- Email: [email protected]
Cytomechanics of cadherin-mediated cell—cell adhesion
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Cytomechanics of cadherin-mediated cell-cell adhesion Cynthia L Adams* and W James Nelsont Cadherin-mediated adhesion regulates transitions from initial cell-cell recognition to loosely adherent cell clusters and ultimately, to strongly compacted groups of cells in colonies. Recent studies have described distinct roles for intermolecular clustering of cadherins as well as interactions of cadherin with the actin cytoskeleton in establishing cell-cell adhesion. Integrating cytomechanical roles of cadherin-mediated adhesion will lead to a greater understanding of how cadherins regulate tissue morphogenesis. Addresses *Cytokinetics, Inc., 280A East Grand Avenue, South San Francisco, CA 94040, USA tDepartment of Molecular and Cellular Physiology, Beckman Center, B121 Stanford University School of Medicine, Stanford, CA 9430554345, USA; e-mail: [email protected] Current Opinion in Cell Biology 1998, 10:572-577
http://biomednet.com/elecref/0955067401000572 © Current Biology Ltd ISSN 0955-0674 Abbreviations CD cytocholasin D MDCK Madin-Darbycanine kidney Introduction
Studies to understand the basis of cell-cell adhesion were underway at the turn of the century [1]. These early studies had a mechanical focus, and aimed to measure the movements and forces underlying the properties of cell-cell interactions. Measurements of forces between cells, chemical requirements for adhesion, and sorting out of heterogeneous populations of cells [2] lead to a greater understanding of some of the physical characteristics of adhesion. When molecules involved in cell-cell adhesion were discovered [3,4,5], the focus shifted to understanding the molecular and biochemical nature of protein-protein interactions and assembly of protein complexes [6]. Recently, however, attention has returned to the problem of how these protein complexes are involved in the physical aspects of cell-cell adhesion. Here we discuss some recent studies on mechanisms involved in cadherin-mediated cell-cell adhesion. Cadherins are a family of single-transmembrane, calciumdependent cell adhesion proteins [6]. Defining mechanisms involved in cadherin-mediated cell-cell adhesion is at the core of understanding a diverse range of cellular phenomena including contact inhibition of cell growth [7,8°,9], condensation and compaction of cells [1,10[, wound healing [11,12], and tumorigenesis [13"]. Cell-cell adhesion requires homotypic binding of the extracellular domain of cadherins on adjacent cells, and the attachment of the intracellular domain of cadherin to the actin cytoskeleton. Cadherins interact with actin through different complexes
composed of cytosolic proteins (see [6]), termed ot-catenin, 13-catenin, plakoglobin, and p120 CAs. Through these proteins, cadherin is attached to the actin cytoskeleton [14",15,16]. Recent characterization of the three-dimensional crystal structure of ]]-catenin [17 °°] has shed new light on how catenins bind to the cytoplasmic domain of cadherin. Much attention has focused on the assembly of cadherin-catenin-actin complexes and the role they play in cell-cell adhesion [6]. Understanding the dynamics of cadherin-catenin-actin complex assembly and the significance of these interactions in stabilizing cell-cell contacts are crucial for defining mechanisms by which tissue integrity is established and maintained. The role of cadherin adhesion
clustering
in c e l l - c e l l
Recently, lateral dimerization and clustering of cadherins have been shown to be involved in cell-cell adhesion in vitro [18,19"] and in vivo [20°1. Crystal structures of cadherins suggest that lateral dimers of cadherin on one cell could bind to lateral dinaers of cadherin on an opposing cell [21]. T h e affinity of homotypic interactions through cadherin extracellular domains is weak (< 1 ~tM, [221) clustering many low affinity binding sites may be required to increase the effective affinity of the cadherin-cadherin interaction. There may be several mechanisms involved in stabilizing a lateral cadherin dimer. A role for interactions between the extracellular domains of neighboring cadherins is supported by a study showing increased cell-cell adhesion in cells engineered to express large aggregrates of the extracellular domain of cadherin without the cytoplasmic domain [20°]. This study indicates that aggregation of the extracellular domain of cadherin is sufficient for cell-cell adhesion. In vivo, however, interactions between the cytosolic domain of cadherin with adjacent cadherin and cytosolic proteins may also be necessary for the formation of lateral cadherin dimers. In one study, cells expressing cadherin lacking the 13catenin binding domain were able to both cluster cadherin and mediate adhesion in suspension, while cells expressing cadherin lacking the juxtamembrane 94 amino acids, a putative p120 CAs binding site, were not [19°'1. This study suggested that p120 CAs binding to cadherin is sufficient for cell--cell adhesion; however, the ability of these cells to adhere in the absence of [3-catenin binding to cadherin is in contrast to many studies that have shown that disruption of E-cadherin binding to [3-catenin decreascs cell-cell adhesion. In particular, it has been shown that a 34 amino acid deletion in the juxtamembranc domain of cadherin had little effect on adhesion, while deletion of the [3-catenin binding site resulted in loss of adhesion [9]. While providing more details about the important molecular interactions, these studies begin to illuminate the difficulty of differentiating
Cytomechanics of cadherin-mediated cell-cell adhesion Adams and Nelson
Figure I Accumulation of E-cadherin into puncta during initial stages of MDCK epithelial cell-cell adhesion. (a-d) Differential interference contrast (DIC) images from a time-lapse video recording and (e) a schematic representation of a developing contact between two MDCK cells are shown. (a-d) The age of the contact prior to fixation is indicated in the upper left hand corner of each frame (in minutes). The vertical lines running down the figure indicate the boundaries of the developing contact at each time point (between red lines, >40 min; red-yellow, 2 5 - 4 0 rain; yellow-green, 10-25 min; and green-blue, 1-10 min). (e) The newly formed length of contact between frames is represented as the horizontal line between the arrows (black, >40 min; red, 2 5 - 4 0 min; blue, 10-25 min; green, 1-10 min). For example, the additional contact length inserted between 40 and 25 min is represented by the area between the vertical red and yellow lines throughout the image, and the red horizontal lines between the arrows in (a). Puncta which are in the region are considered to be 25-40 min old. (f) Retrospective E-cadherin staining of the contact, and (g) in pseudo-color formating. Figure reproduced with permission [18].
(a)
(b)
(c) between the importance of cadherin clustering and cytoskeletal linkage through catenins in regulating adhesion. Catenins probably exert their stabilizing influence on cell-cell contacts through their interactions with actin. 13catenin links cadherin to the actin cytoskeleton via c~-catenin, and this link is thought to be necessary for stabilizing cell contacts; disruption of 0t-catenin binding to the catenin complex inhibits adhesion (see [6]). Significantly, cadherin mediated cell-cell adhesion was inhibited in a dose dependent fashion by overexpressing EpCAM, perhaps by lowering ~-catenin expression [23]. Over-expression of N-cadherin in MDA-MB-468 cells, which lack 0t-catenin expression, however, increased the ability of these cells to adhere [24°]. Vinculin, which has homology to ot-catenin (see [25]), may bind to the cadherin/~-catenin complex thereby compensating for the lack of ~-catenin. Alternatively, an increase in surface expression of N-cadherin may increase adhesion. Changes in expression levels of cadherins and catenins have been shown to affect cell-cell adhesion [26°,27-29]. None of these studies, however, have demonstrated that perturbation of the cadherin clustering mechanism, perse, is responsible for each of these different adhesion phenotypes. T h e exact role of p120 CAs in cell-cell adhesion remains a matter of debate. Expression of the putative p 120 cAs binding site of cadherin, without the catenin binding site or extracellular domain, was first shown to induce an adhesion defect in Xenopus embryos [30]. One explanation of this effect is that pl2OCAS may stabilize a lateral cadherin heterodimer, comprising a mutant and wild-type cadherin, which is nonfunctional. A homodimer cadherin-pl20CAS complex may be necessary to increase the rates of initial homotypic recognition and binding between cadherin molecules on two adhering cells. Subsequently, c~-catenin, 13-catenin and the actin cytoskeleton may stabilize the cadherin-pl20 cAs cluster and, thereby, strengthen cell-cell adhesion. This interpretation would explain why cells over-expressing cadherin which lack the p120 CAs binding
(d)
(e)
(f)
(g)
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574 Cell-to-cellcontactand extraeellularmatrix
region, but maintain the [~-catenin binding region, adhere very slowly [9]. These cells may ultimately form strong, and possibly stronger, contacts by recruiting the actin cytoskeleton to the cell-cell contact site through their catenin linkage to the actin cytoskeleton (see above). Cadherin-catenin complexes immobilized within a cell-cell contact by actin may act as a trap for cadherin freely diffusing within the plasma membrane, as originally envisioned in the diffusion-trap model [31,32]. Over time, freely diffusing E-cadherin may redistribute to the contact (trap) by associating with immobilized E-cadherin or other associated proteins (e.g. p120 Cns, catenins or actin). A consequence of this diffusion would be to increase the local concentration of cadherin at the contact thereby resulting in strengthening of cell-cell adhesion. Analysis of cells prior to cell--cell contact has revealed that E-cadherin has a homogeneous distribution over the plasma membrane [18,33]. After initiation of cell-cell adhesion, Triton X-100 insoluble E-cadherin accumulates along the length of the plasma membrane within the contact. When Triton X-100 soluble E-cadherin is extracted, the relative amount of Triton X-100 insoluble E-cadherin is much greater at cell-cell contacts than in other regions of the plasma membrane, and is associated with actin ([18,33]; see Figure 1). Thus, short-range diffusion trapping around immobilized E-cadherin may be important for clustering E-cadherin within the cell-cell contact. T h e combined effects of long-range diffusion and shortrange clustering of cadherin on cell-cell adhesion may explain discrepancies in the kinetics of adhesion between cells expressing different levels ofcadherin. If clustering of cadherin is important to stabilize the contact, and the level of cadherin protein at a single cluster is critical, then the time required for the appropriate number of cadherin proteins to diffuse into the trap will increase as the number of surface cadherin molecules decreases. Diffusion of cadherin within the plasma membrane has been examined by fluorescence-photobleaching-recovery and single-particletracking techniques [34,35°°]. These studies showed that a small population of E-cadherin is attached to the cytoskeleton in the absence of cell-cell contacts. This population of proteins may play a role in initiating the immobilization of E-cadherin at a developing contact site. A kinetic measurement of cadherin clustering has been performed using time-lapse differential interference contrast microscopy in combination with quantitative retrospective immunocytochemistry ([18]; see Figure 1). T h e distribution and assembly kinetics of Triton X-100 insoluble E-cadherin, c~-catenin, [3-catenin, plakoglobin and actin at newly formed cell-cell contact sites were measured in Madin-Darby canine kidney (MDCK) epithelial cells. A major observation was that during initial stages of cell-cell adhesion, E-cadherin, c~- catenin and I]-catenin exist as uniformly sized and spaced punctate clusters along the length of a cell-cell contact, and accumulate propor-
tionally within 20 minutes of cell-cell contact (see Figure 1). This study dispelled the notion of contact lengthening as a function of continuous E-cadherin zippering. Instead, discrete 'puncta' of E-cadherin, 13-catenin, and ct-catenin are assembled along the contact at a constant average density of ~ 1 puncture per 1.5 lain of contact length. Each of these puncta is associated with actin filaments that appear to split off the circumferential actin belt in the cortex of these previously migrating cells [18].
Clusters of cadherin c o o r d i n a t e a c t i n / m y o s i n t e n s i o n b e t w e e n c o n t a c t i n g cells Two studies shed light on a cytomechanical role for the cadherin-catenin complex in establishing cell-cell adhesion. First, tension transmitted through adherens junctions in Swiss 3T3 fibroblast cells was shown to induce a visceoelastic response in the cytoplasm [36]. Second, the maintenance of the strand-like appearance of the cadherin-catenin complex in fibroblasts was reversibly abolished in the presence of 2,3-butanedione 2-monoxime, a non-specific inhibitor of myosin [37"]. T h e s e data suggest that tension is not just generated through clusters of cadherin, but that tension is required to maintain them. Mechanisms involved in the formation of cytomechanical tension between cells have yet to be identified or measured directly, although much work has been done to understand tension generated by cell-substrate interactions [38"]. Myosin is a good candidate for a force-generating protein important for cell-cell adhesion and cell polarity. Nonmuscle myosin II is a mechanochemical motor protein, which slowly moves from the pointed to the barbed ends of actin filaments in an ATP dependent fashion. In noncontacting edges of cells (lamellae), actin polymerizes at the barbed end and depolymerizes at the pointed end of actin filaments (see reviews [39-41]). There is a continual rearward flow of actin filaments and associated proteins in lamellae, which is referred to as 'treadmilling' (see reviews [39-41]). The rearward flow of actin is due to myosin pulling actin filaments towards the center of the cell [42]. When the barbed ends of actin filaments are firmly attached to the membrane, actin treadmiiling ceases, and movement of myosin along Factin generates tension between the membrane and the cell body [42,43"]. T h e generation of tension allows the cell body to translocate towards the site of attachment using the molecular clutch mechanism proposed for cell-substrate interactions [43"]. It has also been shown that there is no rearward flow of actin in areas behind cell--cell contacts [8°]. T h e role of actin binding to the cadherin--catenin complex may be to strengthen cell-cell adhesion using this molecular clutch mechanism. This mechanism, however, requires that the cadherin--catenin complex binds to the barbed ends of actin filaments. T h e polarity of actin filaments with respect to the cadherin-catenin complex has yet to be determined. Alpha-catenin, the putative linker of cadherin
Cytomechanics of cadherin-mediated cell-cell adhesion Adams and Nelson
to actin, is an actin bundling protein in vitro [15]. Pretreatment of cells with cytochalasin D (CD), an actin capping drug, abolishes the ability of cells to initiate cell--cell adhesion [4,441. But CD does not cause the separation of established cell-cell contacts in polarized epithelia [45]. Together, these results suggest that the cadherin-catenin-actin complex may interact with the barbed ends of actin filaments with a stronger affinity than CD, or that the barbed ends of actin filaments are inaccessible to CD in old contacts. If the cadherin-catenin complex does interact with the barbed ends of actin filaments, it is reasonable to speculate that the myosin machinery, via actin, generates tension between cells via cadherin. While very little is known about the role of myosin during cell--cell adhesion, myosin has been implicated in wound healing which requires the re-establishment of cell-cell contacts. When a polarized monolayer of epithelial cells is wounded by removing a patch of ceils, the monolayer quickly fills in the gap with ceils that surround the wound [11]. One mechanism by which an epithelial monolayer repairs damage is described by the 'purse-string' theory [46]. This theory proposes that circumferential tension is generated by actin cables held together at the peripheral cell-cell contacts of the wound perimeter [47]. This tension causes the actin cables to cinch together, which results in shortening of the wound perimeter, in the absence of cell proliferation, until the wound is filled in by the surrounding cells. The interactions between cadherin, actin, and myosin have been implicated in generating the forces required for wound closure. Actin cables, which circumscribe the wound perimeter, appear continuous from cell to cell, are decorated with myosin II [11,48], and are linked by large clusters of E-cadherin at the cell-cell contact [49]. These E-cadherin clusters may play a role in linking the forces generated by actin cables in adjacent cells. T h e Rho family of small GTPases plays important roles in regulating actin cytoskeleton organization and cell adhesion [49,50°,51"]. Recent studies indicate that epithelial cell-cell adhesion is affected by mutant RhoA and Racl [52°'-54"]. Downstream effectors of RhoA (Rho-kinase [55,56] and its targets, myosin and myosin binding proteins [57-591) and Racl (PAK [60] and IQGAP [61"]) are excellent candidates for regulating assembly of the cadherin-catenin complex and the actin cytoskeleton contractility (myosin). Further insight into structural implications for cadherin binding to the actin cytoskeleton is suggested by studies showing that integrin adhesion proteins are mechanically coupled to the nucleus [62"]. T h e "tensegrity" model postulates that the actin cytoskeleton interconnects extracellular adhesions to the internal organelles [63]. Therefore, stress applied through adhesive complexes is transmitted throughout the cell ultimately coordinating cell shape, motility, and even growth and differentiation. While much work has been done to eh, cidate this conncc-
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tivity in interactions between cells and the extracellular matrix [43"], little is known about similar roles for cell-cell interactions. Studies aimed at understanding the cytomechanical roles of cadherin in this process will be of great interest in the future.
Conclusion T h e structural link between E-cadherin clusters and the actin cytoskeleton is necessary for strengthening cell-cell adhesion. This link may use myosin to generate tension between two contacting cells. T h e tension is redistributed by reorganization of the cortical actin cytoskeleton to the edges of the cell-cell contact, which leads to the formation of strong cell-cell contacts and changes in cell organizations. This tension leads to dramatic changes in cell organization leading to development of cell polarity. Full elucidation of the molecular dynamics underlying the initiation and strengthening of cadherin mediated cell--cell adhesion and the long term reorganization of cells into colonies awaits further study (for further discussion see [64"'1).
Acknowledgements Thank you to Chris Hazuka for his helpful comments and suggestions on the manuscript. Work from the Nelson laboratory was supported by a grant from the National Institutes of Health.
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64. Adams CA, Smith SJ, Nelson WJ: Mechanisms of epithelial cell-cell n adhesion and cell compaction revealed by high resolution tracking of E-cadherin/G FP. J Ce//Bio/1998, 142:1105-1119. The first detailed analysis of E-cadherin dynamics during cell-cell adhesion in living M DCK cells.
Cytomechanics of cadherin-mediated cell-cell adhesion Cynthia L Adams* and W James Nelsont Cadherin-mediated adhesion regulates transitions from initial cell-cell recognition to loosely adherent cell clusters and ultimately, to strongly compacted groups of cells in colonies. Recent studies have described distinct roles for intermolecular clustering of cadherins as well as interactions of cadherin with the actin cytoskeleton in establishing cell-cell adhesion. Integrating cytomechanical roles of cadherin-mediated adhesion will lead to a greater understanding of how cadherins regulate tissue morphogenesis. Addresses *Cytokinetics, Inc., 280A East Grand Avenue, South San Francisco, CA 94040, USA tDepartment of Molecular and Cellular Physiology, Beckman Center, B121 Stanford University School of Medicine, Stanford, CA 9430554345, USA; e-mail: [email protected] Current Opinion in Cell Biology 1998, 10:572-577
http://biomednet.com/elecref/0955067401000572 © Current Biology Ltd ISSN 0955-0674 Abbreviations CD cytocholasin D MDCK Madin-Darbycanine kidney Introduction
Studies to understand the basis of cell-cell adhesion were underway at the turn of the century [1]. These early studies had a mechanical focus, and aimed to measure the movements and forces underlying the properties of cell-cell interactions. Measurements of forces between cells, chemical requirements for adhesion, and sorting out of heterogeneous populations of cells [2] lead to a greater understanding of some of the physical characteristics of adhesion. When molecules involved in cell-cell adhesion were discovered [3,4,5], the focus shifted to understanding the molecular and biochemical nature of protein-protein interactions and assembly of protein complexes [6]. Recently, however, attention has returned to the problem of how these protein complexes are involved in the physical aspects of cell-cell adhesion. Here we discuss some recent studies on mechanisms involved in cadherin-mediated cell-cell adhesion. Cadherins are a family of single-transmembrane, calciumdependent cell adhesion proteins [6]. Defining mechanisms involved in cadherin-mediated cell-cell adhesion is at the core of understanding a diverse range of cellular phenomena including contact inhibition of cell growth [7,8°,9], condensation and compaction of cells [1,10[, wound healing [11,12], and tumorigenesis [13"]. Cell-cell adhesion requires homotypic binding of the extracellular domain of cadherins on adjacent cells, and the attachment of the intracellular domain of cadherin to the actin cytoskeleton. Cadherins interact with actin through different complexes
composed of cytosolic proteins (see [6]), termed ot-catenin, 13-catenin, plakoglobin, and p120 CAs. Through these proteins, cadherin is attached to the actin cytoskeleton [14",15,16]. Recent characterization of the three-dimensional crystal structure of ]]-catenin [17 °°] has shed new light on how catenins bind to the cytoplasmic domain of cadherin. Much attention has focused on the assembly of cadherin-catenin-actin complexes and the role they play in cell-cell adhesion [6]. Understanding the dynamics of cadherin-catenin-actin complex assembly and the significance of these interactions in stabilizing cell-cell contacts are crucial for defining mechanisms by which tissue integrity is established and maintained. The role of cadherin adhesion
clustering
in c e l l - c e l l
Recently, lateral dimerization and clustering of cadherins have been shown to be involved in cell-cell adhesion in vitro [18,19"] and in vivo [20°1. Crystal structures of cadherins suggest that lateral dimers of cadherin on one cell could bind to lateral dinaers of cadherin on an opposing cell [21]. T h e affinity of homotypic interactions through cadherin extracellular domains is weak (< 1 ~tM, [221) clustering many low affinity binding sites may be required to increase the effective affinity of the cadherin-cadherin interaction. There may be several mechanisms involved in stabilizing a lateral cadherin dimer. A role for interactions between the extracellular domains of neighboring cadherins is supported by a study showing increased cell-cell adhesion in cells engineered to express large aggregrates of the extracellular domain of cadherin without the cytoplasmic domain [20°]. This study indicates that aggregation of the extracellular domain of cadherin is sufficient for cell-cell adhesion. In vivo, however, interactions between the cytosolic domain of cadherin with adjacent cadherin and cytosolic proteins may also be necessary for the formation of lateral cadherin dimers. In one study, cells expressing cadherin lacking the 13catenin binding domain were able to both cluster cadherin and mediate adhesion in suspension, while cells expressing cadherin lacking the juxtamembrane 94 amino acids, a putative p120 CAs binding site, were not [19°'1. This study suggested that p120 CAs binding to cadherin is sufficient for cell--cell adhesion; however, the ability of these cells to adhere in the absence of [3-catenin binding to cadherin is in contrast to many studies that have shown that disruption of E-cadherin binding to [3-catenin decreascs cell-cell adhesion. In particular, it has been shown that a 34 amino acid deletion in the juxtamembranc domain of cadherin had little effect on adhesion, while deletion of the [3-catenin binding site resulted in loss of adhesion [9]. While providing more details about the important molecular interactions, these studies begin to illuminate the difficulty of differentiating
Cytomechanics of cadherin-mediated cell-cell adhesion Adams and Nelson
Figure I Accumulation of E-cadherin into puncta during initial stages of MDCK epithelial cell-cell adhesion. (a-d) Differential interference contrast (DIC) images from a time-lapse video recording and (e) a schematic representation of a developing contact between two MDCK cells are shown. (a-d) The age of the contact prior to fixation is indicated in the upper left hand corner of each frame (in minutes). The vertical lines running down the figure indicate the boundaries of the developing contact at each time point (between red lines, >40 min; red-yellow, 2 5 - 4 0 rain; yellow-green, 10-25 min; and green-blue, 1-10 min). (e) The newly formed length of contact between frames is represented as the horizontal line between the arrows (black, >40 min; red, 2 5 - 4 0 min; blue, 10-25 min; green, 1-10 min). For example, the additional contact length inserted between 40 and 25 min is represented by the area between the vertical red and yellow lines throughout the image, and the red horizontal lines between the arrows in (a). Puncta which are in the region are considered to be 25-40 min old. (f) Retrospective E-cadherin staining of the contact, and (g) in pseudo-color formating. Figure reproduced with permission [18].
(a)
(b)
(c) between the importance of cadherin clustering and cytoskeletal linkage through catenins in regulating adhesion. Catenins probably exert their stabilizing influence on cell-cell contacts through their interactions with actin. 13catenin links cadherin to the actin cytoskeleton via c~-catenin, and this link is thought to be necessary for stabilizing cell contacts; disruption of 0t-catenin binding to the catenin complex inhibits adhesion (see [6]). Significantly, cadherin mediated cell-cell adhesion was inhibited in a dose dependent fashion by overexpressing EpCAM, perhaps by lowering ~-catenin expression [23]. Over-expression of N-cadherin in MDA-MB-468 cells, which lack 0t-catenin expression, however, increased the ability of these cells to adhere [24°]. Vinculin, which has homology to ot-catenin (see [25]), may bind to the cadherin/~-catenin complex thereby compensating for the lack of ~-catenin. Alternatively, an increase in surface expression of N-cadherin may increase adhesion. Changes in expression levels of cadherins and catenins have been shown to affect cell-cell adhesion [26°,27-29]. None of these studies, however, have demonstrated that perturbation of the cadherin clustering mechanism, perse, is responsible for each of these different adhesion phenotypes. T h e exact role of p120 CAs in cell-cell adhesion remains a matter of debate. Expression of the putative p 120 cAs binding site of cadherin, without the catenin binding site or extracellular domain, was first shown to induce an adhesion defect in Xenopus embryos [30]. One explanation of this effect is that pl2OCAS may stabilize a lateral cadherin heterodimer, comprising a mutant and wild-type cadherin, which is nonfunctional. A homodimer cadherin-pl20CAS complex may be necessary to increase the rates of initial homotypic recognition and binding between cadherin molecules on two adhering cells. Subsequently, c~-catenin, 13-catenin and the actin cytoskeleton may stabilize the cadherin-pl20 cAs cluster and, thereby, strengthen cell-cell adhesion. This interpretation would explain why cells over-expressing cadherin which lack the p120 CAs binding
(d)
(e)
(f)
(g)
573
574 Cell-to-cellcontactand extraeellularmatrix
region, but maintain the [~-catenin binding region, adhere very slowly [9]. These cells may ultimately form strong, and possibly stronger, contacts by recruiting the actin cytoskeleton to the cell-cell contact site through their catenin linkage to the actin cytoskeleton (see above). Cadherin-catenin complexes immobilized within a cell-cell contact by actin may act as a trap for cadherin freely diffusing within the plasma membrane, as originally envisioned in the diffusion-trap model [31,32]. Over time, freely diffusing E-cadherin may redistribute to the contact (trap) by associating with immobilized E-cadherin or other associated proteins (e.g. p120 Cns, catenins or actin). A consequence of this diffusion would be to increase the local concentration of cadherin at the contact thereby resulting in strengthening of cell-cell adhesion. Analysis of cells prior to cell--cell contact has revealed that E-cadherin has a homogeneous distribution over the plasma membrane [18,33]. After initiation of cell-cell adhesion, Triton X-100 insoluble E-cadherin accumulates along the length of the plasma membrane within the contact. When Triton X-100 soluble E-cadherin is extracted, the relative amount of Triton X-100 insoluble E-cadherin is much greater at cell-cell contacts than in other regions of the plasma membrane, and is associated with actin ([18,33]; see Figure 1). Thus, short-range diffusion trapping around immobilized E-cadherin may be important for clustering E-cadherin within the cell-cell contact. T h e combined effects of long-range diffusion and shortrange clustering of cadherin on cell-cell adhesion may explain discrepancies in the kinetics of adhesion between cells expressing different levels ofcadherin. If clustering of cadherin is important to stabilize the contact, and the level of cadherin protein at a single cluster is critical, then the time required for the appropriate number of cadherin proteins to diffuse into the trap will increase as the number of surface cadherin molecules decreases. Diffusion of cadherin within the plasma membrane has been examined by fluorescence-photobleaching-recovery and single-particletracking techniques [34,35°°]. These studies showed that a small population of E-cadherin is attached to the cytoskeleton in the absence of cell-cell contacts. This population of proteins may play a role in initiating the immobilization of E-cadherin at a developing contact site. A kinetic measurement of cadherin clustering has been performed using time-lapse differential interference contrast microscopy in combination with quantitative retrospective immunocytochemistry ([18]; see Figure 1). T h e distribution and assembly kinetics of Triton X-100 insoluble E-cadherin, c~-catenin, [3-catenin, plakoglobin and actin at newly formed cell-cell contact sites were measured in Madin-Darby canine kidney (MDCK) epithelial cells. A major observation was that during initial stages of cell-cell adhesion, E-cadherin, c~- catenin and I]-catenin exist as uniformly sized and spaced punctate clusters along the length of a cell-cell contact, and accumulate propor-
tionally within 20 minutes of cell-cell contact (see Figure 1). This study dispelled the notion of contact lengthening as a function of continuous E-cadherin zippering. Instead, discrete 'puncta' of E-cadherin, 13-catenin, and ct-catenin are assembled along the contact at a constant average density of ~ 1 puncture per 1.5 lain of contact length. Each of these puncta is associated with actin filaments that appear to split off the circumferential actin belt in the cortex of these previously migrating cells [18].
Clusters of cadherin c o o r d i n a t e a c t i n / m y o s i n t e n s i o n b e t w e e n c o n t a c t i n g cells Two studies shed light on a cytomechanical role for the cadherin-catenin complex in establishing cell-cell adhesion. First, tension transmitted through adherens junctions in Swiss 3T3 fibroblast cells was shown to induce a visceoelastic response in the cytoplasm [36]. Second, the maintenance of the strand-like appearance of the cadherin-catenin complex in fibroblasts was reversibly abolished in the presence of 2,3-butanedione 2-monoxime, a non-specific inhibitor of myosin [37"]. T h e s e data suggest that tension is not just generated through clusters of cadherin, but that tension is required to maintain them. Mechanisms involved in the formation of cytomechanical tension between cells have yet to be identified or measured directly, although much work has been done to understand tension generated by cell-substrate interactions [38"]. Myosin is a good candidate for a force-generating protein important for cell-cell adhesion and cell polarity. Nonmuscle myosin II is a mechanochemical motor protein, which slowly moves from the pointed to the barbed ends of actin filaments in an ATP dependent fashion. In noncontacting edges of cells (lamellae), actin polymerizes at the barbed end and depolymerizes at the pointed end of actin filaments (see reviews [39-41]). There is a continual rearward flow of actin filaments and associated proteins in lamellae, which is referred to as 'treadmilling' (see reviews [39-41]). The rearward flow of actin is due to myosin pulling actin filaments towards the center of the cell [42]. When the barbed ends of actin filaments are firmly attached to the membrane, actin treadmiiling ceases, and movement of myosin along Factin generates tension between the membrane and the cell body [42,43"]. T h e generation of tension allows the cell body to translocate towards the site of attachment using the molecular clutch mechanism proposed for cell-substrate interactions [43"]. It has also been shown that there is no rearward flow of actin in areas behind cell--cell contacts [8°]. T h e role of actin binding to the cadherin--catenin complex may be to strengthen cell-cell adhesion using this molecular clutch mechanism. This mechanism, however, requires that the cadherin--catenin complex binds to the barbed ends of actin filaments. T h e polarity of actin filaments with respect to the cadherin-catenin complex has yet to be determined. Alpha-catenin, the putative linker of cadherin
Cytomechanics of cadherin-mediated cell-cell adhesion Adams and Nelson
to actin, is an actin bundling protein in vitro [15]. Pretreatment of cells with cytochalasin D (CD), an actin capping drug, abolishes the ability of cells to initiate cell--cell adhesion [4,441. But CD does not cause the separation of established cell-cell contacts in polarized epithelia [45]. Together, these results suggest that the cadherin-catenin-actin complex may interact with the barbed ends of actin filaments with a stronger affinity than CD, or that the barbed ends of actin filaments are inaccessible to CD in old contacts. If the cadherin-catenin complex does interact with the barbed ends of actin filaments, it is reasonable to speculate that the myosin machinery, via actin, generates tension between cells via cadherin. While very little is known about the role of myosin during cell--cell adhesion, myosin has been implicated in wound healing which requires the re-establishment of cell-cell contacts. When a polarized monolayer of epithelial cells is wounded by removing a patch of ceils, the monolayer quickly fills in the gap with ceils that surround the wound [11]. One mechanism by which an epithelial monolayer repairs damage is described by the 'purse-string' theory [46]. This theory proposes that circumferential tension is generated by actin cables held together at the peripheral cell-cell contacts of the wound perimeter [47]. This tension causes the actin cables to cinch together, which results in shortening of the wound perimeter, in the absence of cell proliferation, until the wound is filled in by the surrounding cells. The interactions between cadherin, actin, and myosin have been implicated in generating the forces required for wound closure. Actin cables, which circumscribe the wound perimeter, appear continuous from cell to cell, are decorated with myosin II [11,48], and are linked by large clusters of E-cadherin at the cell-cell contact [49]. These E-cadherin clusters may play a role in linking the forces generated by actin cables in adjacent cells. T h e Rho family of small GTPases plays important roles in regulating actin cytoskeleton organization and cell adhesion [49,50°,51"]. Recent studies indicate that epithelial cell-cell adhesion is affected by mutant RhoA and Racl [52°'-54"]. Downstream effectors of RhoA (Rho-kinase [55,56] and its targets, myosin and myosin binding proteins [57-591) and Racl (PAK [60] and IQGAP [61"]) are excellent candidates for regulating assembly of the cadherin-catenin complex and the actin cytoskeleton contractility (myosin). Further insight into structural implications for cadherin binding to the actin cytoskeleton is suggested by studies showing that integrin adhesion proteins are mechanically coupled to the nucleus [62"]. T h e "tensegrity" model postulates that the actin cytoskeleton interconnects extracellular adhesions to the internal organelles [63]. Therefore, stress applied through adhesive complexes is transmitted throughout the cell ultimately coordinating cell shape, motility, and even growth and differentiation. While much work has been done to eh, cidate this conncc-
575
tivity in interactions between cells and the extracellular matrix [43"], little is known about similar roles for cell-cell interactions. Studies aimed at understanding the cytomechanical roles of cadherin in this process will be of great interest in the future.
Conclusion T h e structural link between E-cadherin clusters and the actin cytoskeleton is necessary for strengthening cell-cell adhesion. This link may use myosin to generate tension between two contacting cells. T h e tension is redistributed by reorganization of the cortical actin cytoskeleton to the edges of the cell-cell contact, which leads to the formation of strong cell-cell contacts and changes in cell organizations. This tension leads to dramatic changes in cell organization leading to development of cell polarity. Full elucidation of the molecular dynamics underlying the initiation and strengthening of cadherin mediated cell--cell adhesion and the long term reorganization of cells into colonies awaits further study (for further discussion see [64"'1).
Acknowledgements Thank you to Chris Hazuka for his helpful comments and suggestions on the manuscript. Work from the Nelson laboratory was supported by a grant from the National Institutes of Health.
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