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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.

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Members of the Rho family of small GTPases regulate β3-integrin clustering differentially. B16 β3–GFP cells transfected with myc-epitope–tagged dominant active forms of Cdc42, Rac1, and RhoA were plated overnight on vitronectin (1 μg ml−1)-coated glass coverslips. Cells were fixed and stained for the expression of the myc-epitope (inserts), and GFP fluorescence images were recorded with identical camera settings in order to appreciate qualitative as well as quantitative differences in the integrin localization pattern. (A) Nontransfected control cells displayed the typical pattern of small low-fluorescent focal adhesions in the lamellipodium (profile a) and larger high-fluorescent focal adhesions at lateral borders and rear of the cell (profile b). (B) Dominant active Cdc42 (da-Cdc42) induced the formation of long, streak-like arrays of low-fluorescent β3 integrin focal adhesions mainly localized in the lamella or periphery of the cell. Similarly, dominant active Rac1 (da-Rac1) induced extensive β3-integrin clustering into low-fluorescent adhesion sites at the periphery of the cell (C). In contrast, dominant active RhoA (da-RhoA) induced a retracted cellular morphology with intensively fluorescent β3-integrin focal adhesions at the cell periphery (D). Fluorescence intensity profiles of the indicated traces in A–D are shown in E. Note that the intensity profiles are similar between focal adhesions in the lamellipodium of control cells and cells transfected with dominant active Cdc42 and Rac1. Peak fluorescent intensities of lateral and rear focal adhesions in control cells are consistently higher compared with lamellipodial focal adhesions, but can increase even more after dominant active RhoA induction. A quantification of the β3-integrin density (fluorescence intensity increase over membrane) is shown in F. Bar, 15 μm.
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fig4: Members of the Rho family of small GTPases regulate β3-integrin clustering differentially. B16 β3–GFP cells transfected with myc-epitope–tagged dominant active forms of Cdc42, Rac1, and RhoA were plated overnight on vitronectin (1 μg ml−1)-coated glass coverslips. Cells were fixed and stained for the expression of the myc-epitope (inserts), and GFP fluorescence images were recorded with identical camera settings in order to appreciate qualitative as well as quantitative differences in the integrin localization pattern. (A) Nontransfected control cells displayed the typical pattern of small low-fluorescent focal adhesions in the lamellipodium (profile a) and larger high-fluorescent focal adhesions at lateral borders and rear of the cell (profile b). (B) Dominant active Cdc42 (da-Cdc42) induced the formation of long, streak-like arrays of low-fluorescent β3 integrin focal adhesions mainly localized in the lamella or periphery of the cell. Similarly, dominant active Rac1 (da-Rac1) induced extensive β3-integrin clustering into low-fluorescent adhesion sites at the periphery of the cell (C). In contrast, dominant active RhoA (da-RhoA) induced a retracted cellular morphology with intensively fluorescent β3-integrin focal adhesions at the cell periphery (D). Fluorescence intensity profiles of the indicated traces in A–D are shown in E. Note that the intensity profiles are similar between focal adhesions in the lamellipodium of control cells and cells transfected with dominant active Cdc42 and Rac1. Peak fluorescent intensities of lateral and rear focal adhesions in control cells are consistently higher compared with lamellipodial focal adhesions, but can increase even more after dominant active RhoA induction. A quantification of the β3-integrin density (fluorescence intensity increase over membrane) is shown in F. Bar, 15 μm.

Mentions: Lamellipodia formation, as well as the retraction of cell edges, depends on the reorganization of the actin cytoskeleton. In addition, the transition from small and stationary to larger, retracting focal adhesions was associated with an increase in fluorescence intensity, and hence increased integrin density (Fig. 3). Because signaling through members of the Rho family of small GTPases is known to cause changes in the actin cytoskeleton (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and Hall, 1995), we asked if changes in activation of these GTPases would influence the organization and density of αVβ3-integrin in focal adhesion sites. To answer this question, we transfected dominant active forms of Rac1, Cdc42, and RhoA into B16 β3–GFP cells, and quantified integrin fluorescence and focal adhesion morphology (Fig. 4). Control cells typically displayed a leading lamella with small β3-integrin– positive focal adhesions and larger, fluorescently brighter β3-integrin focal adhesions at the side and rear of the cell (Fig. 4 A). Expression of dominant active Cdc42 (V12) and Rac1 (L61) that are known to induce filopodia and lamellipodia, respectively (Ridley et al., 1992; Nobes and Hall, 1995), led to the appearance of flat and well-spread B16 β3–GFP cells. Compared with control cells the surface area increased by 188 and 248%, respectively (see Materials and methods), and cells exhibited actin-rich filopodia and lamellipodia and many small caliber actin filaments (unpublished data). This phenotype is apparent only after prolonged exposure to dominant active Rac1, and is associated with the formation of many small caliber actin filaments as previously reported (Ridley et al., 1992). In these cells, β3–GFP fluorescence resulted in a streak-like pattern of integrin clusters associated with filopodia (cdc42) or lamellipodia (Rac1) covering large areas of the substratum. These extensive clusters exhibited a granular pattern that resembled assemblies of numerous small focal adhesions (Fig. 4, B and C). The fluorescence intensity profiles indicated in Fig. 4, A–C, revealed that the density of β3-integrin in small focal adhesions in control cells (Fig. 4 E, profile a) corresponded to the densities measured across the integrin clusters of dominant Cdc42- and Rac1-transfected cells (Fig. 4 E, profile da-Cdc42 and da-Rac1). In contrast, measurements of GFP intensity (and hence, integrin densities) in focal adhesions that were localized in retracting cell edges at the rear of control cells were consistently higher (Fig. 4 E, profile b). Moreover, expression of dominant active RhoA (V14) induced robust stress fiber formation and the cells appeared contracted (64% of control cell surface area) with large, even brighter fluorescent β3-integrin focal adhesions (Fig. 4, D and E, profile da-RhoA). From these data we calculated (see Materials and methods) that the relative αVβ3-integrin densities compared with nonclustered integrin in the plasma membrane increased by three- to fivefold in lamellipodial and Rac1- or Cdc42-induced low-density focal adhesions, by five- to eightfold in lateral and rear high-density focal adhesions of control cells, and by 9–14-fold in focal adhesions of dominant RhoA-stimulated cells (Fig. 4 F). Similar to the raise in integrin densities, we also observed a RhoA-dependent increase in anti-vinculin labeling of focal adhesions (unpublished data). These results demonstrate that the Rac1- and RhoA-induced changes in the bundling state of the actin filament network led to changes in the clustering behavior of β3 integrin, i.e., the generation of integrin low- and high-density focal adhesions, respectively.


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)

Members of the Rho family of small GTPases regulate β3-integrin clustering differentially. B16 β3–GFP cells transfected with myc-epitope–tagged dominant active forms of Cdc42, Rac1, and RhoA were plated overnight on vitronectin (1 μg ml−1)-coated glass coverslips. Cells were fixed and stained for the expression of the myc-epitope (inserts), and GFP fluorescence images were recorded with identical camera settings in order to appreciate qualitative as well as quantitative differences in the integrin localization pattern. (A) Nontransfected control cells displayed the typical pattern of small low-fluorescent focal adhesions in the lamellipodium (profile a) and larger high-fluorescent focal adhesions at lateral borders and rear of the cell (profile b). (B) Dominant active Cdc42 (da-Cdc42) induced the formation of long, streak-like arrays of low-fluorescent β3 integrin focal adhesions mainly localized in the lamella or periphery of the cell. Similarly, dominant active Rac1 (da-Rac1) induced extensive β3-integrin clustering into low-fluorescent adhesion sites at the periphery of the cell (C). In contrast, dominant active RhoA (da-RhoA) induced a retracted cellular morphology with intensively fluorescent β3-integrin focal adhesions at the cell periphery (D). Fluorescence intensity profiles of the indicated traces in A–D are shown in E. Note that the intensity profiles are similar between focal adhesions in the lamellipodium of control cells and cells transfected with dominant active Cdc42 and Rac1. Peak fluorescent intensities of lateral and rear focal adhesions in control cells are consistently higher compared with lamellipodial focal adhesions, but can increase even more after dominant active RhoA induction. A quantification of the β3-integrin density (fluorescence intensity increase over membrane) is shown in F. Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Members of the Rho family of small GTPases regulate β3-integrin clustering differentially. B16 β3–GFP cells transfected with myc-epitope–tagged dominant active forms of Cdc42, Rac1, and RhoA were plated overnight on vitronectin (1 μg ml−1)-coated glass coverslips. Cells were fixed and stained for the expression of the myc-epitope (inserts), and GFP fluorescence images were recorded with identical camera settings in order to appreciate qualitative as well as quantitative differences in the integrin localization pattern. (A) Nontransfected control cells displayed the typical pattern of small low-fluorescent focal adhesions in the lamellipodium (profile a) and larger high-fluorescent focal adhesions at lateral borders and rear of the cell (profile b). (B) Dominant active Cdc42 (da-Cdc42) induced the formation of long, streak-like arrays of low-fluorescent β3 integrin focal adhesions mainly localized in the lamella or periphery of the cell. Similarly, dominant active Rac1 (da-Rac1) induced extensive β3-integrin clustering into low-fluorescent adhesion sites at the periphery of the cell (C). In contrast, dominant active RhoA (da-RhoA) induced a retracted cellular morphology with intensively fluorescent β3-integrin focal adhesions at the cell periphery (D). Fluorescence intensity profiles of the indicated traces in A–D are shown in E. Note that the intensity profiles are similar between focal adhesions in the lamellipodium of control cells and cells transfected with dominant active Cdc42 and Rac1. Peak fluorescent intensities of lateral and rear focal adhesions in control cells are consistently higher compared with lamellipodial focal adhesions, but can increase even more after dominant active RhoA induction. A quantification of the β3-integrin density (fluorescence intensity increase over membrane) is shown in F. Bar, 15 μm.
Mentions: Lamellipodia formation, as well as the retraction of cell edges, depends on the reorganization of the actin cytoskeleton. In addition, the transition from small and stationary to larger, retracting focal adhesions was associated with an increase in fluorescence intensity, and hence increased integrin density (Fig. 3). Because signaling through members of the Rho family of small GTPases is known to cause changes in the actin cytoskeleton (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and Hall, 1995), we asked if changes in activation of these GTPases would influence the organization and density of αVβ3-integrin in focal adhesion sites. To answer this question, we transfected dominant active forms of Rac1, Cdc42, and RhoA into B16 β3–GFP cells, and quantified integrin fluorescence and focal adhesion morphology (Fig. 4). Control cells typically displayed a leading lamella with small β3-integrin– positive focal adhesions and larger, fluorescently brighter β3-integrin focal adhesions at the side and rear of the cell (Fig. 4 A). Expression of dominant active Cdc42 (V12) and Rac1 (L61) that are known to induce filopodia and lamellipodia, respectively (Ridley et al., 1992; Nobes and Hall, 1995), led to the appearance of flat and well-spread B16 β3–GFP cells. Compared with control cells the surface area increased by 188 and 248%, respectively (see Materials and methods), and cells exhibited actin-rich filopodia and lamellipodia and many small caliber actin filaments (unpublished data). This phenotype is apparent only after prolonged exposure to dominant active Rac1, and is associated with the formation of many small caliber actin filaments as previously reported (Ridley et al., 1992). In these cells, β3–GFP fluorescence resulted in a streak-like pattern of integrin clusters associated with filopodia (cdc42) or lamellipodia (Rac1) covering large areas of the substratum. These extensive clusters exhibited a granular pattern that resembled assemblies of numerous small focal adhesions (Fig. 4, B and C). The fluorescence intensity profiles indicated in Fig. 4, A–C, revealed that the density of β3-integrin in small focal adhesions in control cells (Fig. 4 E, profile a) corresponded to the densities measured across the integrin clusters of dominant Cdc42- and Rac1-transfected cells (Fig. 4 E, profile da-Cdc42 and da-Rac1). In contrast, measurements of GFP intensity (and hence, integrin densities) in focal adhesions that were localized in retracting cell edges at the rear of control cells were consistently higher (Fig. 4 E, profile b). Moreover, expression of dominant active RhoA (V14) induced robust stress fiber formation and the cells appeared contracted (64% of control cell surface area) with large, even brighter fluorescent β3-integrin focal adhesions (Fig. 4, D and E, profile da-RhoA). From these data we calculated (see Materials and methods) that the relative αVβ3-integrin densities compared with nonclustered integrin in the plasma membrane increased by three- to fivefold in lamellipodial and Rac1- or Cdc42-induced low-density focal adhesions, by five- to eightfold in lateral and rear high-density focal adhesions of control cells, and by 9–14-fold in focal adhesions of dominant RhoA-stimulated cells (Fig. 4 F). Similar to the raise in integrin densities, we also observed a RhoA-dependent increase in anti-vinculin labeling of focal adhesions (unpublished data). These results demonstrate that the Rac1- and RhoA-induced changes in the bundling state of the actin filament network led to changes in the clustering behavior of β3 integrin, i.e., the generation of integrin low- and high-density focal adhesions, respectively.

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