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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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FRAP reveals different β3-integrin exchange rates in high- versus low-density focal adhesions. (A) Nontransfected or dominant active Rac1 transfected B16 β3–GFP cells were cultured overnight on serum-coated glass coverslips and FRAP was performed on focal adhesions localized to the edge of cells. The bleached area of each series is circled in the first frame and the recovery time (seconds after completion of bleach) indicated to the left. In control cells, immobile (first series) and inward sliding (second series) high-density focal adhesions show almost complete recovery (MF >80%). In cells transfected with dominant active Rac1 (third series) in which low-density focal adhesions were formed, fluorescence recovery was only partial (50% MF), reaching fluorescent levels just slightly above fluorescence intensities of nonclustered β3–GFP-integrin present in the plasma membrane (visible on the right hand side of the frame). Qualitative FRAP curves from several cells (5–8) are displayed in B. Each data point is the median of three to five individual focal adhesions. Bar, 10 μm.
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fig7: FRAP reveals different β3-integrin exchange rates in high- versus low-density focal adhesions. (A) Nontransfected or dominant active Rac1 transfected B16 β3–GFP cells were cultured overnight on serum-coated glass coverslips and FRAP was performed on focal adhesions localized to the edge of cells. The bleached area of each series is circled in the first frame and the recovery time (seconds after completion of bleach) indicated to the left. In control cells, immobile (first series) and inward sliding (second series) high-density focal adhesions show almost complete recovery (MF >80%). In cells transfected with dominant active Rac1 (third series) in which low-density focal adhesions were formed, fluorescence recovery was only partial (50% MF), reaching fluorescent levels just slightly above fluorescence intensities of nonclustered β3–GFP-integrin present in the plasma membrane (visible on the right hand side of the frame). Qualitative FRAP curves from several cells (5–8) are displayed in B. Each data point is the median of three to five individual focal adhesions. Bar, 10 μm.

Mentions: In the previous experiments, we determined that the density of αVβ3-integrin and the apparent sliding of focal adhesions are critically linked to the tension created by the actin cytoskeleton in a Rac1/RhoA-dependent fashion. During cell migration, newly formed, small, stationary, low-density focal adhesions firmly anchor the cell to the substratum. At the cell rear, retraction and apparent sliding of high-density focal adhesions require a more flexible interaction with the substrate. To understand the mechanical differences between these two types of adhesion sites, it is necessary to determine the dynamics of integrins within these substrate contacts. Therefore, we performed FRAP of β3–GFP-integrins within high-density focal adhesions and compared it with the recovery of integrins in low-density focal adhesions. FRAP measurements of β3–GFP-integrins in high-density focal contacts revealed a fast exchange (Fig. 7 A). Within 120 s, 50% of the integrin fluorescence recovered in the bleached contacts, whereas the exchange of all the β3-integrins was completed within 5–10 min (mobile fraction [MF] = 80–100%) (Fig. 7 B). This exchange was independent whether a high-density focal adhesion was stationary or sliding (Fig. 7 A). Due to the transient nature of low-density focal adhesions within an advancing lamellipodia, it was impossible to measure FRAP (Fig. 2). Therefore, we analyzed low-density focal adhesions formed upon transfection with dominant active Rac1 (Fig. 4). These low-density focal adhesions revealed a 2.5× slower recovery of integrins than high-density focal adhesions (Fig. 7 B). In addition, during the time of observation recovery reached only 50% (MF), suggesting that half the integrins in low-density focal adhesions are immobilized. Our experiments showed that although the integrin density in Rac1-induced focal adhesions was lower (see above), the retention time of integrins was dramatically increased compared with high-density focal adhesions. These data also suggest that an increase in intracellular tension, and hence the activation of RhoA, increases the turnover rate of integrins in focal adhesions.


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)

FRAP reveals different β3-integrin exchange rates in high- versus low-density focal adhesions. (A) Nontransfected or dominant active Rac1 transfected B16 β3–GFP cells were cultured overnight on serum-coated glass coverslips and FRAP was performed on focal adhesions localized to the edge of cells. The bleached area of each series is circled in the first frame and the recovery time (seconds after completion of bleach) indicated to the left. In control cells, immobile (first series) and inward sliding (second series) high-density focal adhesions show almost complete recovery (MF >80%). In cells transfected with dominant active Rac1 (third series) in which low-density focal adhesions were formed, fluorescence recovery was only partial (50% MF), reaching fluorescent levels just slightly above fluorescence intensities of nonclustered β3–GFP-integrin present in the plasma membrane (visible on the right hand side of the frame). Qualitative FRAP curves from several cells (5–8) are displayed in B. Each data point is the median of three to five individual focal adhesions. Bar, 10 μm.
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fig7: FRAP reveals different β3-integrin exchange rates in high- versus low-density focal adhesions. (A) Nontransfected or dominant active Rac1 transfected B16 β3–GFP cells were cultured overnight on serum-coated glass coverslips and FRAP was performed on focal adhesions localized to the edge of cells. The bleached area of each series is circled in the first frame and the recovery time (seconds after completion of bleach) indicated to the left. In control cells, immobile (first series) and inward sliding (second series) high-density focal adhesions show almost complete recovery (MF >80%). In cells transfected with dominant active Rac1 (third series) in which low-density focal adhesions were formed, fluorescence recovery was only partial (50% MF), reaching fluorescent levels just slightly above fluorescence intensities of nonclustered β3–GFP-integrin present in the plasma membrane (visible on the right hand side of the frame). Qualitative FRAP curves from several cells (5–8) are displayed in B. Each data point is the median of three to five individual focal adhesions. Bar, 10 μm.
Mentions: In the previous experiments, we determined that the density of αVβ3-integrin and the apparent sliding of focal adhesions are critically linked to the tension created by the actin cytoskeleton in a Rac1/RhoA-dependent fashion. During cell migration, newly formed, small, stationary, low-density focal adhesions firmly anchor the cell to the substratum. At the cell rear, retraction and apparent sliding of high-density focal adhesions require a more flexible interaction with the substrate. To understand the mechanical differences between these two types of adhesion sites, it is necessary to determine the dynamics of integrins within these substrate contacts. Therefore, we performed FRAP of β3–GFP-integrins within high-density focal adhesions and compared it with the recovery of integrins in low-density focal adhesions. FRAP measurements of β3–GFP-integrins in high-density focal contacts revealed a fast exchange (Fig. 7 A). Within 120 s, 50% of the integrin fluorescence recovered in the bleached contacts, whereas the exchange of all the β3-integrins was completed within 5–10 min (mobile fraction [MF] = 80–100%) (Fig. 7 B). This exchange was independent whether a high-density focal adhesion was stationary or sliding (Fig. 7 A). Due to the transient nature of low-density focal adhesions within an advancing lamellipodia, it was impossible to measure FRAP (Fig. 2). Therefore, we analyzed low-density focal adhesions formed upon transfection with dominant active Rac1 (Fig. 4). These low-density focal adhesions revealed a 2.5× slower recovery of integrins than high-density focal adhesions (Fig. 7 B). In addition, during the time of observation recovery reached only 50% (MF), suggesting that half the integrins in low-density focal adhesions are immobilized. Our experiments showed that although the integrin density in Rac1-induced focal adhesions was lower (see above), the retention time of integrins was dramatically increased compared with high-density focal adhesions. These data also suggest that an increase in intracellular tension, and hence the activation of RhoA, increases the turnover rate of integrins in focal adhesions.

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