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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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Dynamics of β3–GFP-integrin in stable transfected B16 F1 cells. Time-lapse analysis of a B16 β3–GFP cell plated overnight on vitronectin (1 μg ml−1) revealed the transient β3–GFP-integrin clustering and subsequent dispersal in the advancing lamellipodium. A typical β3-integrin cluster (A, circled) appeared close to the leading edge (8′) and remained stationary (12′) until it began to gradually disappear (16′–24′). In retracting parts of the cells, integrin clusters began to slide inward (arrow). To appreciate the relative movement of the different integrin clusters during this time-lapse, an overlay revealed the stationary nature of focal adhesions in the lamellipodia (arrowhead) and the streak-like pattern of sliding focal adhesions in retracting parts of the cell (arrow). In B, a higher temporal and spatial resolution of the boxed area in A (12′) revealed the polymorphic appearance of the stationary integrin clusters (small box as reverence). Although shapes were variable, the fate of the clusters were identical. Arrowheads in B mark the smoothly advancing leading edge of the lamellipodium. Bar, 18 μm.
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fig2: Dynamics of β3–GFP-integrin in stable transfected B16 F1 cells. Time-lapse analysis of a B16 β3–GFP cell plated overnight on vitronectin (1 μg ml−1) revealed the transient β3–GFP-integrin clustering and subsequent dispersal in the advancing lamellipodium. A typical β3-integrin cluster (A, circled) appeared close to the leading edge (8′) and remained stationary (12′) until it began to gradually disappear (16′–24′). In retracting parts of the cells, integrin clusters began to slide inward (arrow). To appreciate the relative movement of the different integrin clusters during this time-lapse, an overlay revealed the stationary nature of focal adhesions in the lamellipodia (arrowhead) and the streak-like pattern of sliding focal adhesions in retracting parts of the cell (arrow). In B, a higher temporal and spatial resolution of the boxed area in A (12′) revealed the polymorphic appearance of the stationary integrin clusters (small box as reverence). Although shapes were variable, the fate of the clusters were identical. Arrowheads in B mark the smoothly advancing leading edge of the lamellipodium. Bar, 18 μm.

Mentions: This αVβ3–GFP tool permitted now the direct observation of integrin clustering and turnover in adhesion sites of migrating or stationary cells. Therefore, we performed time-lapse experiments with stably β3–GFP-transfected, fast-migrating B16 F1 melanoma cells, or stationary 3T3 fibroblasts (B16 β3–GFP or 3T3 β3–GFP, respectively). In B16 β3–GFP cells, we observed the formation of small integrin clusters just behind the leading edge of the advancing lamellipodia (Fig. 2, A and B). These clusters remained stationary with respect to the substratum, whereas the cell moved forward. When GFP-containing focal adhesions reached a distance of 10 μm from the leading edge, they began to shrink and finally disappeared (Fig. 2, A, circled, and B, boxed; Video1, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). We noted that some of the focal adhesions in the smoothly protruding lamellipodia assumed an elongated shape. Although we never observed actin stress fibers in actively protruding lamellipodia, the presence of radially oriented actin ribs within the lamellipodia was frequent (Ballestrem et al., 1998). Continuous appearance and disappearance of stationary β3-integrin focal adhesions occurred within a restricted area in the advancing lamella. We refer to this area as the zone of transient integrin clustering. In posterior regions of the cell, integrin-containing focal adhesions moved in relation to the substratum during retraction (Fig. 2 A, arrow; Video2, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). To examine the possibility that integrin contacts in highly migratory melanoma cells might behave differently from stationary or slow moving fibroblasts (3T3 β3–GFP cells), we compared the appearance of GFP fluorescence in transfected B16 and 3T3 cells. 3T3 cells displayed continuous cycles of lamellipodia formation followed by retraction, and they showed comparable integrin cluster dynamics to what had been seen in B16 β3–GFP cells (Fig. 3; Video3, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). However, during collapse of lamellipodia, small focal adhesions in 3T3 cells transformed into larger, fluorescently brighter focal adhesions (Fig. 3 B). During retraction, these focal adhesions began to move in relation to the substratum (Fig. 3, B and C). In conclusion, our data show that αVβ3-integrins aggregate into stationary focal adhesions within the zone of transient integrin clustering during the protrusion of lamellipodia. After the collapse of lamellipodia and subsequent retraction, small stationary focal adhesions transform into inwards sliding larger 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)

Dynamics of β3–GFP-integrin in stable transfected B16 F1 cells. Time-lapse analysis of a B16 β3–GFP cell plated overnight on vitronectin (1 μg ml−1) revealed the transient β3–GFP-integrin clustering and subsequent dispersal in the advancing lamellipodium. A typical β3-integrin cluster (A, circled) appeared close to the leading edge (8′) and remained stationary (12′) until it began to gradually disappear (16′–24′). In retracting parts of the cells, integrin clusters began to slide inward (arrow). To appreciate the relative movement of the different integrin clusters during this time-lapse, an overlay revealed the stationary nature of focal adhesions in the lamellipodia (arrowhead) and the streak-like pattern of sliding focal adhesions in retracting parts of the cell (arrow). In B, a higher temporal and spatial resolution of the boxed area in A (12′) revealed the polymorphic appearance of the stationary integrin clusters (small box as reverence). Although shapes were variable, the fate of the clusters were identical. Arrowheads in B mark the smoothly advancing leading edge of the lamellipodium. Bar, 18 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199321&req=5

fig2: Dynamics of β3–GFP-integrin in stable transfected B16 F1 cells. Time-lapse analysis of a B16 β3–GFP cell plated overnight on vitronectin (1 μg ml−1) revealed the transient β3–GFP-integrin clustering and subsequent dispersal in the advancing lamellipodium. A typical β3-integrin cluster (A, circled) appeared close to the leading edge (8′) and remained stationary (12′) until it began to gradually disappear (16′–24′). In retracting parts of the cells, integrin clusters began to slide inward (arrow). To appreciate the relative movement of the different integrin clusters during this time-lapse, an overlay revealed the stationary nature of focal adhesions in the lamellipodia (arrowhead) and the streak-like pattern of sliding focal adhesions in retracting parts of the cell (arrow). In B, a higher temporal and spatial resolution of the boxed area in A (12′) revealed the polymorphic appearance of the stationary integrin clusters (small box as reverence). Although shapes were variable, the fate of the clusters were identical. Arrowheads in B mark the smoothly advancing leading edge of the lamellipodium. Bar, 18 μm.
Mentions: This αVβ3–GFP tool permitted now the direct observation of integrin clustering and turnover in adhesion sites of migrating or stationary cells. Therefore, we performed time-lapse experiments with stably β3–GFP-transfected, fast-migrating B16 F1 melanoma cells, or stationary 3T3 fibroblasts (B16 β3–GFP or 3T3 β3–GFP, respectively). In B16 β3–GFP cells, we observed the formation of small integrin clusters just behind the leading edge of the advancing lamellipodia (Fig. 2, A and B). These clusters remained stationary with respect to the substratum, whereas the cell moved forward. When GFP-containing focal adhesions reached a distance of 10 μm from the leading edge, they began to shrink and finally disappeared (Fig. 2, A, circled, and B, boxed; Video1, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). We noted that some of the focal adhesions in the smoothly protruding lamellipodia assumed an elongated shape. Although we never observed actin stress fibers in actively protruding lamellipodia, the presence of radially oriented actin ribs within the lamellipodia was frequent (Ballestrem et al., 1998). Continuous appearance and disappearance of stationary β3-integrin focal adhesions occurred within a restricted area in the advancing lamella. We refer to this area as the zone of transient integrin clustering. In posterior regions of the cell, integrin-containing focal adhesions moved in relation to the substratum during retraction (Fig. 2 A, arrow; Video2, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). To examine the possibility that integrin contacts in highly migratory melanoma cells might behave differently from stationary or slow moving fibroblasts (3T3 β3–GFP cells), we compared the appearance of GFP fluorescence in transfected B16 and 3T3 cells. 3T3 cells displayed continuous cycles of lamellipodia formation followed by retraction, and they showed comparable integrin cluster dynamics to what had been seen in B16 β3–GFP cells (Fig. 3; Video3, available at http://www.jcb.org/cgi/content/full/jcb.200107107/DC1). However, during collapse of lamellipodia, small focal adhesions in 3T3 cells transformed into larger, fluorescently brighter focal adhesions (Fig. 3 B). During retraction, these focal adhesions began to move in relation to the substratum (Fig. 3, B and C). In conclusion, our data show that αVβ3-integrins aggregate into stationary focal adhesions within the zone of transient integrin clustering during the protrusion of lamellipodia. After the collapse of lamellipodia and subsequent retraction, small stationary focal adhesions transform into inwards sliding larger 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