Limits...
Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Ballestrem C, Hinz B, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2001)

Bottom Line: We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain.Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature.In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Centre Médical Universitaire, Geneva, Switzerland.

ABSTRACT
Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Show MeSH

Related in: MedlinePlus

The GFP-tagged β3-integrin chain forms functional heterodimers with endogenous αV. (A) Scheme of the αVβ3–GFP-integrin heterodimer. The GFP protein is tagged COOH-terminally to the cytoplasmic domain of the β3 subunit. (B) Immunoprecipitations of cell extracts from surface biotinylated B16 β3–GFP cells. Extracts were precipitated with the indicated antibodies (c, control) and separated under reducing conditions by PAGE followed by transfer onto nitrocellulose membranes. Revelation with either streptavidin coupled horseradish peroxidase (SA-HPO, top) or anti-GFP antibodies (GFP, bottom) demonstrated the typical double-band pattern for integrin heterodimers and the coprecipitated GFP-tagged β3-integrin subunit, respectively. The position of the molecular mass markers is indicated to the left of the blots. (C) Substrate-specific clustering of the αVβ3–GFP-integrin into adhesions sites. B16 β3–GFP cells were plated overnight on glass coverslips, previously coated with 5 μg ml−1 laminin-1 (LN), 5 μg ml−1 fibronectin (FN), or 1 μg ml−1 vitronectin (VN). Cells were subsequently fixed and substrate adhesion sites were revealed by immunohistochemical detection of vinculin. Note that β3–GFP-integrin–positive adhesion sites were only found on fibronectin and vitronectin, which are ligands for αVβ3-integrin. In contrast, β3–GFP-integrin did not cluster on laminin-1, for which it is not a ligand. Because B16 cells use a different type of integrin receptor (α6β1) to adhere to LN than to FN or VN (α5β1, αVβ3), their morphology and migration behavior is different between these substrates (Ballestrem et al., 1998). (D) FACS analysis of nontransfected, β3-, and β3–GFP-transfected CHO cells with a Kistrin–CD31 fusion construct (SKI-7) (Legler et al., 2001). Note that the β3–GFP-transfected CHO clone is not homogeneous, exhibiting cells that lost β3-GFP expression, which reduces their SKI-7 reactivity to endogenous αVβ3-integrin levels (gate 1) (ctr; SKI-7, unpublished data). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199321&req=5

fig1: The GFP-tagged β3-integrin chain forms functional heterodimers with endogenous αV. (A) Scheme of the αVβ3–GFP-integrin heterodimer. The GFP protein is tagged COOH-terminally to the cytoplasmic domain of the β3 subunit. (B) Immunoprecipitations of cell extracts from surface biotinylated B16 β3–GFP cells. Extracts were precipitated with the indicated antibodies (c, control) and separated under reducing conditions by PAGE followed by transfer onto nitrocellulose membranes. Revelation with either streptavidin coupled horseradish peroxidase (SA-HPO, top) or anti-GFP antibodies (GFP, bottom) demonstrated the typical double-band pattern for integrin heterodimers and the coprecipitated GFP-tagged β3-integrin subunit, respectively. The position of the molecular mass markers is indicated to the left of the blots. (C) Substrate-specific clustering of the αVβ3–GFP-integrin into adhesions sites. B16 β3–GFP cells were plated overnight on glass coverslips, previously coated with 5 μg ml−1 laminin-1 (LN), 5 μg ml−1 fibronectin (FN), or 1 μg ml−1 vitronectin (VN). Cells were subsequently fixed and substrate adhesion sites were revealed by immunohistochemical detection of vinculin. Note that β3–GFP-integrin–positive adhesion sites were only found on fibronectin and vitronectin, which are ligands for αVβ3-integrin. In contrast, β3–GFP-integrin did not cluster on laminin-1, for which it is not a ligand. Because B16 cells use a different type of integrin receptor (α6β1) to adhere to LN than to FN or VN (α5β1, αVβ3), their morphology and migration behavior is different between these substrates (Ballestrem et al., 1998). (D) FACS analysis of nontransfected, β3-, and β3–GFP-transfected CHO cells with a Kistrin–CD31 fusion construct (SKI-7) (Legler et al., 2001). Note that the β3–GFP-transfected CHO clone is not homogeneous, exhibiting cells that lost β3-GFP expression, which reduces their SKI-7 reactivity to endogenous αVβ3-integrin levels (gate 1) (ctr; SKI-7, unpublished data). Bar, 20 μm.

Mentions: To study and quantify αVβ3-integrin dynamics in living cells, we generated a fusion protein of the β3 integrin subunit with GFP (Fig. 1 A). To determine whether this β3–GFP-integrin chain formed heterodimers with the endogenous αV subunit, we surface biotinylated stable β3–GFP-integrin–transfected cells (B16 F1 melanoma and 3T3 fibroblasts), and performed immunoprecipitations with antibodies against either the αV- or the β3-integrin subunits (Fig. 1 B). After precipitation of the integrin and subsequent Western blotting, both α- and β-integrin subunits could be detected with avidin-peroxidase (Fig. 1 B). Bands for the β3–GFP-integrin fusion protein were only detected in precipitations with anti–αV- or –β3-integrin subunits, but not with control rat serum nor with anti–α6-integrin subunit which forms heterodimers with the β1 and β4 chains (Fig. 1 B, bottom). These experiments clearly demonstrated that β3–GFP-integrin was expressed on the cell surface as a heterodimeric complex in association with the endogenous αV chain.


Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Ballestrem C, Hinz B, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2001)

The GFP-tagged β3-integrin chain forms functional heterodimers with endogenous αV. (A) Scheme of the αVβ3–GFP-integrin heterodimer. The GFP protein is tagged COOH-terminally to the cytoplasmic domain of the β3 subunit. (B) Immunoprecipitations of cell extracts from surface biotinylated B16 β3–GFP cells. Extracts were precipitated with the indicated antibodies (c, control) and separated under reducing conditions by PAGE followed by transfer onto nitrocellulose membranes. Revelation with either streptavidin coupled horseradish peroxidase (SA-HPO, top) or anti-GFP antibodies (GFP, bottom) demonstrated the typical double-band pattern for integrin heterodimers and the coprecipitated GFP-tagged β3-integrin subunit, respectively. The position of the molecular mass markers is indicated to the left of the blots. (C) Substrate-specific clustering of the αVβ3–GFP-integrin into adhesions sites. B16 β3–GFP cells were plated overnight on glass coverslips, previously coated with 5 μg ml−1 laminin-1 (LN), 5 μg ml−1 fibronectin (FN), or 1 μg ml−1 vitronectin (VN). Cells were subsequently fixed and substrate adhesion sites were revealed by immunohistochemical detection of vinculin. Note that β3–GFP-integrin–positive adhesion sites were only found on fibronectin and vitronectin, which are ligands for αVβ3-integrin. In contrast, β3–GFP-integrin did not cluster on laminin-1, for which it is not a ligand. Because B16 cells use a different type of integrin receptor (α6β1) to adhere to LN than to FN or VN (α5β1, αVβ3), their morphology and migration behavior is different between these substrates (Ballestrem et al., 1998). (D) FACS analysis of nontransfected, β3-, and β3–GFP-transfected CHO cells with a Kistrin–CD31 fusion construct (SKI-7) (Legler et al., 2001). Note that the β3–GFP-transfected CHO clone is not homogeneous, exhibiting cells that lost β3-GFP expression, which reduces their SKI-7 reactivity to endogenous αVβ3-integrin levels (gate 1) (ctr; SKI-7, unpublished data). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199321&req=5

fig1: The GFP-tagged β3-integrin chain forms functional heterodimers with endogenous αV. (A) Scheme of the αVβ3–GFP-integrin heterodimer. The GFP protein is tagged COOH-terminally to the cytoplasmic domain of the β3 subunit. (B) Immunoprecipitations of cell extracts from surface biotinylated B16 β3–GFP cells. Extracts were precipitated with the indicated antibodies (c, control) and separated under reducing conditions by PAGE followed by transfer onto nitrocellulose membranes. Revelation with either streptavidin coupled horseradish peroxidase (SA-HPO, top) or anti-GFP antibodies (GFP, bottom) demonstrated the typical double-band pattern for integrin heterodimers and the coprecipitated GFP-tagged β3-integrin subunit, respectively. The position of the molecular mass markers is indicated to the left of the blots. (C) Substrate-specific clustering of the αVβ3–GFP-integrin into adhesions sites. B16 β3–GFP cells were plated overnight on glass coverslips, previously coated with 5 μg ml−1 laminin-1 (LN), 5 μg ml−1 fibronectin (FN), or 1 μg ml−1 vitronectin (VN). Cells were subsequently fixed and substrate adhesion sites were revealed by immunohistochemical detection of vinculin. Note that β3–GFP-integrin–positive adhesion sites were only found on fibronectin and vitronectin, which are ligands for αVβ3-integrin. In contrast, β3–GFP-integrin did not cluster on laminin-1, for which it is not a ligand. Because B16 cells use a different type of integrin receptor (α6β1) to adhere to LN than to FN or VN (α5β1, αVβ3), their morphology and migration behavior is different between these substrates (Ballestrem et al., 1998). (D) FACS analysis of nontransfected, β3-, and β3–GFP-transfected CHO cells with a Kistrin–CD31 fusion construct (SKI-7) (Legler et al., 2001). Note that the β3–GFP-transfected CHO clone is not homogeneous, exhibiting cells that lost β3-GFP expression, which reduces their SKI-7 reactivity to endogenous αVβ3-integrin levels (gate 1) (ctr; SKI-7, unpublished data). Bar, 20 μm.
Mentions: To study and quantify αVβ3-integrin dynamics in living cells, we generated a fusion protein of the β3 integrin subunit with GFP (Fig. 1 A). To determine whether this β3–GFP-integrin chain formed heterodimers with the endogenous αV subunit, we surface biotinylated stable β3–GFP-integrin–transfected cells (B16 F1 melanoma and 3T3 fibroblasts), and performed immunoprecipitations with antibodies against either the αV- or the β3-integrin subunits (Fig. 1 B). After precipitation of the integrin and subsequent Western blotting, both α- and β-integrin subunits could be detected with avidin-peroxidase (Fig. 1 B). Bands for the β3–GFP-integrin fusion protein were only detected in precipitations with anti–αV- or –β3-integrin subunits, but not with control rat serum nor with anti–α6-integrin subunit which forms heterodimers with the β1 and β4 chains (Fig. 1 B, bottom). These experiments clearly demonstrated that β3–GFP-integrin was expressed on the cell surface as a heterodimeric complex in association with the endogenous αV chain.

Bottom Line: We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain.Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature.In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Centre Médical Universitaire, Geneva, Switzerland.

ABSTRACT
Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Show MeSH
Related in: MedlinePlus