells (B and C) was documented by using video–differential interference contrast microscopy. B and C represent images of the same cells taken with a 16-min interval. During contact formation in control IAR-2 cells (A), free cell edges adjacent to the contact zone appeared stretched and contained few lamellipodia. Edges of PMA-treated cells contained numerous lamellipodia, which continued to form even after the establishment of the initial cell–cell contact (B). A new leading lamella (arrow) formed at the free edge of a PMA-treated cell resulting in cell reorientation even after contact expansion and increased cell–cell overlapping (C). (Bar = 10 μm.)

Figure 3

Organization of the actin cytoskeleton and cell–cell contacts in PMA-treated cells. IAR-2 cells were allowed to form new cell–cell contacts in the presence of PMA alone (A_–_C) or PMA and p160 Rho-dependent kinase inhibitor HA-1077 (D_–_F). Cells were fixed and double-stained for actin filaments (A and D) and β-catenin (B and E). C and F are superimposed images of actin and β-catenin staining in which actin is shown in red and β-catenin in green. Yellow color indicates areas in which actin and β-catenin are colocalized. (A_–_C) PMA-treated cells contained numerous lamellipodia and thick bundles of actin filaments radiating outward from the cell center (A). Cell–cell contacts formed in the presence of PMA consisted of parallel radial strands located in the lamellae that overlapped the surface of the adjacent cell (B, arrow). Radial strands of β-catenin often colocalized with the ends of actin filament bundles (C). (D–F) Inhibition of myosin contractility with HA-1077 resulted in the loss of actin filament bundles (D) and loss of radial cell–cell contacts (E). Thin, scallop-shaped linear cell–cell contacts in cells treated with HA-1077 often formed at the base (F, arrow) of lamellae (F, arrowhead) that overlapped with the surface of an adjacent cell. (Bar = 10 μm.)

Figure 4

Localization of E-cadherin to cell–cell contacts in control and PMA-treated epithelial cells. IAR-2 cells forming new cell–cell contacts either in the absence (A) or presence (B) of PMA were fixed and stained with anti-E-cadherin antibodies. In control cells, E-cadherin accumulated as a thin tangential cell–cell contact (A) whereas in PMA-treated cells, E-cadherin was localized to numerous radial strands. (Bars = 10 μm.)

Figure 5

Organization of cell–cell contacts and the actin cytoskeleton after treatment with nocodazole. Monolayers of IAR-2 cells were treated with nocodazole for 1 hr either in the absence (A_–_C) or presence (D) of HA-1077. Cells were fixed and either were double-stained for actin filaments (A) and β-catenin (B) or were single-labeled with anti-E-cadherin antibodies (C and D). In cells treated with nocodazole, the ends of the apical bundles of actin filaments were thickened, and they appeared to colocalize with β-catenin (compare arrowheads in A and B). Additionally, there were numerous contact strands extending radially away from cell–cell contacts (C) and the tangential cell–cell contacts appeared somewhat fragmented (C, arrow). Inhibition of p160 Rho-dependent kinase using HA-1077 resulted in disappearance of radial strands and restoration of tangential contacts (D). (Bars = 10 μm.)

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Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell-cell contacts in cultured epitheliocytes - PubMed (original) (raw)