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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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Block of intracellular tension reduces focal adhesion density. Time-lapse analysis of β3-integrin fluorescence of focal adhesions in B16 β3–GFP cells after addition of Y-27632 (20 μM) (A, 5′ before addition; B, 60′ after addition). (C) Higher magnification of the boxed area in A demonstrates (a) the reduction in β3-integrin density (fluorescence intensity) during the first 10 min of treatment and (b) the further dispersal of compact β3-integrin focal adhesions into irregularly shaped β3- integrin clusters (arrowhead). (D) The average peak β3-integrin integrin density in the peripheral focal adhesions was measured before and after the addition of the inhibitor. The indicated time refers to the addition of inhibitor. Bar, 20 μm.
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fig6: Block of intracellular tension reduces focal adhesion density. Time-lapse analysis of β3-integrin fluorescence of focal adhesions in B16 β3–GFP cells after addition of Y-27632 (20 μM) (A, 5′ before addition; B, 60′ after addition). (C) Higher magnification of the boxed area in A demonstrates (a) the reduction in β3-integrin density (fluorescence intensity) during the first 10 min of treatment and (b) the further dispersal of compact β3-integrin focal adhesions into irregularly shaped β3- integrin clusters (arrowhead). (D) The average peak β3-integrin integrin density in the peripheral focal adhesions was measured before and after the addition of the inhibitor. The indicated time refers to the addition of inhibitor. Bar, 20 μm.

Mentions: What causes this RhoA-induced increase in integrin density? Previously it has been shown that activated RhoA increases myosin-dependent contraction of the actin cytoskeleton, leading to intracellular tension (Chrzanowska-Wodnicka and Burridge, 1996; Amano et al., 1997). Integrins in focal adhesion sites are anchored within the actin cytoskeleton, such that contraction of this actin filament backbone induced by myosin activity may lead to increased integrin density, i.e., the transition of lamellipodial to lateral focal adhesions. To test this, we determined whether intracellular tension induced by RhoA was correlated with this transition. Intracellular tension was measured by plating 3T3 fibroblasts on flexible silicone rubber that formed wrinkles in response to cellular contraction (Harris et al., 1980). Activation of RhoA by lysophosphatidic acid (LPA) (10 μM) within these cells increased the number of wrinkles (Fig. 5 A; Video4, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1) (Amano et al., 1997). RhoA activates Rho-kinase, which blocks myosin light chain phosphatase, resulting in myosin-dependent actin contraction (Kimura et al., 1996). Treatment of cells with the Rho-kinase inhibitor Y-27632 (10 μM) removed the wrinkles (Fig. 5 B; Video5, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1) (Uehata et al., 1997). Similarly, blocking of myosin light chain kinase with the wide-spectrum protein kinase inhibitor staurosporine (50 nM) resulted also in the disappearance of wrinkles (Fig. 5 C; Video6, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). Using these modulators of intracellular tension, the formation of high- and low-density focal adhesions was analyzed. Treatment of 3T3 fibroblasts with LPA increased β3-integrin compaction and formed high-density focal adhesions (Fig. 5 E). In contrast, cells treated with Y-27632 or staurosporine displayed the disappearance of high-density focal adhesions, whereas low-density focal adhesions remained in the periphery of the cells in the lamellipodia (Fig. 5, F and G). To correlate the changes in intracellular tension with that of the integrin density and focal adhesion size, we displayed the relative peak integrin density (compared with the integrin density in the membrane) and the respective area of focal adhesions (see Materials and methods). For each experimental condition, we analyzed ∼500 focal adhesions from different cells. Control cells displayed a significant number of small-sized, low-density focal adhesions that were mainly associated with protruding lamellipodia (Fig. 5 H). In addition, a considerable number of large-sized, generally high-density focal adhesions were found (Fig. 5 H) (average relative integrin density and size: 5.14-fold, 1.15 μm2). The stimulation of the RhoA pathway with LPA led to a shift to denser and generally larger focal adhesions (Fig. 5 I) (average relative integrin density and size: 6.77-fold, 1.69 μm2). In contrast, treatment with either Y-27632 or staurosporine resulted in a drop in integrin densities (Fig. 5, J and K). Interestingly, the average size of the contacts in Y-27632–treated cells was only slightly lower compared with control cells, whereas the average size of staurosporine treated contacts was dramatically reduced (average relative integrin density and size: Y-27632, 4.39-fold, 0.87 μm2; staurosporine, 4.49-fold, 0.43 μm2). Furthermore, the dynamic transition of high- to low-density focal adhesions was analyzed in living B16 F1 cells by time-lapse microscopy (Fig. 6; Video7, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). Release of intracellular tension by Y-27632 induced the rapid decrease in integrin densities in peripheral focal adhesions (Fig. 6, B and C, arrowhead). During the first 10 min of treatment, neither the shape of the contacts nor that of the cell did change. With time, the cell developed large lamellipodia that contained numerous dot-like tension-independent focal adhesions. In addition, the Y-27632–induced low-density focal adhesions began to change shape but remained undispersed during the entire time of observation (up to 90 min) (Video7, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). These observations suggest that high intracellular tension correlates with the formation and maintenance of high-density αVβ3-integrin focal adhesions. The formation of low-density αVβ3-integrin focal adhesions is favored under conditions of low intracellular tension.


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)

Block of intracellular tension reduces focal adhesion density. Time-lapse analysis of β3-integrin fluorescence of focal adhesions in B16 β3–GFP cells after addition of Y-27632 (20 μM) (A, 5′ before addition; B, 60′ after addition). (C) Higher magnification of the boxed area in A demonstrates (a) the reduction in β3-integrin density (fluorescence intensity) during the first 10 min of treatment and (b) the further dispersal of compact β3-integrin focal adhesions into irregularly shaped β3- integrin clusters (arrowhead). (D) The average peak β3-integrin integrin density in the peripheral focal adhesions was measured before and after the addition of the inhibitor. The indicated time refers to the addition of inhibitor. Bar, 20 μm.
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Related In: Results  -  Collection

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fig6: Block of intracellular tension reduces focal adhesion density. Time-lapse analysis of β3-integrin fluorescence of focal adhesions in B16 β3–GFP cells after addition of Y-27632 (20 μM) (A, 5′ before addition; B, 60′ after addition). (C) Higher magnification of the boxed area in A demonstrates (a) the reduction in β3-integrin density (fluorescence intensity) during the first 10 min of treatment and (b) the further dispersal of compact β3-integrin focal adhesions into irregularly shaped β3- integrin clusters (arrowhead). (D) The average peak β3-integrin integrin density in the peripheral focal adhesions was measured before and after the addition of the inhibitor. The indicated time refers to the addition of inhibitor. Bar, 20 μm.
Mentions: What causes this RhoA-induced increase in integrin density? Previously it has been shown that activated RhoA increases myosin-dependent contraction of the actin cytoskeleton, leading to intracellular tension (Chrzanowska-Wodnicka and Burridge, 1996; Amano et al., 1997). Integrins in focal adhesion sites are anchored within the actin cytoskeleton, such that contraction of this actin filament backbone induced by myosin activity may lead to increased integrin density, i.e., the transition of lamellipodial to lateral focal adhesions. To test this, we determined whether intracellular tension induced by RhoA was correlated with this transition. Intracellular tension was measured by plating 3T3 fibroblasts on flexible silicone rubber that formed wrinkles in response to cellular contraction (Harris et al., 1980). Activation of RhoA by lysophosphatidic acid (LPA) (10 μM) within these cells increased the number of wrinkles (Fig. 5 A; Video4, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1) (Amano et al., 1997). RhoA activates Rho-kinase, which blocks myosin light chain phosphatase, resulting in myosin-dependent actin contraction (Kimura et al., 1996). Treatment of cells with the Rho-kinase inhibitor Y-27632 (10 μM) removed the wrinkles (Fig. 5 B; Video5, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1) (Uehata et al., 1997). Similarly, blocking of myosin light chain kinase with the wide-spectrum protein kinase inhibitor staurosporine (50 nM) resulted also in the disappearance of wrinkles (Fig. 5 C; Video6, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). Using these modulators of intracellular tension, the formation of high- and low-density focal adhesions was analyzed. Treatment of 3T3 fibroblasts with LPA increased β3-integrin compaction and formed high-density focal adhesions (Fig. 5 E). In contrast, cells treated with Y-27632 or staurosporine displayed the disappearance of high-density focal adhesions, whereas low-density focal adhesions remained in the periphery of the cells in the lamellipodia (Fig. 5, F and G). To correlate the changes in intracellular tension with that of the integrin density and focal adhesion size, we displayed the relative peak integrin density (compared with the integrin density in the membrane) and the respective area of focal adhesions (see Materials and methods). For each experimental condition, we analyzed ∼500 focal adhesions from different cells. Control cells displayed a significant number of small-sized, low-density focal adhesions that were mainly associated with protruding lamellipodia (Fig. 5 H). In addition, a considerable number of large-sized, generally high-density focal adhesions were found (Fig. 5 H) (average relative integrin density and size: 5.14-fold, 1.15 μm2). The stimulation of the RhoA pathway with LPA led to a shift to denser and generally larger focal adhesions (Fig. 5 I) (average relative integrin density and size: 6.77-fold, 1.69 μm2). In contrast, treatment with either Y-27632 or staurosporine resulted in a drop in integrin densities (Fig. 5, J and K). Interestingly, the average size of the contacts in Y-27632–treated cells was only slightly lower compared with control cells, whereas the average size of staurosporine treated contacts was dramatically reduced (average relative integrin density and size: Y-27632, 4.39-fold, 0.87 μm2; staurosporine, 4.49-fold, 0.43 μm2). Furthermore, the dynamic transition of high- to low-density focal adhesions was analyzed in living B16 F1 cells by time-lapse microscopy (Fig. 6; Video7, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). Release of intracellular tension by Y-27632 induced the rapid decrease in integrin densities in peripheral focal adhesions (Fig. 6, B and C, arrowhead). During the first 10 min of treatment, neither the shape of the contacts nor that of the cell did change. With time, the cell developed large lamellipodia that contained numerous dot-like tension-independent focal adhesions. In addition, the Y-27632–induced low-density focal adhesions began to change shape but remained undispersed during the entire time of observation (up to 90 min) (Video7, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). These observations suggest that high intracellular tension correlates with the formation and maintenance of high-density αVβ3-integrin focal adhesions. The formation of low-density αVβ3-integrin focal adhesions is favored under conditions of low intracellular tension.

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