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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 associates with F-actin in response to EGF. A431 cells were plated on laminin-1 for 1 h before stimulation with EGF (1 ng/ml) for 15 min. The cells were extracted with a Triton X-100–containing buffer and then fixed as described in Materials and Methods. The fixed cells were double-stained for indirect immunofluorescence analysis using (A and C) GoH3 mAb and (B and D) FITC-phalloidin. Arrows indicate points of colocalization. Bars, 10 μm.
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Figure 4: The α6β4 integrin associates with F-actin in response to EGF. A431 cells were plated on laminin-1 for 1 h before stimulation with EGF (1 ng/ml) for 15 min. The cells were extracted with a Triton X-100–containing buffer and then fixed as described in Materials and Methods. The fixed cells were double-stained for indirect immunofluorescence analysis using (A and C) GoH3 mAb and (B and D) FITC-phalloidin. Arrows indicate points of colocalization. Bars, 10 μm.

Mentions: The localization of α6β4 in membrane ruffles and lamellipodia that form in response to EGF stimulation prompted us to explore the possibility of its association with F-actin in these structures because we had previously observed such an association in colon carcinoma cells 44. We found that the colocalization of α6β4 with F-actin in cell protrusions detected by immunofluorescence was retained in a significant number of protrusions after extraction of EGF-stimulated cells with a Triton X-100 buffer that preserves the actin cytoskeleton (Fig. 4A and Fig. B). However, extraction of these EGF-stimulated cells with the Tween-40/DOC buffer described above eliminated both the F-actin and α6β4 staining (data not shown). Taken together, these findings indicate that EGF stimulates a dissociation of α6β4 from cytokeratin-associated hemidesmosomes, as well as the formation of lamellipodia and ruffles that contain α6β4 in association with F-actin.


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 associates with F-actin in response to EGF. A431 cells were plated on laminin-1 for 1 h before stimulation with EGF (1 ng/ml) for 15 min. The cells were extracted with a Triton X-100–containing buffer and then fixed as described in Materials and Methods. The fixed cells were double-stained for indirect immunofluorescence analysis using (A and C) GoH3 mAb and (B and D) FITC-phalloidin. Arrows indicate points of colocalization. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The α6β4 integrin associates with F-actin in response to EGF. A431 cells were plated on laminin-1 for 1 h before stimulation with EGF (1 ng/ml) for 15 min. The cells were extracted with a Triton X-100–containing buffer and then fixed as described in Materials and Methods. The fixed cells were double-stained for indirect immunofluorescence analysis using (A and C) GoH3 mAb and (B and D) FITC-phalloidin. Arrows indicate points of colocalization. Bars, 10 μm.
Mentions: The localization of α6β4 in membrane ruffles and lamellipodia that form in response to EGF stimulation prompted us to explore the possibility of its association with F-actin in these structures because we had previously observed such an association in colon carcinoma cells 44. We found that the colocalization of α6β4 with F-actin in cell protrusions detected by immunofluorescence was retained in a significant number of protrusions after extraction of EGF-stimulated cells with a Triton X-100 buffer that preserves the actin cytoskeleton (Fig. 4A and Fig. B). However, extraction of these EGF-stimulated cells with the Tween-40/DOC buffer described above eliminated both the F-actin and α6β4 staining (data not shown). Taken together, these findings indicate that EGF stimulates a dissociation of α6β4 from cytokeratin-associated hemidesmosomes, as well as the formation of lamellipodia and ruffles that contain α6β4 in association with F-actin.

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