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A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo Y - J. Cell Biol. (2001)

Bottom Line: When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia.Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers.The coexpression of ILK enhances this effect.

View Article: PubMed Central - PubMed

Affiliation: The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.

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Distribution of affixin during the early stages of the cell spreading process. CHO cells (A, C, and E) or those cotransfected with T7-tagged affixin and Flag-tagged ILK (G and I) were replated on fibronectin-coated coverslips and 1 h later fixed and stained with antiaffixin or anti-T7 antibodies as indicated. Cells were stained simultaneously with anti-ILK (B), antivinculin (D), anti-FAK (F), anti-Flag (H) antibodies, or FITC-phalloidin (J). Note that affixin is concentrated in peripheral blebs (A–F, arrowheads), whereas in well-spread cells dot-like staining is observed from which F-actin bundles emanate (I and J). Fixation is 100% methanol (A–F), 2% paraformaldehyde (G–J). Bars, 25 μm.
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Figure 8: Distribution of affixin during the early stages of the cell spreading process. CHO cells (A, C, and E) or those cotransfected with T7-tagged affixin and Flag-tagged ILK (G and I) were replated on fibronectin-coated coverslips and 1 h later fixed and stained with antiaffixin or anti-T7 antibodies as indicated. Cells were stained simultaneously with anti-ILK (B), antivinculin (D), anti-FAK (F), anti-Flag (H) antibodies, or FITC-phalloidin (J). Note that affixin is concentrated in peripheral blebs (A–F, arrowheads), whereas in well-spread cells dot-like staining is observed from which F-actin bundles emanate (I and J). Fixation is 100% methanol (A–F), 2% paraformaldehyde (G–J). Bars, 25 μm.

Mentions: Because the leading edge is a site where the formation and growth of de novo cell–substrate adhesions actively occur, the above results on the distribution of affixin and ILK at the tip of the leading edge suggest the possibility that their interaction plays a role in the initial phase of FA formation. This notion was supported by analyzing their localization in reseeded CHO cells that are actively spreading. When CHO cells are harvested in trypsin/EDTA solution and reseeded on fibronectin-coated coverslips, their cell–substrate adhesions are gradually restored, and their shape changes from round to spread within 4 h (Bauer et al. 1993). During the early stages of this spreading process (1 h after replating) when the cells attach to the substrate with a limited central area of round cell body, most cells transiently develop many spherical out-pouchings of the plasma membrane, blebs, which are ultimately replaced by flat ruffles or small lamellipodia. As shown in Fig. 8A–C, high concentrations of affixin and ILK are observed in these blebs during the early stages of cell spreading when the cells still show irregular dot-like staining of vinculin in their inner area (Fig. 8 D). Overexpressed affixin and ILK also show similar distribution in blebs (Fig. 8G and Fig. H). On the other hand, FAK was not observed in these peripheral membrane protrusions even when blebs have developed into more flattened lammelipodia-like structures (Fig. 8E and Fig. F). Actin filament identified by rhodamine-phalloidin staining was observed only in restricted numbers of blebs (data not shown), but at later stages when cells have started to spread, short actin bundles started to appear in the peripheral lamellae from the dot-like structures at which affixin localizes (Fig. 8I and Fig. J). These results indicate that affixin and ILK are recruited into nascent cell–substrate adhesion structures at a very early stage of cell spreading faster than FAK and vinculin from which mature FAs and SFs develop.


A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Yamaji S, Suzuki A, Sugiyama Y, Koide Y, Yoshida M, Kanamori H, Mohri H, Ohno S, Ishigatsubo Y - J. Cell Biol. (2001)

Distribution of affixin during the early stages of the cell spreading process. CHO cells (A, C, and E) or those cotransfected with T7-tagged affixin and Flag-tagged ILK (G and I) were replated on fibronectin-coated coverslips and 1 h later fixed and stained with antiaffixin or anti-T7 antibodies as indicated. Cells were stained simultaneously with anti-ILK (B), antivinculin (D), anti-FAK (F), anti-Flag (H) antibodies, or FITC-phalloidin (J). Note that affixin is concentrated in peripheral blebs (A–F, arrowheads), whereas in well-spread cells dot-like staining is observed from which F-actin bundles emanate (I and J). Fixation is 100% methanol (A–F), 2% paraformaldehyde (G–J). Bars, 25 μm.
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Figure 8: Distribution of affixin during the early stages of the cell spreading process. CHO cells (A, C, and E) or those cotransfected with T7-tagged affixin and Flag-tagged ILK (G and I) were replated on fibronectin-coated coverslips and 1 h later fixed and stained with antiaffixin or anti-T7 antibodies as indicated. Cells were stained simultaneously with anti-ILK (B), antivinculin (D), anti-FAK (F), anti-Flag (H) antibodies, or FITC-phalloidin (J). Note that affixin is concentrated in peripheral blebs (A–F, arrowheads), whereas in well-spread cells dot-like staining is observed from which F-actin bundles emanate (I and J). Fixation is 100% methanol (A–F), 2% paraformaldehyde (G–J). Bars, 25 μm.
Mentions: Because the leading edge is a site where the formation and growth of de novo cell–substrate adhesions actively occur, the above results on the distribution of affixin and ILK at the tip of the leading edge suggest the possibility that their interaction plays a role in the initial phase of FA formation. This notion was supported by analyzing their localization in reseeded CHO cells that are actively spreading. When CHO cells are harvested in trypsin/EDTA solution and reseeded on fibronectin-coated coverslips, their cell–substrate adhesions are gradually restored, and their shape changes from round to spread within 4 h (Bauer et al. 1993). During the early stages of this spreading process (1 h after replating) when the cells attach to the substrate with a limited central area of round cell body, most cells transiently develop many spherical out-pouchings of the plasma membrane, blebs, which are ultimately replaced by flat ruffles or small lamellipodia. As shown in Fig. 8A–C, high concentrations of affixin and ILK are observed in these blebs during the early stages of cell spreading when the cells still show irregular dot-like staining of vinculin in their inner area (Fig. 8 D). Overexpressed affixin and ILK also show similar distribution in blebs (Fig. 8G and Fig. H). On the other hand, FAK was not observed in these peripheral membrane protrusions even when blebs have developed into more flattened lammelipodia-like structures (Fig. 8E and Fig. F). Actin filament identified by rhodamine-phalloidin staining was observed only in restricted numbers of blebs (data not shown), but at later stages when cells have started to spread, short actin bundles started to appear in the peripheral lamellae from the dot-like structures at which affixin localizes (Fig. 8I and Fig. J). These results indicate that affixin and ILK are recruited into nascent cell–substrate adhesion structures at a very early stage of cell spreading faster than FAK and vinculin from which mature FAs and SFs develop.

Bottom Line: When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia.Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers.The coexpression of ILK enhances this effect.

View Article: PubMed Central - PubMed

Affiliation: The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.

Show MeSH
Related in: MedlinePlus