Cadherin-based cell–cell contacts undergo distinct morphological transitions both in vivo and in vitro, even after cells have established contact with one another. The challenge is to define specific molecular mechanisms responsible for the morphogenesis of these cell–cell interactions. Our current work identifies myosin VI as an important factor that interacts with cadherin adhesion to control the process by which early discontinuous interactions become organized into cohesive, linear contacts as epithelial monolayers mature in culture. We found that myosin VI binds to E-cadherin and localizes at cell–cell contacts as they undergo the transition from discontinous to coherent and continuous. This appears to reflect a regulated recruitment process, as cellular levels of myosin VI do not change during this period. Importantly, the disruption of myosin VI function by RNAi or expression of a dominant-negative mutant perturbed the cohesive integrity of cell–cell contacts and reduced cadherin adhesion. Moreover, myosin VI depletion perturbed the integrity of tight junctions and desmosomes, which is consistent with the central role for E-cadherin function in junctional biogenesis (Gumbiner et al., 1988
). In contrast, myosin VI had no effect on integrin-based cell adhesion to fibronectin. Thus, in cultured mammalian cells, myosin VI acts at a relatively late stage in epithelial maturation to generate cohesive cell–cell interactions.
Our findings support and extend earlier evidence from Drosophila
that implicated myosin VI in cadherin-based cell–cell interactions. Myosin VI cooperates with DE-cadherin to support morphogenetic movement in the fly egg chamber (Geisbrecht and Montell, 2002
). Specifically, myosin VI is expressed and forms a complex with DE-cadherin and armadillo in migrating border cells. Importantly, myosin VI is necessary for this cadherin-dependent form of cell-on-cell migration, which is consistent with our finding that myosin VI supports cadherin adhesion and aspects of cellular morphogenesis in mammalian epithelia. Similarly, the disruption of myosin VI perturbed intercellular cohesion, DE-cadherin localization, and dorsal closure during early fly morphogenesis (Millo et al., 2004
). Collectively, our findings in mammalian cells, taken with these earlier precedents in invertebrates, suggest an important conserved contribution of myosin VI to cadherin function.
How might myosin VI regulate cadherin contacts and adhesion in mammalian epithelia? This unconventional motor has been implicated in two broad cellular processes: membrane transport and actin filament organization. Myosin VI is recruited to clathrin-coated pits and persists on the subsequent uncoated vesicles, likely through interaction with several adaptor proteins (Buss et al., 2001
; Aschenbrenner et al., 2003
). This, taken with its minus end–directed movement, has suggested a role in endocytosis (Buss et al., 2002
). Myosin VI also associates with the Golgi apparatus and can support exocytotic transport (Warner et al., 2003
). Indeed, in MDCK cells, myosin VI was necessary for the basolateral delivery of proteins that contain tyrosine-based motifs sorted by the AP-1B clathrin adaptor complex (Au et al., 2007
). However, myosin VI did not affect protein sorting that depended on dileucine motifs, including E-cadherin (Bryant and Stow, 2004
). Similarly, we found no substantial change in either the total or surface levels of E-cadherin in myosin VI KD cells, nor could we detect changes in the transport of E-cadherin to the cell surface (unpublished data). Thus, although myosin VI may play a more subtle role in cadherin trafficking, this pathway does not readily account for our results.
Instead, we favor the notion that myosin VI participates in cadherin–actin cooperation. We found that the perijunctional actin cytoskeleton was clearly disrupted by myosin VI depletion. As epithelial monolayers matured, reorganization of the perijunctional actin cytoskeleton accompanied the appearance of linear, cohesive cadherin contacts, an initially loose distribution of F-actin being replaced by dense staining concentrated in the immediate vicinity of cell–cell contacts. In myosin VI KD cells, the dense perijunctional packing of F-actin was replaced by a looser organization, without any concomitant change in total cellular F-actin levels. Similarly, earlier studies reported that myosin VI can stabilize actin filament networks (Noguchi et al., 2006
) and potentially filaments themselves (Naccache and Hasson, 2006
), characteristically promoting the dense packing and accumulation of filaments. For example, during spermatid individualization in Drosophila
, myosin VI is necessary to organize the actin cones that separate the syncytial spermatids. Notably, the density of filaments in the actin cones is substantially reduced in myosin VI mutant testes without apparent changes in filament turnover, suggesting that myosin VI participates in packing and organizing actin filament networks (Noguchi et al., 2006
). The impact of myosin VI on organization of the perijunctional actin cytoskeleton implies that a similar contribution may occur in epithelial cells.
Importantly, several lines of evidence identify the actin-binding protein vinculin as a downstream effector for myosin VI at cadherin adhesions. (1) Myosin VI was necessary for vinculin to stably associate with E-cadherin in mature epithelial monolayers, which was assessed by both coimmunoprecipitation and immunofluorescence analysis. Vinculin was also selectively lost from cell–cell contacts but not focal adhesions in myosin VI KD cells. However, the ability of myosin VI to interact with E-cadherin was not affected by vinculin KD, implying that vinculin recruitment to E-cadherin is downstream of myosin VI. (2) Vinculin KD disrupted the integrity of cohesive E-cadherin contacts in a manner similar to myosin VI KD. (3) Membrane-targeted vinculin fragments effectively restored the cohesive integrity of cadherin contacts in myosin VI KD cells. This implied that reconstitution of vinculin function at E-cadherin adhesions could compensate for the global loss of myosin VI. (4) Vinculin was necessary for myosin VI to regulate junctional integrity. Exogenous myosin VI could not rescue contact integrity in cells depleted of vinculin as well as endogenous myosin VI, indicating that vinculin was necessary for the reconstitution of myosin VI to be effective. (5) Myosin VI could not compensate for the loss of vinculin, as the overexpression of myosin VI did not rescue contact integrity in vinculin KD cells. This argues against the possibility that vinculin and myosin VI are in parallel pathways. Collectively, these findings indicate that vinculin is an important effector for myosin VI at cadherin contacts.
Vinculin has long been recognized to accumulate at cell–cell contacts, where it is thought to incorporate into adherens junctions (Geiger et al., 1980
). However, it is notable that previous studies implicated α-catenin in recruiting vinculin to cadherin adhesions (Watabe-Uchida et al., 1998
; Weiss et al., 1998
). We, too, found that vinculin stains in early cell–cell contacts (unpublished data) before myosin VI is readily detected, a process that therefore may entail α-catenin. Yet, in our experience, myosin VI was necessary for vinculin to stably incorporate into mature cadherin adhesions. This suggests that two mechanisms participate in localizing vinculin to cadherin adhesions, with myosin VI being dominant at a later stage than α-catenin.
Thus, our findings identify myosin VI and vinculin as part of a molecular apparatus responsible for generating the cohesive cell–cell contacts that distinguish epithelial biogenesis in vitro. We postulate that myosin VI and vinculin cooperate to reorganize the perijunctional actin cytoskeleton, leading to the generation of cohesive, linear cadherin contacts. Precisely how vinculin participates in this process has yet to be defined at a molecular level. Vinculin can both bind and bundle actin filaments (Jockusch and Isenberg, 1981
; Menkel et al., 1994
; Huttelmaier et al., 1997
; Janssen et al., 2006
), so may provide a mechanism to organize and compact actin filament meshworks at cadherin adhesions. The actin-binding site has been mapped to the tail region of the vinculin molecule (Johnson and Craig, 2000
). Therefore, it was interesting to note that the head fragment could also rescue contact integrity. The mechanism for this effect remains to be determined.
However, our data further indicate that myosin VI does not act only by recruiting vinculin into a cadherin-based complex. Although the myosin VI tail alone can coimmunoprecipitate both E-cadherin and vinculin and localize to cell–cell contacts (unpublished data), this fragment did not rescue the cohesive integrity of those contacts in myosin VI KD cells. Therefore, the actin-binding activity of the myosin VI head domain must functionally cooperate with vinculin for myosin VI to support cohesive cell–cell contacts. The nature of this cooperation remains to be determined. Our data do not exclude the possibility that other proteins participate in this myosin VI–based effector pathway. One speculative possibility is that myosin VI may serve as an actin-based anchor (Altman et al., 2004
), cooperating with vinculin's ability to organize actin in order to link cadherin complexes onto perijunctional actin filaments. Perhaps this cooperative interaction with actin also serves to stabilize vinculin at contacts, accounting for the role of myosin VI to stably localize vinculin in mature contacts.
Irrespective of the deep molecular mechanism, our findings highlight the concept that multiple actin regulators operate at cadherin adhesive contacts, and they reinforce the notion that the functional expression of individual mechanisms is likely to be tightly regulated by cellular context. Understanding the regulated recruitment of myosin VI will be important if we are to elucidate the varied molecular mechanisms that cadherin adhesion uses to regulate the actin cytoskeleton.