Rapid cell migration requires the efficient regulated formation and breakdown of adhesions and cycling of components from the rear to the front. Several models have been proposed for adhesion formation, but less is known about the breakdown of adhesions. One set of studies suggest a hierarchical model for adhesive assembly (Miyamoto et al. 1995a
,Miyamoto et al. 1995b
; Yamada and Miyamoto 1995
), whereas other studies suggest that large preformed cytoskeletal complexes are stabilized by their association with integrins bound to the substratum (DePasquale and Izzard 1987
; Izzard 1988
). Adhesion breakdown may occur by a reversal of the mechanisms for assembly, specific enzymatic modifications, or mechanical stresses that lead to the fracture of specific interactions in the cytoskeletal–integrin linkage. Mechanisms have been proposed for cycling components that accumulate at the rear to the cell front, including directed vesicle trafficking, movement of adhesive complexes, and directed molecular movements.
In this study, we evaluated the relative contributions of these mechanisms to adhesion dynamics by directly visualizing α5 integrin-, paxillin-, and α-actinin–GFP as adhesions formed and dispersed in migrating cells. Our data support hierarchical models for the formation of initial adhesive complexes. We provide evidence that classes of adhesive components enter adhesions serially. Our observations further suggest that signaling components such as paxillin enter adhesions early and turn over readily with prominent accumulations of structural molecules such as α-actinin subsequently joining the adhesion. We also support nucleation rather than the clustering of smaller minicomplexes, as reported previously for the formation of E-cadherin junctions (Adams et al. 1998
), as the mechanism by which small adhesions (putative focal complexes) form. By contrast, we do not have evidence supporting the stabilization of large preformed cytoplasmic complexes. After adhesions broke down in the rear, α5-GFP was found in fibrous structures behind the cell, whereas α-actinin–GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dispersed.
The leading edge of membrane protrusions is a site where new adhesions form. Of the three fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell near the leading edge. It appeared in a wave of fluorescent intensity and then concentrated in visible focal complex–like structures. Interestingly, α5 integrin and prominent α-actinin though present at the leading edge are not detectable in these paxillin-rich complexes. Thus, paxillin recruitment to these contact sites is an early event in the formation of adhesions. Since paxillin serves an adaptor function in recruiting several signaling components to the membrane, it follows that these newly forming adhesions likely serve signaling roles. Consistent with this hypothesis, other studies have suggested that tyrosine phosphorylation of paxillin occurs early in focal adhesion assembly (Richardson et al. 1997
). Tyrosine phosphorylation of paxillin can create at least two SH2 domains, which can function as binding sites for other signaling molecules such as members of the Crk family (Bellis et al. 1995
; Schaller and Parsons 1995
; Richardson et al. 1997
The absence of clearly visible α5 integrin in these complexes suggests that either the α5 integrin is not involved in the formation of new adhesions or newly forming adhesions are initiated and/or nucleated by α5 concentrations that are too low to be detected as discrete visible complexes in the light microscope. Although it is possible that other molecules, including other integrins, layilin, or syndecan play this role, it is also clear that these cells require α5 to adhere and migrate (Borowsky and Hynes 1998
; Longley et al. 1999
). The CHO B2 cells, which have almost no endogenous α5 integrin, do not adhere and migrate unless they ectopically express α5 integrin. In addition, adhesion-perturbing antibodies directed against the α5 integrin or Fn inhibited the organization of paxillin, which was also not seen in cells plated on poly-l
-lysine. Despite reports that there are few other integrins in these cells, we stained for αv, β1, and β3 subunits and were unable to detect organized adhesions. We repeated all of these studies in WI38 cells, which tend to have more highly organized adhesions. The result was similar in that the forming adhesions show clearly organized paxillin but not α5 integrin. It is also evident that α5 binding to Fn is necessary for the formation of these adhesions since they were not observed when the integrin–Fn interaction was disrupted with a function-blocking antibody. Finally, as visualized with antibody staining or GFP probes in cells with well-developed focal adhesions the amount of α5 in the adhesions is considerably less than that seen for cytoskeletal markers such as paxillin or vinculin. Furthermore, not all adhesions that stain with cytoskeletal markers colocalize with visibly organized integrin. From this, we conclude that the newly forming adhesions are initiated by α5 engagement, since it is required for organization of paxillin. These putative α5 integrin-containing nucleation sites are not organized as large visible complexes, though present in a concentration sufficient to stimulate robust paxillin recruitment. As with other amplification cascades that typify many other signaling pathways, ligand activation of only a small number of integrins may be sufficient to stimulate the assembly of a large macromolecular adhesive complex.
Once protrusions stabilized, α-actinin began to colocalize with paxillin in small foci at the edge of the former lamellipodium. These small α-actinin–containing foci grew in size and extended small fiber-like structures toward the cell body, which is consistent with a recent study (Edlund et al. 2001
). Once formed, some of the α-actinin adhesions slid inward from the cell perimeter and stabilized the paxillin, which no longer turned over. Thus, both paxillin and α-actinin appeared to enter newly forming adhesions before visible α5 integrin. However, once visibly organized α5 integrin entered the adhesions they remained relatively stationary and did not move toward the cell center; these adhesions also contained paxillin and α-actinin. Taken together, this suggests that not all adhesions contain clearly visible α5 integrin, but when the integrin was present the adhesions remained stationary. Previous studies have also reported a sliding of adhesions in cells (Smilenov et al. 1999
; Pankov et al. 2000
; Zamir et al. 2000
). In one study, the membrane-spanning and cytoplasmic domain of β1 integrin was fused to GFP such that the GFP region resided on the outside of the cell (Smilenov et al. 1999
). Interestingly, they reported that in stationary cells adhesions moved toward the cell center, whereas in migrating cells the adhesions were stationary (Smilenov et al. 1999
). Although we observed adhesions moving toward the cell center in migrating cells, those with visible α5 integrin complexes were stationary.
The α5-GFP and α-actinin–GFP localized prominently at the leading edge in membrane ruffles and protrusions. Since membrane-bound but not soluble GFP was also seen in ruffles, this reflects their membrane localization. However, α-actinin is an intracellular molecule that has no membrane-targeting signal and thus must rely on intermolecular interactions to localize it to the membrane. Since neither soluble GFP nor paxillin was observed at the leading edge, the interactions mediating α-actinin targeting are specific. A possible mechanism for α-actinin targeting to membrane protrusions is through its interaction with integrins, since α-actinin binds directly to the cytoplasmic domain of the β1 subunit in vitro (Otey et al. 1990
). Consistent with this mechanism, α-actinin colocalized with α5 integrin in the membrane ruffles. When the head and rod domains of α-actinin fused to GFP were expressed in the CHO K1 cells, the rod domain, which contains the β1 integrin–binding site (Otey et al. 1993
), localized to membrane protrusions; however, the head domain, which contains the actin-binding site, appeared in highly organized fiber-like structures. This observation is consistent with a mechanism by which an interaction with integrins may function to localize α-actinin to membrane protrusions, but cytotoxicity precludes a firm conclusion. Alternatively, other α-actinin–binding partners such as phosphatidylinositol bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate may play a role in its recruitment to membrane protrusions (Fukami et al. 1994
; Greenwood et al. 2000
The movement of the cell over stable adhesions suggests that adhesive components will tend to concentrate away from the leading edge toward the cell rear. One hypothesis proposes that integrins are recycled from the rear of the cell to the leading edge, thus providing a supply of integrins to newly forming adhesions (Bretscher 1989
; Dalton et al. 1995
; Lawson and Maxfield 1995
; Pierini et al. 2000
). The most direct observation supporting this mechanism comes from staining integrins in fixed neutrophils (Lawson and Maxfield 1995
; Pierini et al. 2000
). In these studies, vesicles are seen at the rear in cells whose migration was inhibited by calcium buffering and in a polarized perinuclear area in migrating cells. However, these cells are too small to discern spatially the biosynthetic and recycling compartments from the base of the protrusion. Furthermore, some mechanisms used by neutrophils are not readily apparent in other cells such as fibroblasts and are specific for certain integrins (Lawson and Maxfield 1995
; Pierini et al. 2000
). Our studies with larger cells and direct visualization at high temporal resolution intervals provide a far more complete picture.
In CHO cells with robust protrusive activity, we observed α5 integrin in vesicle-like structures that emanated from membrane protrusions and congregated in a perinuclear region where it colocalized with endogenous transferrin receptor. Although the fate of these internalized integrins is not known, this observation suggests that at least a fraction of these molecules are delivered to a large recycling compartment. Alternatively, some of the internalized integrin vesicles may be degraded in lysosomal compartments. A small fraction of the vesicles moved from the perinuclear area toward the cell front, but they disappeared at or before reaching the lamellipodial base; none were observed in the lamellipodium or at the leading edge. Cells migrating under conditions in which they exhibited minimal membrane ruffling presented a complementary picture. In these cells, α5 vesicles moved from the perinuclear region to the base of the lamellipodia, whereas vesicles moving from the front were seen only infrequently. In all cells, we observed vesicles moving from the cell rear to the perinuclear area in agreement with previous observations in fibroblasts and neutrophils (Regen and Horwitz 1992
; Lawson and Maxfield 1995
; Palecek et al. 1996
; Pierini et al. 2000
). However, vesicles moving from the rear to the front were not observed possibly due to the density of fluorescent material in the perinuclear area.
Thus, two endocytic pathways may be used by integrins. One may function to remove unligated integrin from the membrane in highly protrusive regions of the cell. In support of this, fewer vesicles were observed emanating from membrane protrusions as the substrate concentration increased. A second pathway removes integrins at the cell rear and delivers them either to the lysosomal compartment or to the cell front for formation of new adhesions. The movement of integrin-containing vesicles from the perinuclear area to the base of protrusions is consistent with previous studies (Lawson and Maxfield 1995
; Pierini et al. 2000
). It also complements particle-tracking studies that reported the directed movement of integrins to the leading edge in lamellipodia (Schmidt et al. 1993
). Finally, we do not observe α-actinin on any of the α5-containing vesicles, suggesting that the integrins are not trafficked in α-actinin–containing complexes.
Cleavage of the linkage between integrin and other cytoskeletal components may initiate the release of adhesions. The integrins are seen in fibers behind migrating cells without visible α-actinin or paxillin, whereas adhesive complexes containing α-actinin and paxillin without highly organized integrin translocate from the rear by sliding along the cell edge. Unlike adhesion formation, α-actinin and paxillin were not observed to depart the adhesive clusters serially, but instead the complexes were seen to disperse. This suggests that adhesion breakdown is not simply a reversal of the mechanisms of formation.
We propose the following working model based on our observations of adhesion formation and turnover in migrating cells. Integrin, membrane-bound α-actinin (possibly complexed to the integrin), and cytoplasmic paxillin are all present in new protrusions. The binding of integrins to the ECM initiates, perhaps in conjunction with other receptors, the recruitment of signaling molecules such as paxillin to newly forming contact sites. Although substrate-bound integrins may also serve as the nucleation sites for these new adhesions, it is also possible that other molecules serve this role. The unligated integrins are rapidly endocytosed and traffic to a perinuclear region. These paxillin-rich sites are highly dynamic and tend to turn over at the base of the protrusion and cycle to the leading edge as new adhesions form. Structural molecules like α-actinin are subsequently recruited to this site, although small quantities may reside with the initial putative integrin foci. These developing adhesion complexes grow in size and molecular complexity as α-actinin first enters them and then forms stress fiber–like extensions that grow toward the cell body. The presence of α-actinin serves to stabilize the paxillin, which does not turn over in adhesions containing prominent α-actinin; its presence also coincides with the centripetal movement of adhesions. Subsequently, visible concentrations of integrin enter the adhesive complex and function to stabilize the centripetal movement. At the cell rear, cleavage of the integrin–cytoskeletal linkage at a site proximal to the integrin is a prominent mechanism to initiate breakdown of adhesions. The remaining paxillin- and α-actinin–containing complexes move toward the cell body and then disperse rapidly. Although some integrin is left behind on the substrate, some integrin also appears in vesicles that move toward the cell body where they are either degraded or cycled to the cell front for incorporation into new adhesions.