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Pli Selon Pli
CHAPTER THIRTY-SEVEN
Pli Selon Pli: Mechanochemical Feedback and the Morphogenetic Role of Contractility at Cadherin Cell–Cell Junctions Bipul R. Acharya, Alpha S. Yap1 Division of Cell Biology and Molecular Medicine, Program in Membrane Interface Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Cadherins and the Contractile Cytoskeleton 3. The Elements of the System 3.1 Molecular Basis of Cadherin–F-Actin Binding 3.2 Incorporating Myosin II 3.3 Upstream Regulation by Cell Signaling 4. Mechanochemical Feedback Among the Elements of the Cadherin–Actomyosin System 5. Emergent Properties of Mechanochemical Networks 5.1 Generating Contractile Pulsatility 5.2 Generating Sustained Tension 5.3 Morphogenetic Implications 6. Concluding Comments Acknowledgments References
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Abstract Cellular contractility, driven by actomyosin networks coupled to cadherin cell–cell adhesion junctions, is a major determinant of cellular rearrangement during morphogenesis. It now emerges that contractility arises as the emergent property of a mechanochemical feedback system that encompasses the signals that regulate contractility and the elements of the actomyosin network itself.
Current Topics in Developmental Biology, Volume 117 ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2015.10.021
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2016 Elsevier Inc. All rights reserved.
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1. INTRODUCTION The development of multicellular organisms and tissues operates at many levels. Their constituent cells integrate many molecular mechanisms that allow them to serve as the building blocks of tissues. Yet interactions between cells also generate complex, emergent properties at the tissue level. Thus, a comprehensive understanding of development presents the great challenge of understanding how these different (molecular, cellular, tissue) levels are integrated, despite the substantial differences in length and timescales at which each level operates. The challenges, and our progress in responding to them, are well illustrated by the classical cadherins, whose founding member, E-cadherin, was first discovered by Vestweber and Kemler as a protein necessary for compaction of the early mouse embryo (Vestweber & Kemler, 1984, 1985) and by Takeichi as a mediator of adhesion between cultured cells (Takeichi, 1977). Since then, the tissue-specific expression of different classical cadherins has been comprehensively characterized, and their contributions to development, morphogenesis, and organogenesis have been established in a range of invertebrate and vertebrate model systems (Tepass, Truong, Godt, Ikura, & Peifer, 2000). In parallel, we have learnt much about how cadherins operate and are regulated at the molecular and cellular levels (Niessen, Leckband, & Yap, 2011; Takeichi, 2014). In this essay, we will discuss how some recent insights into the cellular mechanisms of cadherin biology can influence the way we think about the processes that drive epithelial morphogenesis. We focus on how adhesion cooperates with the actin cytoskeleton.
2. CADHERINS AND THE CONTRACTILE CYTOSKELETON We have long believed that cadherins cooperate, functionally and physically, with the actin cytoskeleton. Much of the early evidence for this derived from cell culture, such as the observation that inhibitors of F-actin integrity disrupt cadherin adhesion ( Jaffe et al., 1990). Cellular and biochemical studies further suggested that cadherins could interact physically with actin filaments (Nagafuchi & Takeichi, 1988; Ozawa, Ringwald, & Kemler, 1990). Indeed, many actin-binding proteins (ABPs) have now been identified as capable of interacting with classical cadherins (Ratheesh & Yap, 2012), and recent screens using proximity-biotinylation techniques have
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identified a host of other potential cadherin-associated ABPs (Guo et al., 2014; Van Itallie et al., 2014). An important advance in understanding the functional impact of cadherin–actin cooperation has come from the in vivo demonstration that mechanical forces exerted by the actin cytoskeleton are transmitted to cadherin junctions. In epithelia, this can take the form of pulsatile movements of junctions that are driven by pulsatile contractions of the cytoskeleton (Martin, Kaschube, & Wieschaus, 2009; Munjal, Philippe, Munro, & Lecuit, 2015; Rauzi, Lenne, & Lecuit, 2010; Roh-Johnson et al., 2012; Wu et al., 2014) or sustained contractile tension that is found in the junctions (Ratheesh et al., 2012; Wu et al., 2014). These different patterns of contractility often distinguish different morphogenetic processes and are associated with different actomyosin networks that associate with junctions. Pulsatile contraction is commonly seen with apical constriction and invagination (Martin et al., 2009; Roh-Johnson et al., 2012) and is driven by two-dimensional (2D) actomyosin networks found at the apical poles of cells (often called medial–apical networks) that undergo periodic condensation (Martin et al., 2009; Munjal et al., 2015). In contrast, sustained junctional tension is associated with processes such as neighbor exchange, boundary formation, and tissue homeostasis (Fernandez-Gonzalez, Simoes Sde, Roper, Eaton, & Zallen, 2009; Monier, Pelissier-Monier, Brand, & Sanson, 2009; Wu et al., 2014); it is attributed to circumferential actomyosin bundles that run parallel to, and associate with, cadherin junctions, especially the apically positioned zonulae adherente (ZA). However, these two modes of contractile organization can coexist within the same cell (Rauzi et al., 2010; Wu et al., 2014; Fig. 1). This fact—that cadherin junctions are sites where the cytoskeleton generates force—carries several important implications. First, this mechanical connection implies that there must be physical connections between the cadherins and the actin cytoskeleton. Second, since contractile forces are generated by nonmuscle Myosin II (NMII) acting on actin filaments, cadherins interact with an actomyosin contractile apparatus. This is further supported by the observation that mechanical stress exerted on cadherins can cause the cytoskeleton to stiffen (Bazellieres et al., 2015; le Duc et al., 2010), a process that requires both F-actin and myosin. Third, it alters the way we approach the biology of cell–cell adhesion: rather than principally being a mechanism that passively resists detachment forces to preserve tissue integrity, cadherins serve to generate tensile forces by coupling contractile cortices of neighboring cells together (Maitre et al., 2012). Furthermore,
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A
Junctional Medial–apical
F-actin NMIIA B
Zonula adherens
Lateral junction
Figure 1 Patterns of actomyosin contractility at epithelial cell–cell junctions. (A) Apical actomyosin networks include two-dimensional medial–apical networks and junctional actomyosin bundles, as found in epithelial cells undergoing apical constriction. (B) In polarized epithelia, apical bundles at the zonula adherens (ZA) can coexist with twodimensional networks found at the lateral junctions, below the ZA.
cadherins can promote assembly and homeostasis of the contractile apparatus itself, while actomyosin feeds back to promote adhesion (Shewan et al., 2005; Smutny et al., 2010). Taken together, these observations suggest that at the cellular level the functional interrelationship between cadherin and actomyosin represents an integrated cellular system. As we shall now describe, considerable recent progress has been made in characterizing the elements that contribute to cadherin–actin cooperation. In the process, we have begun to discover feedback interactions between these elements that integrate them into a complex network whose emergent properties have implications for understanding morphogenesis.
3. THE ELEMENTS OF THE SYSTEM Integrating cadherin adhesion with contractility involves several components. These include the physical association of adhesion with
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actomyosin, the building of a contractile actomyosin apparatus that is linked to cadherin, and signaling pathways which coordinate these individual elements.
3.1 Molecular Basis of Cadherin–F-Actin Binding At a basic level, junctional contractility arises from a physical association of cadherin adhesion complexes with actomyosin. To date, the mechanical coupling of contractility to cadherin adhesion appears to be mediated by the F-actin elements of the actomyosin apparatus; there is no evidence that NMII interacts directly with the cadherin complex. Three broad pathways are available to couple cadherins to F-actin. First, cadherins may associate with actin filaments through α-catenin, a ubiquitous component of the core cadherin–catenin complex. Although free α-catenin can directly bind F-actin (Pokutta, Drees, Takai, Nelson, & Weis, 2002; Rimm, Koslov, Kebriaei, Cianci, & Morrow, 1995), its role as a linker between cadherins and F-actin became controversial when it proved difficult to reconstitute F-actin binding when α-catenin was incorporated into a minimal cadherin/ catenin complex in vitro (Drees, Pokutta, Yamada, Nelson, & Weis, 2005; Yamada, Pokutta, Drees, Weis, & Nelson, 2005). This conundrum has been recently resolved by the demonstration that minimal cadherin–catenin complexes will bind F-actin when under force (Buckley et al., 2014), whereas the earlier reconstitution studies were performed in solution. Second, α-catenin can recruit other ABPs to the cadherin complex (Abe & Takeichi, 2008). This is exemplified by vinculin, which bears multiple F-actin-binding sites (Ziegler, Liddington, & Critchley, 2006) and can bind directly to α-catenin itself (Choi et al., 2012). Junctional vinculin is increased when force is stimulated and decreased when forces are relaxed (Leerberg et al., 2014; Peng, Cuff, Lawton, & DeMali, 2010; Yonemura, Wada, Watanabe, Nagafuchi, & Shibata, 2010). This appears to reflect an ability of force to reveal cryptic vinculin-binding sites that are present in α-catenin (Yao et al., 2014; Yonemura et al., 2010). Thus, force appears to play an integral role in controlling how α-catenin may mediate interactions between cadherins and actin filaments. Finally, other proteins can mediate physical association between cadherins and actin filaments. For example, Myosin VI is an actin-binding motor that can directly interact with the E-cadherin cytoplasmic tail and is found at mature cell–cell junctions (Maddugoda, Crampton, Shewan, & Yap, 2007). However, Myosin VI is recruited to E-cadherin relatively late in the process of junctional biogenesis, where it is necessary for assembly of
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the ZA and organization of its associated actin cytoskeleton through vinculin. Together, these observations reinforce the notion that cadherins associate with F-actin through multiple pathways. The ability of α-catenin to directly bind F-actin is likely to constitute a ubiquitous mechanism that operates when cadherins come under force to link them with cortical actin (Chen et al., 2015). This may represent an elemental mechanism that can then be built upon by the force-dependent association of proteins like vinculin to α-catenin and the context-dependent recruitment of additional ABPs, such as Myosin VI.
3.2 Incorporating Myosin II Cellular contractility in nonmuscle cells is generated by the motor action of NMII exerted upon F-actin networks (Murrell, Oakes, Lenz, & Gardel, 2015). Consistent with this central cell-biological role, NMII is found decorating the medial–apical and junctional F-actin networks that are thought to exert contractile forces on cell–cell junctions (Mason, Tworoger, & Martin, 2013; Smutny et al., 2010). Considering that cadherin-bound actin filaments are the scaffolds upon which actomyosin is assembled, the incorporation of NMII at junctions involves two parallel processes. First, NMII must be activated. A dominant pathway involves the phosphorylation of the regulatory light chain of myosin through pathways mediated by kinases such as myosin light chain kinase (MLCK) and Rhodependent kinase (ROCK) (Heissler & Manstein, 2013). This is thought to promote F-actin binding, mini-filament assembly, and hence formation of an effective contractile unit. Consistent with this, both MLCK and ROCK support junctional contractility and the recruitment of NMII to the junctional cytoskeleton (Smutny et al., 2010) and to medial–apical networks (Mason et al., 2013; Munjal et al., 2015). Second, activated NMII must interact with F-actin networks. Of note, cadherin junctions are sites of dynamic actin assembly, initiated by actin nucleators such as Arp2/3 and formins, and modulated by postnucleation regulators such as Ena/VASP proteins and α-actinin-4 (Ratheesh & Yap, 2012). Their contribution to recruitment of NMII is illustrated by evidence that inhibiting Arp2/3 or its upstream activator, WAVE2, impaired the junctional recruitment of NMII and reduced junctional tension (Verma et al., 2012). This implies that the assembly of actin filaments at the junctional cortex serves to recruit activated NMII to the junctions, thereby allowing the contractile apparatus to assemble. However, the role of nucleators
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such as Arp2/3 carries a potential paradox. Arp2/3 generates branched actin networks that are thought to be poorly fitted for either stable NMII binding or contractile force generation. In part, this is because NMII tends to cause severing of branched networks, thereby turning over the actin scaffolds necessary for stable NMII localization (Reymann et al., 2012). Despite this, the cell clearly has developed strategies that compensate for this potential paradox, which may include stabilization of nascent filaments (Kovacs et al., 2011; Wu et al., 2014) and reorganization of actin networks to allow them to better sustain contractile stress (Murrell et al., 2015). While many mechanistic details have yet to be resolved, these observations carry the important implication that regulation of junctional F-actin (kinetics, organization) may constitute a pathway to influence contractility that operates orthogonal to that of the classical mode of NMII activation.
3.3 Upstream Regulation by Cell Signaling It will be apparent from the discussion above that cell signaling plays a central role in coordinating the many elements of cadherin–actin cooperation. Key signals include canonical regulators of the actin cytoskeleton, notably the GTPases Rac and Rho, as well as many protein and lipid kinases which are found at cell–cell junctions (Niessen et al., 2011). Rather than focusing on the many details of the mechanisms, we would highlight the following concepts. First, cadherin adhesion often plays an important role in establishing the junctional signaling pathways that control actomyosin. For example, depleting cadherin impairs the ability of cells to sustain both Rac and Rho signaling (Kovacs, Ali, McCormack, & Yap, 2002; Lampugnani et al., 2002; Priya, Yap, & Gomez, 2013). This dependence can reflect different paradigms for signal activation. In some instances, cadherins appear to behave like adhesion-activated signaling receptors. This is exemplified by Rac, whose activity is acutely stimulated by cadherin adhesion (Kovacs et al., 2002). In other cases, cadherins act more indirectly, as is seen with Rho. E-cadherin is necessary for Rho signaling to be established at cell–cell junctions (Priya et al., 2013), but acute ligation of cadherins can actually downregulate Rho, through feedback from Rac (Noren, Arthur, & Burridge, 2003; Noren, Niessen, Gumbiner, & Burridge, 2001). Here, cadherin adhesion appears to serve less as a ligand-activated receptor than as a membrane platform that is necessary for signaling zones to be established and maintained.
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Second, key signals, such as Rac and Rho, reflect networks of upstream molecular activators and inhibitors, whose balanced action determines the expression of signaling. For example, Rho signaling at cadherin junctions is activated by exchange factors (GEFs), such as Ect2 (Ratheesh et al., 2012), and also potentially inhibited by GTPase-activating proteins (GAPs) such as Myosin XIA (Omelchenko & Hall, 2012) and p190 RhoGAP (Priya et al., 2015). The spatial control of GEFs is important to initiate Rho signaling (Ratheesh et al., 2012), and the complementary inhibition of GAPs can play an important role in sustaining signaling (Priya et al., 2015).
4. MECHANOCHEMICAL FEEDBACK AMONG THE ELEMENTS OF THE CADHERIN–ACTOMYOSIN SYSTEM We conventionally think of the above elements of cadherin– actomyosin cooperativity as reflecting linear pathways that potentially work in parallel to one another. However, it is increasingly evident that these pathways are linked to ultimately form a network of chemical signals and mechanical effectors. One of the most recent examples involves Rho and NMII. As noted above, Rho (Rho A in vertebrates and Rho1 in Drosophila) is a canonical activator of NMII that is necessary for the various forms of contractility that impinge on cell–cell junctions. Thus, inhibiting Rho blocked the pulsatile contractility that is seen in the medial–apical actomyosin networks during Drosophila germband extension (Munjal et al., 2015) and that is found at the lateral junctions between mammalian epithelial cells (Wu et al., 2014). Inhibiting Rho, or its upstream activators, also degrades the sustained junctional tension of the ZA, which is associated with perijunctional actomyosin bundles (Fernandez-Gonzalez et al., 2009; Ratheesh et al., 2012). In all these instances, the impact of Rho is mediated by ROCK. Indeed, ligation of E-cadherin alone is sufficient to activate NMII in a ROCK-dependent fashion (Shewan et al., 2005). Together, these observations are consistent with a canonical linear pathway that leads from Rho to NMII via ROCK. The close functional link between the elements of the Rho–ROCK– NMII pathway is emphasized by the coaccumulation of these components at sites of contractility. This is especially striking in pulsatile medial–lateral networks in Drosophila. Here, not only do GTP-Rho and ROCK
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coaccumulate with actomyosin, but they also condense with the contracting medial–apical networks (Munjal et al., 2015; Vasquez, Tworoger, & Martin, 2014), suggesting that the dynamic spatial distribution of these signals is closely linked with that of NMII. Similarly, if less dramatically, these signals concentrate at the contractile ZA of polarized epithelial cells along with actomyosin (Priya et al., 2015, 2013; Ratheesh et al., 2012; Simoes Sde, Mainieri, & Zallen, 2014). At first, these patterns of coaccumulation fit well with a conventional linear signaling pathway. Surprisingly, however, not only does inhibiting Rho–ROCK degrade contractility, but directly inhibiting NMII itself also perturbs upstream Rho–ROCK signaling. Blocking contractility in gastrulating Drosophila embryonic cells, both prevented the pulsatile condensation of actomyosin seen in the medial–apical networks of these cells and also displaced GTP-Rho and ROCK from the apical cortex (Munjal et al., 2015). Similarly, inhibiting NMII directly in cultured mammalian epithelial cells reduced GTP-Rho and ROCK at the apical ZA of these cells (Priya et al., 2015). Together, these findings indicate that NMII can feedback to support Rho–ROCK signaling at cellular cortices. Importantly, both these instances reflect modes of mechanochemical feedback. In the pulsatile medial–apical networks of Drosophila germband cells, Rho and ROCK appear to undergo advection, being concentrated by a process of active bulk flow in the cellular cortex when the medial– apical actomyosin networks contracted and condensed. Such concentration may promote the interaction of Rho activators, thereby supporting positive feedback (Howard, Grill, & Bois, 2011). Consistent with this, inhibition of pulsatile contractility, which blocks advection, also decreased the amplitude of Rho and ROCK pulses (Munjal et al., 2015). In the case of the mammalian ZA, the mechanism involved anchorage of ROCK by physical association with NMII. When anchored at the ZA, ROCK then antagonized the cortical localization of a Rho inhibitor, p190B RhoGAP, to thereby support Rho signaling (Priya et al., 2015). Computational modeling predicted that in this network the local concentration of NMII would stabilize zones of activated Rho by preventing its premature inactivation. These observations add to the increasing realization that mechanochemical factors influence contractility at junctions. Indeed, tension can stabilize junctional NMII itself (Fernandez-Gonzalez et al., 2009), which might now be predicted to feedback upstream to affect Rho signaling.
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5. EMERGENT PROPERTIES OF MECHANOCHEMICAL NETWORKS A common consequence of networked control systems is the generation of emergent properties, features that are not predictable from analysis of the individual components but arise from their interactions at the higherorder network level. Could feedback from NMII to Rho yield emergent properties that are morphogenetically relevant? Here, an intriguing question is how different patterns of contractility—pulsed contractions associated with 2D networks versus the more sustained contractility of junctional bundles—can be generated from the same principal components (Rho, ROCK, NMII, and F-actin). Some potential answers to these questions lie in considering how mechanochemical feedback acts in these different contexts.
5.1 Generating Contractile Pulsatility By its very nature, pulsatility in actomyosin networks involves cycles of contraction/condensation and relaxation. While these coincide with pulsed fluctuations in Rho signaling found at the same sites as these actomyosin networks, contractile pulsatility is not simply an entrained response to those changes in Rho, as inhibiting contractility downregulates Rho signaling itself (Munjal et al., 2015). Instead, feedback within the Rho–NMII network contributes to generating pulses. One form of feedback occurs during the contraction phase when mechanochemical feedback by advection can promote local Rho signaling, thereby potentiating contractility (Munjal et al., 2015). Such mechanochemical feedback also affects the relaxation phase. One mechanism to induce contractile relaxation is inactivation of the NMII motor itself. This is mediated by myosin phosphatase, which is necessary for pulsatility to occur (Mason et al., 2013; Munjal et al., 2015). Interestingly, NMII was necessary for the cortical localization of myosin phosphatase, which also coaccumulated with condensed actomyosin networks when they contracted (Mason et al., 2013; Munjal et al., 2015). This suggests that actomyosin contractility promotes the local recruitment of a mechanism (myosin phosphatase) that leads to its inactivation, a sequence which would be predicted to promote pulsatile behavior. This mechanism for myosin inactivation is complemented by other changes in the contractile apparatus. Thus, advection declined during the
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transition from contraction to relaxation (Munjal et al., 2015), which constitutes a mechanism for negative feedback that is predicted to be necessary for disassembly of pulsed contractions. This was attributable to the increased density of actomyosin networks that developed during condensation, which can increase friction to oppose advection. In addition, NMII turnover increased (Munjal et al., 2015), suggesting that its association with actin networks became less stable. This can reflect inactivation of NMII as well the potential for stress to sever actin filaments and promote their turnover (Wu et al., 2014). Indeed, blocking contractility stabilized F-actin at lateral epithelial cell–cell junctions, regions of adhesion where pulsatile contraction also occurs (Wu et al., 2014). Therefore, rather than simply reflecting entrained responses to upstream signals, many factors, encompassing regulatory signals and the structural (F-actin) elements of the contractile apparatus, collaborate to drive the assembly and disassembly phases of contractile pulsatility.
5.2 Generating Sustained Tension How, then, can a system capable of generating pulsed contractions be converted into one that generates sustained tension? One possibility is that signaling by upstream activators of NMII behaves in a sustained, rather than a pulsatile, fashion (Munjal et al., 2015). Indeed, this is found at the apical ZA of mammalian epithelial cells, where GTP-Rho forms a defined micronsized signaling zone whose intensity and size is relatively stable over timescales (10 s of minutes) that are much longer than the dynamics of its constituent molecules (Priya et al., 2015) or the cycles associated with pulsatile actomyosin networks (100 s) (Rauzi et al., 2010; Wu et al., 2014). This zone depends on NMII and reflects the myosin-based feedback network described above. Of note, computational analysis of the feedback network predicted that the system was capable of generating bistable outcomes (Priya et al., 2015), meaning that once NMII was activated and localized to the junctional cortex, the feedback network that it set up could stably sustain signaling within the population of molecules found at the ZA zone. Sustained Rho signaling can then reflect the impact of the mechanochemical feedback network implemented by NMII. Computational analysis further predicted that surface stabilization of NMII plays an important role in establishing a stable Rho zone (Priya et al., 2015). Consistent with this, NMII was more stable at the ZA, where the stable Rho zone forms, than elsewhere at cell–cell junctions
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(Priya et al., 2015). This differential cortical stability of NMII may be a response to Rho signaling (Priya et al., 2015), but it can also be influenced by the actin networks with which it interacts (Wu et al., 2014). For example, F-actin is more stable at the ZA than elsewhere at cell–cell junctions, an effect of the actin regulator, N-WASP (Kovacs et al., 2011; Wu et al., 2014), and this is necessary for strong junctional tension to be generated at the ZA (Wu et al., 2014). Potentially, regulators of F-actin that promote association of NMII may then influence NMII–Rho feedback at junctions.
5.3 Morphogenetic Implications Ultimately, an important question is how these cellular properties of the cadherin–actomyosin system help us understand their impact on morphogenesis. Potential insight comes from considering the mechanical impact of pulsatile versus sustained contractile systems. Pulsatile contractile systems essentially contain the capacity to dissipate the stresses that they generate during their contractile phases. Their ability to support apical constriction is thought to arise from a rachet-like mechanism which allows the constricted adhesive junctions to be stabilized, i.e., to fail to relax when the medial–apical networks relax (Martin & Goldstein, 2014). Such relaxation may be important to coordinate apical constriction across populations of cells that undergo tissue invaginations (Vasquez et al., 2014). The mechanism responsible for rachet-like stabilization is not yet clear but coincides with the accumulation of actomyosin at junctions (Rauzi et al., 2010). As these are sites of sustained junctional tension, it is tempting to speculate that this contributes to generating the rachet. In contrast, the pulsatile networks found at the lateral junctions of polarized epithelial cells (i.e., below the apical ZA; Fig. 1) do not appear to be coupled to a rachet-like mechanism: the cadherin clusters with which they are associated also relax when the condensed actomyosin networks dissipate and relax. This limits the contractile tension that can be generated at these regions of junctions (Wu et al., 2014). One consequence of reducing junctional tension in these lateral regions may be to minimize mechanical barriers to hydrodynamic flow between the cytoplasmic compartments of cells, something that is also important for apical constriction to occur during development (He, Doubrovinski, Polyakov, & Wieschaus, 2014).
6. CONCLUDING COMMENTS Overall, these recent findings indicate that distinct contractile outputs can be generated by the core actomyosin apparatus, depending on different
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settings within the mechanochemical feedback network that encompasses Rho, NMII, and actin at cell–cell junctions. It seems likely that we have only begun to appreciate the versatility that can be generated by this Rhoactomyosin network. However, a key outstanding question is the role that the cadherin system may play in this network. To date, adhesion has principally been treated as the mechanism that mechanically integrates the contractile cortices of cells together (Maitre et al., 2012). This is also consistent with current focus on understanding the molecular mechanisms that physically couple cadherin adhesion complexes to F-actin. However, as we have discussed, cadherin adhesion also supports the biogenesis of junctional actomyosin, through processes such as actin assembly (Verma et al., 2012) and Rho signaling (Ratheesh et al., 2012). This active feedback from adhesion to contractility might be predicted to influence morphogenetic outcomes by tuning contractile outcomes. Further, junctional actomyosin promotes the accumulation and stability of cadherins at junctions and can influence cadherin clustering to promote adhesion (Priya et al., 2013; Smutny et al., 2010; Wu, Kanchanawong, & Zaidel-Bar, 2015). It is tempting to speculate that this might affect mechanical coupling between cells and even the degree of friction that adhesion presents as cells slide across each other during morphogenetic events such as neighbor exchange. If so, then, the physically coupled cadherin system would constitute another active feedback element in the mechanochemical network that allows cellular contractility to drive morphogenesis.
ACKNOWLEDGMENTS We thank all our lab colleagues for feedback and endlessly stimulating conversations. Our work is funded by the Human Frontiers Science Program, The Australian Research Council, and the National Health and Medical Research Council of Australia (1044041, 1037320, and 1067405).
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Roh-Johnson, M., Shemer, G., Higgins, C. D., McClellan, J. H., Werts, A. D., Tulu, U. S., et al. (2012). Triggering a cell shape change by exploiting preexisting actomyosin contractions. Science, 335, 1232–1235. Shewan, A. M., Maddugoda, M., Kraemer, A., Stehbens, S. J., Verma, S., Kovacs, E. M., et al. (2005). Myosin 2 is a key Rho kinase target necessary for the local concentration of E-cadherin at cell-cell contacts. Molecular Biology of the Cell, 16, 4531–4532. Simoes Sde, M., Mainieri, A., & Zallen, J. A. (2014). Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension. The Journal of Cell Biology, 204, 575–589. Smutny, M., Cox, H. L., Leerberg, J. M., Kovacs, E. M., Conti, M. A., Ferguson, C., et al. (2010). Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nature Cell Biology, 12, 696–702. Takeichi, M. (1977). Functional correlation between cell adhesive properties and some cell surface proteins. The Journal of Cell Biology, 75, 464–474. Takeichi, M. (2014). Dynamic contacts: Rearranging adherens junctions to drive epithelial remodelling. Nature Reviews. Molecular Cell Biology, 15, 397–410. Tepass, U., Truong, K., Godt, D., Ikura, M., & Peifer, M. (2000). Cadherins in embryonic and neural morphogenesis. Nature Reviews. Molecular Cell Biology, 1, 91–100. Van Itallie, C. M., Tietgens, A. J., Aponte, A., Fredriksson, K., Fanning, A. S., Gucek, M., et al. (2014). Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion. Journal of Cell Science, 127, 885–895. Vasquez, C. G., Tworoger, M., & Martin, A. C. (2014). Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis. The Journal of Cell Biology, 206, 435–450. Verma, S., Han, S. P., Michael, M., Gomez, G. A., Yang, Z., Teasdale, R. D., et al. (2012). A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens. Molecular Biology of the Cell, 23, 4601–4610. Vestweber, D., & Kemler, R. (1984). Rabbit antiserum against a purified surface glycoprotein decompacts mouse preimplantation embryos and reacts with specific adult tissues. Experimental Cell Research, 152, 169–178. Vestweber, D., & Kemler, R. (1985). Identification of a putative cell adhesion domain of uvomorulin. The EMBO Journal, 4, 3393–3398. Wu, S. K., Gomez, G. A., Michael, M., Verma, S., Cox, H. L., Lefevre, J. G., et al. (2014). Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia. Nature Cell Biology, 16, 167–178. Wu, Y., Kanchanawong, P., & Zaidel-Bar, R. (2015). Actin-delimited adhesionindependent clustering of E-cadherin forms the nanoscale building blocks of adherens junctions. Developmental Cell, 32, 139–154. Yamada, S., Pokutta, S., Drees, F., Weis, W. I., & Nelson, W. J. (2005). Deconstructing the cadherin-catenin-actin complex. Cell, 123, 889–901. Yao, M., Qiu, W., Liu, R., Efremov, A. K., Cong, P., Seddiki, R., et al. (2014). Forcedependent conformational switch of alpha-catenin controls vinculin binding. Nature Communications, 5, 4525. Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A., & Shibata, M. (2010). alpha-Catenin as a tension transducer that induces adherens junction development. Nature Cell Biology, 12, 533–542. Ziegler, W. H., Liddington, R. C., & Critchley, D. R. (2006). The structure and regulation of vinculin. Trends in Cell Biology, 16, 453–460.
Pli Selon Pli: Mechanochemical Feedback and the Morphogenetic Role of Contractility at Cadherin Cell–Cell Junctions Bipul R. Acharya, Alpha S. Yap1 Division of Cell Biology and Molecular Medicine, Program in Membrane Interface Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Cadherins and the Contractile Cytoskeleton 3. The Elements of the System 3.1 Molecular Basis of Cadherin–F-Actin Binding 3.2 Incorporating Myosin II 3.3 Upstream Regulation by Cell Signaling 4. Mechanochemical Feedback Among the Elements of the Cadherin–Actomyosin System 5. Emergent Properties of Mechanochemical Networks 5.1 Generating Contractile Pulsatility 5.2 Generating Sustained Tension 5.3 Morphogenetic Implications 6. Concluding Comments Acknowledgments References
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Abstract Cellular contractility, driven by actomyosin networks coupled to cadherin cell–cell adhesion junctions, is a major determinant of cellular rearrangement during morphogenesis. It now emerges that contractility arises as the emergent property of a mechanochemical feedback system that encompasses the signals that regulate contractility and the elements of the actomyosin network itself.
Current Topics in Developmental Biology, Volume 117 ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2015.10.021
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1. INTRODUCTION The development of multicellular organisms and tissues operates at many levels. Their constituent cells integrate many molecular mechanisms that allow them to serve as the building blocks of tissues. Yet interactions between cells also generate complex, emergent properties at the tissue level. Thus, a comprehensive understanding of development presents the great challenge of understanding how these different (molecular, cellular, tissue) levels are integrated, despite the substantial differences in length and timescales at which each level operates. The challenges, and our progress in responding to them, are well illustrated by the classical cadherins, whose founding member, E-cadherin, was first discovered by Vestweber and Kemler as a protein necessary for compaction of the early mouse embryo (Vestweber & Kemler, 1984, 1985) and by Takeichi as a mediator of adhesion between cultured cells (Takeichi, 1977). Since then, the tissue-specific expression of different classical cadherins has been comprehensively characterized, and their contributions to development, morphogenesis, and organogenesis have been established in a range of invertebrate and vertebrate model systems (Tepass, Truong, Godt, Ikura, & Peifer, 2000). In parallel, we have learnt much about how cadherins operate and are regulated at the molecular and cellular levels (Niessen, Leckband, & Yap, 2011; Takeichi, 2014). In this essay, we will discuss how some recent insights into the cellular mechanisms of cadherin biology can influence the way we think about the processes that drive epithelial morphogenesis. We focus on how adhesion cooperates with the actin cytoskeleton.
2. CADHERINS AND THE CONTRACTILE CYTOSKELETON We have long believed that cadherins cooperate, functionally and physically, with the actin cytoskeleton. Much of the early evidence for this derived from cell culture, such as the observation that inhibitors of F-actin integrity disrupt cadherin adhesion ( Jaffe et al., 1990). Cellular and biochemical studies further suggested that cadherins could interact physically with actin filaments (Nagafuchi & Takeichi, 1988; Ozawa, Ringwald, & Kemler, 1990). Indeed, many actin-binding proteins (ABPs) have now been identified as capable of interacting with classical cadherins (Ratheesh & Yap, 2012), and recent screens using proximity-biotinylation techniques have
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identified a host of other potential cadherin-associated ABPs (Guo et al., 2014; Van Itallie et al., 2014). An important advance in understanding the functional impact of cadherin–actin cooperation has come from the in vivo demonstration that mechanical forces exerted by the actin cytoskeleton are transmitted to cadherin junctions. In epithelia, this can take the form of pulsatile movements of junctions that are driven by pulsatile contractions of the cytoskeleton (Martin, Kaschube, & Wieschaus, 2009; Munjal, Philippe, Munro, & Lecuit, 2015; Rauzi, Lenne, & Lecuit, 2010; Roh-Johnson et al., 2012; Wu et al., 2014) or sustained contractile tension that is found in the junctions (Ratheesh et al., 2012; Wu et al., 2014). These different patterns of contractility often distinguish different morphogenetic processes and are associated with different actomyosin networks that associate with junctions. Pulsatile contraction is commonly seen with apical constriction and invagination (Martin et al., 2009; Roh-Johnson et al., 2012) and is driven by two-dimensional (2D) actomyosin networks found at the apical poles of cells (often called medial–apical networks) that undergo periodic condensation (Martin et al., 2009; Munjal et al., 2015). In contrast, sustained junctional tension is associated with processes such as neighbor exchange, boundary formation, and tissue homeostasis (Fernandez-Gonzalez, Simoes Sde, Roper, Eaton, & Zallen, 2009; Monier, Pelissier-Monier, Brand, & Sanson, 2009; Wu et al., 2014); it is attributed to circumferential actomyosin bundles that run parallel to, and associate with, cadherin junctions, especially the apically positioned zonulae adherente (ZA). However, these two modes of contractile organization can coexist within the same cell (Rauzi et al., 2010; Wu et al., 2014; Fig. 1). This fact—that cadherin junctions are sites where the cytoskeleton generates force—carries several important implications. First, this mechanical connection implies that there must be physical connections between the cadherins and the actin cytoskeleton. Second, since contractile forces are generated by nonmuscle Myosin II (NMII) acting on actin filaments, cadherins interact with an actomyosin contractile apparatus. This is further supported by the observation that mechanical stress exerted on cadherins can cause the cytoskeleton to stiffen (Bazellieres et al., 2015; le Duc et al., 2010), a process that requires both F-actin and myosin. Third, it alters the way we approach the biology of cell–cell adhesion: rather than principally being a mechanism that passively resists detachment forces to preserve tissue integrity, cadherins serve to generate tensile forces by coupling contractile cortices of neighboring cells together (Maitre et al., 2012). Furthermore,
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Junctional Medial–apical
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Figure 1 Patterns of actomyosin contractility at epithelial cell–cell junctions. (A) Apical actomyosin networks include two-dimensional medial–apical networks and junctional actomyosin bundles, as found in epithelial cells undergoing apical constriction. (B) In polarized epithelia, apical bundles at the zonula adherens (ZA) can coexist with twodimensional networks found at the lateral junctions, below the ZA.
cadherins can promote assembly and homeostasis of the contractile apparatus itself, while actomyosin feeds back to promote adhesion (Shewan et al., 2005; Smutny et al., 2010). Taken together, these observations suggest that at the cellular level the functional interrelationship between cadherin and actomyosin represents an integrated cellular system. As we shall now describe, considerable recent progress has been made in characterizing the elements that contribute to cadherin–actin cooperation. In the process, we have begun to discover feedback interactions between these elements that integrate them into a complex network whose emergent properties have implications for understanding morphogenesis.
3. THE ELEMENTS OF THE SYSTEM Integrating cadherin adhesion with contractility involves several components. These include the physical association of adhesion with
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actomyosin, the building of a contractile actomyosin apparatus that is linked to cadherin, and signaling pathways which coordinate these individual elements.
3.1 Molecular Basis of Cadherin–F-Actin Binding At a basic level, junctional contractility arises from a physical association of cadherin adhesion complexes with actomyosin. To date, the mechanical coupling of contractility to cadherin adhesion appears to be mediated by the F-actin elements of the actomyosin apparatus; there is no evidence that NMII interacts directly with the cadherin complex. Three broad pathways are available to couple cadherins to F-actin. First, cadherins may associate with actin filaments through α-catenin, a ubiquitous component of the core cadherin–catenin complex. Although free α-catenin can directly bind F-actin (Pokutta, Drees, Takai, Nelson, & Weis, 2002; Rimm, Koslov, Kebriaei, Cianci, & Morrow, 1995), its role as a linker between cadherins and F-actin became controversial when it proved difficult to reconstitute F-actin binding when α-catenin was incorporated into a minimal cadherin/ catenin complex in vitro (Drees, Pokutta, Yamada, Nelson, & Weis, 2005; Yamada, Pokutta, Drees, Weis, & Nelson, 2005). This conundrum has been recently resolved by the demonstration that minimal cadherin–catenin complexes will bind F-actin when under force (Buckley et al., 2014), whereas the earlier reconstitution studies were performed in solution. Second, α-catenin can recruit other ABPs to the cadherin complex (Abe & Takeichi, 2008). This is exemplified by vinculin, which bears multiple F-actin-binding sites (Ziegler, Liddington, & Critchley, 2006) and can bind directly to α-catenin itself (Choi et al., 2012). Junctional vinculin is increased when force is stimulated and decreased when forces are relaxed (Leerberg et al., 2014; Peng, Cuff, Lawton, & DeMali, 2010; Yonemura, Wada, Watanabe, Nagafuchi, & Shibata, 2010). This appears to reflect an ability of force to reveal cryptic vinculin-binding sites that are present in α-catenin (Yao et al., 2014; Yonemura et al., 2010). Thus, force appears to play an integral role in controlling how α-catenin may mediate interactions between cadherins and actin filaments. Finally, other proteins can mediate physical association between cadherins and actin filaments. For example, Myosin VI is an actin-binding motor that can directly interact with the E-cadherin cytoplasmic tail and is found at mature cell–cell junctions (Maddugoda, Crampton, Shewan, & Yap, 2007). However, Myosin VI is recruited to E-cadherin relatively late in the process of junctional biogenesis, where it is necessary for assembly of
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the ZA and organization of its associated actin cytoskeleton through vinculin. Together, these observations reinforce the notion that cadherins associate with F-actin through multiple pathways. The ability of α-catenin to directly bind F-actin is likely to constitute a ubiquitous mechanism that operates when cadherins come under force to link them with cortical actin (Chen et al., 2015). This may represent an elemental mechanism that can then be built upon by the force-dependent association of proteins like vinculin to α-catenin and the context-dependent recruitment of additional ABPs, such as Myosin VI.
3.2 Incorporating Myosin II Cellular contractility in nonmuscle cells is generated by the motor action of NMII exerted upon F-actin networks (Murrell, Oakes, Lenz, & Gardel, 2015). Consistent with this central cell-biological role, NMII is found decorating the medial–apical and junctional F-actin networks that are thought to exert contractile forces on cell–cell junctions (Mason, Tworoger, & Martin, 2013; Smutny et al., 2010). Considering that cadherin-bound actin filaments are the scaffolds upon which actomyosin is assembled, the incorporation of NMII at junctions involves two parallel processes. First, NMII must be activated. A dominant pathway involves the phosphorylation of the regulatory light chain of myosin through pathways mediated by kinases such as myosin light chain kinase (MLCK) and Rhodependent kinase (ROCK) (Heissler & Manstein, 2013). This is thought to promote F-actin binding, mini-filament assembly, and hence formation of an effective contractile unit. Consistent with this, both MLCK and ROCK support junctional contractility and the recruitment of NMII to the junctional cytoskeleton (Smutny et al., 2010) and to medial–apical networks (Mason et al., 2013; Munjal et al., 2015). Second, activated NMII must interact with F-actin networks. Of note, cadherin junctions are sites of dynamic actin assembly, initiated by actin nucleators such as Arp2/3 and formins, and modulated by postnucleation regulators such as Ena/VASP proteins and α-actinin-4 (Ratheesh & Yap, 2012). Their contribution to recruitment of NMII is illustrated by evidence that inhibiting Arp2/3 or its upstream activator, WAVE2, impaired the junctional recruitment of NMII and reduced junctional tension (Verma et al., 2012). This implies that the assembly of actin filaments at the junctional cortex serves to recruit activated NMII to the junctions, thereby allowing the contractile apparatus to assemble. However, the role of nucleators
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such as Arp2/3 carries a potential paradox. Arp2/3 generates branched actin networks that are thought to be poorly fitted for either stable NMII binding or contractile force generation. In part, this is because NMII tends to cause severing of branched networks, thereby turning over the actin scaffolds necessary for stable NMII localization (Reymann et al., 2012). Despite this, the cell clearly has developed strategies that compensate for this potential paradox, which may include stabilization of nascent filaments (Kovacs et al., 2011; Wu et al., 2014) and reorganization of actin networks to allow them to better sustain contractile stress (Murrell et al., 2015). While many mechanistic details have yet to be resolved, these observations carry the important implication that regulation of junctional F-actin (kinetics, organization) may constitute a pathway to influence contractility that operates orthogonal to that of the classical mode of NMII activation.
3.3 Upstream Regulation by Cell Signaling It will be apparent from the discussion above that cell signaling plays a central role in coordinating the many elements of cadherin–actin cooperation. Key signals include canonical regulators of the actin cytoskeleton, notably the GTPases Rac and Rho, as well as many protein and lipid kinases which are found at cell–cell junctions (Niessen et al., 2011). Rather than focusing on the many details of the mechanisms, we would highlight the following concepts. First, cadherin adhesion often plays an important role in establishing the junctional signaling pathways that control actomyosin. For example, depleting cadherin impairs the ability of cells to sustain both Rac and Rho signaling (Kovacs, Ali, McCormack, & Yap, 2002; Lampugnani et al., 2002; Priya, Yap, & Gomez, 2013). This dependence can reflect different paradigms for signal activation. In some instances, cadherins appear to behave like adhesion-activated signaling receptors. This is exemplified by Rac, whose activity is acutely stimulated by cadherin adhesion (Kovacs et al., 2002). In other cases, cadherins act more indirectly, as is seen with Rho. E-cadherin is necessary for Rho signaling to be established at cell–cell junctions (Priya et al., 2013), but acute ligation of cadherins can actually downregulate Rho, through feedback from Rac (Noren, Arthur, & Burridge, 2003; Noren, Niessen, Gumbiner, & Burridge, 2001). Here, cadherin adhesion appears to serve less as a ligand-activated receptor than as a membrane platform that is necessary for signaling zones to be established and maintained.
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Second, key signals, such as Rac and Rho, reflect networks of upstream molecular activators and inhibitors, whose balanced action determines the expression of signaling. For example, Rho signaling at cadherin junctions is activated by exchange factors (GEFs), such as Ect2 (Ratheesh et al., 2012), and also potentially inhibited by GTPase-activating proteins (GAPs) such as Myosin XIA (Omelchenko & Hall, 2012) and p190 RhoGAP (Priya et al., 2015). The spatial control of GEFs is important to initiate Rho signaling (Ratheesh et al., 2012), and the complementary inhibition of GAPs can play an important role in sustaining signaling (Priya et al., 2015).
4. MECHANOCHEMICAL FEEDBACK AMONG THE ELEMENTS OF THE CADHERIN–ACTOMYOSIN SYSTEM We conventionally think of the above elements of cadherin– actomyosin cooperativity as reflecting linear pathways that potentially work in parallel to one another. However, it is increasingly evident that these pathways are linked to ultimately form a network of chemical signals and mechanical effectors. One of the most recent examples involves Rho and NMII. As noted above, Rho (Rho A in vertebrates and Rho1 in Drosophila) is a canonical activator of NMII that is necessary for the various forms of contractility that impinge on cell–cell junctions. Thus, inhibiting Rho blocked the pulsatile contractility that is seen in the medial–apical actomyosin networks during Drosophila germband extension (Munjal et al., 2015) and that is found at the lateral junctions between mammalian epithelial cells (Wu et al., 2014). Inhibiting Rho, or its upstream activators, also degrades the sustained junctional tension of the ZA, which is associated with perijunctional actomyosin bundles (Fernandez-Gonzalez et al., 2009; Ratheesh et al., 2012). In all these instances, the impact of Rho is mediated by ROCK. Indeed, ligation of E-cadherin alone is sufficient to activate NMII in a ROCK-dependent fashion (Shewan et al., 2005). Together, these observations are consistent with a canonical linear pathway that leads from Rho to NMII via ROCK. The close functional link between the elements of the Rho–ROCK– NMII pathway is emphasized by the coaccumulation of these components at sites of contractility. This is especially striking in pulsatile medial–lateral networks in Drosophila. Here, not only do GTP-Rho and ROCK
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coaccumulate with actomyosin, but they also condense with the contracting medial–apical networks (Munjal et al., 2015; Vasquez, Tworoger, & Martin, 2014), suggesting that the dynamic spatial distribution of these signals is closely linked with that of NMII. Similarly, if less dramatically, these signals concentrate at the contractile ZA of polarized epithelial cells along with actomyosin (Priya et al., 2015, 2013; Ratheesh et al., 2012; Simoes Sde, Mainieri, & Zallen, 2014). At first, these patterns of coaccumulation fit well with a conventional linear signaling pathway. Surprisingly, however, not only does inhibiting Rho–ROCK degrade contractility, but directly inhibiting NMII itself also perturbs upstream Rho–ROCK signaling. Blocking contractility in gastrulating Drosophila embryonic cells, both prevented the pulsatile condensation of actomyosin seen in the medial–apical networks of these cells and also displaced GTP-Rho and ROCK from the apical cortex (Munjal et al., 2015). Similarly, inhibiting NMII directly in cultured mammalian epithelial cells reduced GTP-Rho and ROCK at the apical ZA of these cells (Priya et al., 2015). Together, these findings indicate that NMII can feedback to support Rho–ROCK signaling at cellular cortices. Importantly, both these instances reflect modes of mechanochemical feedback. In the pulsatile medial–apical networks of Drosophila germband cells, Rho and ROCK appear to undergo advection, being concentrated by a process of active bulk flow in the cellular cortex when the medial– apical actomyosin networks contracted and condensed. Such concentration may promote the interaction of Rho activators, thereby supporting positive feedback (Howard, Grill, & Bois, 2011). Consistent with this, inhibition of pulsatile contractility, which blocks advection, also decreased the amplitude of Rho and ROCK pulses (Munjal et al., 2015). In the case of the mammalian ZA, the mechanism involved anchorage of ROCK by physical association with NMII. When anchored at the ZA, ROCK then antagonized the cortical localization of a Rho inhibitor, p190B RhoGAP, to thereby support Rho signaling (Priya et al., 2015). Computational modeling predicted that in this network the local concentration of NMII would stabilize zones of activated Rho by preventing its premature inactivation. These observations add to the increasing realization that mechanochemical factors influence contractility at junctions. Indeed, tension can stabilize junctional NMII itself (Fernandez-Gonzalez et al., 2009), which might now be predicted to feedback upstream to affect Rho signaling.
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5. EMERGENT PROPERTIES OF MECHANOCHEMICAL NETWORKS A common consequence of networked control systems is the generation of emergent properties, features that are not predictable from analysis of the individual components but arise from their interactions at the higherorder network level. Could feedback from NMII to Rho yield emergent properties that are morphogenetically relevant? Here, an intriguing question is how different patterns of contractility—pulsed contractions associated with 2D networks versus the more sustained contractility of junctional bundles—can be generated from the same principal components (Rho, ROCK, NMII, and F-actin). Some potential answers to these questions lie in considering how mechanochemical feedback acts in these different contexts.
5.1 Generating Contractile Pulsatility By its very nature, pulsatility in actomyosin networks involves cycles of contraction/condensation and relaxation. While these coincide with pulsed fluctuations in Rho signaling found at the same sites as these actomyosin networks, contractile pulsatility is not simply an entrained response to those changes in Rho, as inhibiting contractility downregulates Rho signaling itself (Munjal et al., 2015). Instead, feedback within the Rho–NMII network contributes to generating pulses. One form of feedback occurs during the contraction phase when mechanochemical feedback by advection can promote local Rho signaling, thereby potentiating contractility (Munjal et al., 2015). Such mechanochemical feedback also affects the relaxation phase. One mechanism to induce contractile relaxation is inactivation of the NMII motor itself. This is mediated by myosin phosphatase, which is necessary for pulsatility to occur (Mason et al., 2013; Munjal et al., 2015). Interestingly, NMII was necessary for the cortical localization of myosin phosphatase, which also coaccumulated with condensed actomyosin networks when they contracted (Mason et al., 2013; Munjal et al., 2015). This suggests that actomyosin contractility promotes the local recruitment of a mechanism (myosin phosphatase) that leads to its inactivation, a sequence which would be predicted to promote pulsatile behavior. This mechanism for myosin inactivation is complemented by other changes in the contractile apparatus. Thus, advection declined during the
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transition from contraction to relaxation (Munjal et al., 2015), which constitutes a mechanism for negative feedback that is predicted to be necessary for disassembly of pulsed contractions. This was attributable to the increased density of actomyosin networks that developed during condensation, which can increase friction to oppose advection. In addition, NMII turnover increased (Munjal et al., 2015), suggesting that its association with actin networks became less stable. This can reflect inactivation of NMII as well the potential for stress to sever actin filaments and promote their turnover (Wu et al., 2014). Indeed, blocking contractility stabilized F-actin at lateral epithelial cell–cell junctions, regions of adhesion where pulsatile contraction also occurs (Wu et al., 2014). Therefore, rather than simply reflecting entrained responses to upstream signals, many factors, encompassing regulatory signals and the structural (F-actin) elements of the contractile apparatus, collaborate to drive the assembly and disassembly phases of contractile pulsatility.
5.2 Generating Sustained Tension How, then, can a system capable of generating pulsed contractions be converted into one that generates sustained tension? One possibility is that signaling by upstream activators of NMII behaves in a sustained, rather than a pulsatile, fashion (Munjal et al., 2015). Indeed, this is found at the apical ZA of mammalian epithelial cells, where GTP-Rho forms a defined micronsized signaling zone whose intensity and size is relatively stable over timescales (10 s of minutes) that are much longer than the dynamics of its constituent molecules (Priya et al., 2015) or the cycles associated with pulsatile actomyosin networks (100 s) (Rauzi et al., 2010; Wu et al., 2014). This zone depends on NMII and reflects the myosin-based feedback network described above. Of note, computational analysis of the feedback network predicted that the system was capable of generating bistable outcomes (Priya et al., 2015), meaning that once NMII was activated and localized to the junctional cortex, the feedback network that it set up could stably sustain signaling within the population of molecules found at the ZA zone. Sustained Rho signaling can then reflect the impact of the mechanochemical feedback network implemented by NMII. Computational analysis further predicted that surface stabilization of NMII plays an important role in establishing a stable Rho zone (Priya et al., 2015). Consistent with this, NMII was more stable at the ZA, where the stable Rho zone forms, than elsewhere at cell–cell junctions
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(Priya et al., 2015). This differential cortical stability of NMII may be a response to Rho signaling (Priya et al., 2015), but it can also be influenced by the actin networks with which it interacts (Wu et al., 2014). For example, F-actin is more stable at the ZA than elsewhere at cell–cell junctions, an effect of the actin regulator, N-WASP (Kovacs et al., 2011; Wu et al., 2014), and this is necessary for strong junctional tension to be generated at the ZA (Wu et al., 2014). Potentially, regulators of F-actin that promote association of NMII may then influence NMII–Rho feedback at junctions.
5.3 Morphogenetic Implications Ultimately, an important question is how these cellular properties of the cadherin–actomyosin system help us understand their impact on morphogenesis. Potential insight comes from considering the mechanical impact of pulsatile versus sustained contractile systems. Pulsatile contractile systems essentially contain the capacity to dissipate the stresses that they generate during their contractile phases. Their ability to support apical constriction is thought to arise from a rachet-like mechanism which allows the constricted adhesive junctions to be stabilized, i.e., to fail to relax when the medial–apical networks relax (Martin & Goldstein, 2014). Such relaxation may be important to coordinate apical constriction across populations of cells that undergo tissue invaginations (Vasquez et al., 2014). The mechanism responsible for rachet-like stabilization is not yet clear but coincides with the accumulation of actomyosin at junctions (Rauzi et al., 2010). As these are sites of sustained junctional tension, it is tempting to speculate that this contributes to generating the rachet. In contrast, the pulsatile networks found at the lateral junctions of polarized epithelial cells (i.e., below the apical ZA; Fig. 1) do not appear to be coupled to a rachet-like mechanism: the cadherin clusters with which they are associated also relax when the condensed actomyosin networks dissipate and relax. This limits the contractile tension that can be generated at these regions of junctions (Wu et al., 2014). One consequence of reducing junctional tension in these lateral regions may be to minimize mechanical barriers to hydrodynamic flow between the cytoplasmic compartments of cells, something that is also important for apical constriction to occur during development (He, Doubrovinski, Polyakov, & Wieschaus, 2014).
6. CONCLUDING COMMENTS Overall, these recent findings indicate that distinct contractile outputs can be generated by the core actomyosin apparatus, depending on different
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settings within the mechanochemical feedback network that encompasses Rho, NMII, and actin at cell–cell junctions. It seems likely that we have only begun to appreciate the versatility that can be generated by this Rhoactomyosin network. However, a key outstanding question is the role that the cadherin system may play in this network. To date, adhesion has principally been treated as the mechanism that mechanically integrates the contractile cortices of cells together (Maitre et al., 2012). This is also consistent with current focus on understanding the molecular mechanisms that physically couple cadherin adhesion complexes to F-actin. However, as we have discussed, cadherin adhesion also supports the biogenesis of junctional actomyosin, through processes such as actin assembly (Verma et al., 2012) and Rho signaling (Ratheesh et al., 2012). This active feedback from adhesion to contractility might be predicted to influence morphogenetic outcomes by tuning contractile outcomes. Further, junctional actomyosin promotes the accumulation and stability of cadherins at junctions and can influence cadherin clustering to promote adhesion (Priya et al., 2013; Smutny et al., 2010; Wu, Kanchanawong, & Zaidel-Bar, 2015). It is tempting to speculate that this might affect mechanical coupling between cells and even the degree of friction that adhesion presents as cells slide across each other during morphogenetic events such as neighbor exchange. If so, then, the physically coupled cadherin system would constitute another active feedback element in the mechanochemical network that allows cellular contractility to drive morphogenesis.
ACKNOWLEDGMENTS We thank all our lab colleagues for feedback and endlessly stimulating conversations. Our work is funded by the Human Frontiers Science Program, The Australian Research Council, and the National Health and Medical Research Council of Australia (1044041, 1037320, and 1067405).
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