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Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

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The α6β4 integrin redistributes from hemidesmosomes to lamellipodia and ruffles after EGF stimulation. Indirect immunofluorescence analysis of A431 cells plated on laminin-1–coated dishes for 2 h and either stimulated with EGF (E and F; 1 ng/ml) or left without treatment (A–D). All cells were processed for double-staining with the α6-specific rat mAb GoH3 (red) and either a mouse mAb specific for one of the following hemidesmosome components (green): BPAG-1 (A), BPAG-2 (B), HD1/plectin (C and F), or (green) FITC-phalloidin (D and E). In these photomicrographs, the yellow color is indicative of colocalization of two antigens. All images correspond to confocal sections taken at the basal plane of the cell except E that was taken at a suprabasal plane. Thick arrows indicate hemidesmosomes, arrowheads indicate lamellipodia and ruffles, and thin arrow indicates F-actin stress fibers. Bar, 10 μm.
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Figure 1: The α6β4 integrin redistributes from hemidesmosomes to lamellipodia and ruffles after EGF stimulation. Indirect immunofluorescence analysis of A431 cells plated on laminin-1–coated dishes for 2 h and either stimulated with EGF (E and F; 1 ng/ml) or left without treatment (A–D). All cells were processed for double-staining with the α6-specific rat mAb GoH3 (red) and either a mouse mAb specific for one of the following hemidesmosome components (green): BPAG-1 (A), BPAG-2 (B), HD1/plectin (C and F), or (green) FITC-phalloidin (D and E). In these photomicrographs, the yellow color is indicative of colocalization of two antigens. All images correspond to confocal sections taken at the basal plane of the cell except E that was taken at a suprabasal plane. Thick arrows indicate hemidesmosomes, arrowheads indicate lamellipodia and ruffles, and thin arrow indicates F-actin stress fibers. Bar, 10 μm.

Mentions: Indirect immunofluorescence microscopy revealed that the α6β4 integrin on the ventral surface of A431 cells plated on either laminin-1 (Fig. 1A–D) or collagen (not shown) is localized primarily in discrete structures and plaques in areas that exclude stress fibers. This pattern of staining for α6β4 is characteristic of its localization in hemidesmosomes 1146. Moreover, a distinct colocalization of α6β4 with the hemidesmosomal components BPAG-1 (Fig. 1 A), BPAG-2 (Fig. 1 B), and HD1/plectin (Fig. 1 C) was evident in these structures. These data establish that α6β4 is localized in structures that are characteristic of hemidesmosomes on the ventral surface of adherent A431 cells.


Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

The α6β4 integrin redistributes from hemidesmosomes to lamellipodia and ruffles after EGF stimulation. Indirect immunofluorescence analysis of A431 cells plated on laminin-1–coated dishes for 2 h and either stimulated with EGF (E and F; 1 ng/ml) or left without treatment (A–D). All cells were processed for double-staining with the α6-specific rat mAb GoH3 (red) and either a mouse mAb specific for one of the following hemidesmosome components (green): BPAG-1 (A), BPAG-2 (B), HD1/plectin (C and F), or (green) FITC-phalloidin (D and E). In these photomicrographs, the yellow color is indicative of colocalization of two antigens. All images correspond to confocal sections taken at the basal plane of the cell except E that was taken at a suprabasal plane. Thick arrows indicate hemidesmosomes, arrowheads indicate lamellipodia and ruffles, and thin arrow indicates F-actin stress fibers. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169473&req=5

Figure 1: The α6β4 integrin redistributes from hemidesmosomes to lamellipodia and ruffles after EGF stimulation. Indirect immunofluorescence analysis of A431 cells plated on laminin-1–coated dishes for 2 h and either stimulated with EGF (E and F; 1 ng/ml) or left without treatment (A–D). All cells were processed for double-staining with the α6-specific rat mAb GoH3 (red) and either a mouse mAb specific for one of the following hemidesmosome components (green): BPAG-1 (A), BPAG-2 (B), HD1/plectin (C and F), or (green) FITC-phalloidin (D and E). In these photomicrographs, the yellow color is indicative of colocalization of two antigens. All images correspond to confocal sections taken at the basal plane of the cell except E that was taken at a suprabasal plane. Thick arrows indicate hemidesmosomes, arrowheads indicate lamellipodia and ruffles, and thin arrow indicates F-actin stress fibers. Bar, 10 μm.
Mentions: Indirect immunofluorescence microscopy revealed that the α6β4 integrin on the ventral surface of A431 cells plated on either laminin-1 (Fig. 1A–D) or collagen (not shown) is localized primarily in discrete structures and plaques in areas that exclude stress fibers. This pattern of staining for α6β4 is characteristic of its localization in hemidesmosomes 1146. Moreover, a distinct colocalization of α6β4 with the hemidesmosomal components BPAG-1 (Fig. 1 A), BPAG-2 (Fig. 1 B), and HD1/plectin (Fig. 1 C) was evident in these structures. These data establish that α6β4 is localized in structures that are characteristic of hemidesmosomes on the ventral surface of adherent A431 cells.

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

Show MeSH
Related in: MedlinePlus