Adherens Junction Assembly
- Introduction to the Assembly of Adherens Junctions
- The 'Fork Initiation and Zipper' Model
- Cadherin Recruitment
- Spontaneous recruitment of cadherins to adherens junctions
- Recruitment of plaque proteins to adherens junctions
- Linking the cytoskeleton to adhesions sites
- The role of adaptor proteins in the stabilization and function of core AJ components
In C.elegans embryos, sealing of the epidermal sheets around the entirety of the embryo in a process termed ventral enclosure, also utilizes filopodia. Moreover, α-catenin was shown to rapidly accumulate at filopodial contact points in a cadherin-dependent manner and in doing so promote junction formation. In cells at the leading edge made mutant by removing cadherin expression, filopodial protrusions were still extended, however catenin accumulation and subsequent junction formation failed. The term ‘filopodial priming’ was coined to describe the formation of nascent junctions via the recruitment of α-catenin to filopodial tips engaged in cell-cell contact via their cell surface cadherins. This process leads to further recruitment of cadherin-catenin complexes in order to strengthen the nascent junction .
Another family of proteins that have been detected at nascent junctions are the nectins and nectin-like (Necl) proteins (see Figure below). These proteins have been suggested to initiate AJ formation prior to the involvement of cadherins in some circumstances, as evidenced predominantly from mammalian cell culture studies (as reviewed in ). Nectins comprise a single extracellular domain of three Ig-like loops, a transmembrane domain and a cytoplasmic domain capable of binding the actin-binding protein afadin (as reviewed in ). Nectins, like cadherins, can interact with each other in trans between apposing cells and in cis on the same cell. Nectins utilize their first and outermost Ig-like loop to engage in trans interactions and their second Ig-like loop to engage in cis interactions, which is a pre-requisite to the subsequent formation of trans interactions [9, 10]. In contrast to cadherins, these interactions do not require calcium.
Cdc42 activation leads to increased filopodia formation and activation of the Rho family GTPase Rac that leads to increased lamellipodia formation. It should be noted that in this context Rac activation requires both Cdc42 and c-Src acting via the Rac GEF Vav2. The increase in filopodia enhances cell-cell contact, whilst the increase in lamellipodia promotes the closure of gaps between contact sites through increased cell surface receptor interactions . These morphological events can therefore be described as ‘fork initiation’ (multiple filopodia extending outwards) and ‘zippering’ (lamellipodial protrusions sealing the gaps). This signaling cascade ultimately leads to the recruitment of cadherins to the nectin-based cell-cell contact in order to stabilize the formation of the adhesive junction (as reviewed in ).
It is currently unknown precisely how the cadherin-catenin complex is recruited to sites of cell-cell contact, with several different models being posited including, but not limited to; nectin-based recruitment and spontaneous recruitment.
14]). In cell culture scenarios various proteins are suggested to bridge the gap between the nectin-afadin complex and the cadherin-catenin complex.
During early junction formation vinculin and ponsin are suggested to link the nectins to the cadherins. Vinculin is known to bind α-catenin that can bind β-catenin, which directly interacts with cadherin. In vitro experiments show ponsin is able to bind both afadin and vinculin, however these interactions are competitive, suggesting additional proteins are needed in vivo to allow ponsin and vinculin to recruit cadherins to nectin-based adhesion sites .
Other protein complexes connecting nectin-afadin to cadherin-catenin include LMO7-α-actinin  and ADIP-α-actinin . However it is unclear at what point during junction formation these proteins come into play, with evidence suggesting LMO7 is only involved at a more mature stage of junction development .
The role of cis interactions between cadherins is less established, with no clear consensus on precisely which domains are engaged in these interactions (as reviewed in). Recent evidence however suggests that these interactions are not required to facilitate trans interactions , whereas trans interactions are required in order for cisinteractions to occur . Computational modeling has been carried out to determine how these interactions, both trans and cis, result in the formation of a stable junction.
These in silico experiments showed that cadherins freely floating in the plasma membrane are not able to successfully form trans dimers unless their diffusion is slowed down in some manner. The introduction of a diffusion trap permitted the formation oftrans dimers, however without subsequent cis interactions these trans dimers were unable to coalesce to form an ordered structure. Though weak, cis interactions were nonetheless shown to be vital for junction formation. Moreover, the ordered formation of multiple trans dimers into a junction was shown to be polarized such that each transdimer had a specific orientation, resulting in the production of a directional lattice .
In the absence of the formation of a stable adhesion site, the cadherins at the plasma membrane are removed. This can occur through endocytosis, which has been observed in established AJs upon the release of cell-cell contact (see Figure below and ) and is known to regulate E-cadherin distribution in mature AJs . Endocytosis has been proposed as a means to prevent cadherin accumulation at the plasma membrane in the absence of cell-cell contact, which would otherwise occur due to the natural and spontaneous accumulation of cadherins 
The accumulation of cadherins, in the presence of cell-cell contact stimulates events at the cytoplasmic side of the adhesion site, including reorganisation of the cortical actin cytoskeleton through effectors downstream of Rho GTPases and through linker proteins in complex with nectins and cadherins. Collectively these processes establish the gross architecture of the adherens junction.
Different types of adhesion complexes exist, each with their own composition. For example, while the classical cadherins in adherens junctions (AJs) are bound to catenins such as β-catenin and p120 catenin which subsequently bind to the F-actin cytoskeletal network, desmosomal cadherins, bind to distinct cytoplasmic proteins such as plakoglobin (also known as γ-catenin) which in turn link to intermediate filaments. This variation exists despite desmosmal cadherins possessing a similar domain organization to the classical cadherins . In this case the adhesion complex, known as a desmosome, is found in tissues subjected to higher levels of mechanical stress .
All cell-cell adhesion complexes are composed of intercellular and cytoplasmic components. Intercellular interactions are generally facilitated by cell-adhesion molecules (CAMs) such as cadherin or nectin, often in a calcium-dependent process. The complexity of the cytoplasmic region on the other hand has yet to be fully defined. This region, which has been visualized as a dense plaque of proteins (reviewed in ) that contains a vast array of structural and scaffolding proteins as well as regulatory proteins. These regulatory proteins mediate adhesion dynamics, including assembly and disassembly and will be discussed in greater detail in step 4 – Regulation of Adhesion Dynamics.
In its most simplistic arrangement, the cytoplasmic component of AJs can be described as containing several core components. These include p120-catenin, which is bound to cadherin at the juxtamembrane position  and β-catenin or plakoglobin, which are bound to the cytoplasmic tail . α-catenin, which also binds to β-catenin, then links the complex to the actin cytoskeletal network. Although these proteins form the core components of AJs, and have been shown to be indispensable for adhesion function, a substantial body of research indicates a far greater complexity.
Interactions between cadherin and β-catenin, which occur shortly after the proteins are translated and before cadherin integrates into the membrane [37, 2], facilitate further interactions with other adaptor proteins. These interactions may influence the activity of regulatory proteins such as GAPs and GEFs, tyrosine and serine/threonine kinases and phosphatases however they also facilitate the assembly of a physical link that is capable of mechno-signal and force transduction. The structural composition of the plaque region is discussed below. It should be noted despite significant advancements in the field in recent years, the specifics of AJ composition and recruitment have yet to be fully defined.
Indeed, the importance of α-catenin in this link was highlighted after chimeric cadherin-αE-catenin fusion proteins were introduced into fibroblasts lacking cadherin, and a rescue of cadherin function was observed. This was noted to be dependent on the inclusion of the αE-catenin C-terminus . α-catenin’s importance was again highlighted in vivo when a similar study was performed using Drosophila. In this case the DE-cadherin-α-catenin fusion protein was found to rescue adhesion defects independently of β-catenin .
Despite the clear evidence that α-catenin is essential in this link, it is also clear that a more complex arrangement of proteins localize to the AJ plaque region. This was proposed by the Weis and Nelson groups after in vitro biochemical experiments showed that α-catenin-β-catenin heterodimers have a low affinity for actin [58, 59]. α-catenin binds more readily to actin as a homodimer, yet due to the β-catenin binding site overlapping with heterodimer interface, α-catenin can only bind to the AJ as a monomer. Recently, this was attributed to β-catenin disrupting interactions that exist between four α-helix bundles of α-catenin and confer its asymmetric homodimer arrangement. These findings were determined through crystallization of a near-complete human α-catenin dimer . Furthermore, in an earlier study a novel assay using isolated cadherin-containing membrane patches to which β-catenin was added also showed that although α-catenin could bind the cadherin-β-catenin complex, subsequent addition of G- or F-actin failed to bind to this new complex of cadherin-β-catenin-α-catenin . Similarly, although the cadherin-β-catenin-α-catenin complex has successfully been isolated from cells, this has not yet been achieved with actin also bound. It is important to note, however, that both of these are basically negative results.
Additional biochemical and in vitro studies by the Weis and Nelson groups further indicate that a direct link between the cytoskeleton and α-catenin is unlikely to occur in vivo, and instead, is likely mediated by additional adaptor proteins. For example, observations from FRAP experiments revealed contrasting dynamics of actin and α-catenin at cell-cell contact sites. Here, α-catenin was seen to exhibit a significantly slower depletion rate when compared to actin, which underwent rapid exchange with a cytoplasmic pool of actin [58, 59]. The dynamics of actin and α-catenin were also explored using Drosophila epithelial cells and two separate populations of actin were identified, each with different rates of turnover. Whilst the majority of actin was highly dynamic, and was proposed to serve in preventing the lateral movement of homophilic E-cadherin clusters within the membrane, a smaller pool of actin was highly stable and this was correlated to adhesion stability. Interestingly α-catenin was found to be essential for tethering the cadherin clusters to the dynamic actin filament network but was not required for complex stability . It was concluded in this study that unidentified adaptor proteins likely ensure stability in mobile AJs.
Importantly, adaptor proteins localized at AJs may also facilitate the recruitment of other proteins that function in associated mechanisms such as cytoskeletal organization. It is known for example that AJ adaptor proteins recruit the Arp2/3 complex, formins and other proteins involved in the regulation of the actin cytoskeleton[79, 80, 81]. Overall, the complex and highly dynamic arrangement of the AJ may enable it to endure the transient yet strong forces produced during processes such as apical constriction, where the actin cytoskeleton pulls upon the adhesion complex.
It is unlikely that any single protein is responsible for linking the catenin-cadherin complex with the actin cytoskeleton. Instead, eplin and vinculin, as well as additional candidate proteins such as ZO-1, α-actinin, afadin  are likely to contribute in a manner that is cell type dependent. The interactions that facilitate this link are also likely to be transient which is consistent with both adaptor protein turnover rate, which is within minutes, and the dynamic nature of the cortical actin cytoskeleton as revealed by FRAP experiments . Whilst this makes detection of any link difficult using current experimental techniques it should be noted that even numerous weak, transient interactions could still promote strong linkage between cadherins and the actin cytoskeleton through sheer numbers (as reviewed in . These interactions may be cell type specific and given their importance in maintaining tissue integrity may also exhibit protein redundancy.