The role of apical cell-cell junctions and associated cytoskeleton in

1. Review papers

1.2. The role of apical cell-cell junctions and associated cytoskeleton in

Sluysmans S, Vasileva E, Spadaro D, Shah J, Rouaud F, Citi S. Biol Cell. 2017.

Cell-cell junctions are a prerequisite for the establishment of multicellular organisms. They provide physical anchoring between adjacent cells and define distinct tissue compartments.

They also act as essential platforms in the transmission of signals. Specifically, cell-cell junctions can perceive intra- and extracellular mechanical cues and convert them into intracellular signals to respond and adapt to stimuli, i. e. they carry out mechanotransduction.

The apical junctional complex in vertebrates comprises TJs and AJs and is connected to the actin and microtubule cytoskeletons. Several cytoskeleton-associated junctional proteins have been implicated in mechanotransduction. In this review, the organization of actin filaments and microtubules is described, focusing especially on their association with cell-cell junctions and how it impacts cellular physiology. The molecular machinery linking the cytoskeleton to TJs and AJs is also detailed, and the molecular basis of how junctions perceive and transduce mechanical stimulation are explained, thus reviewing junctional mechanotransduction.

My participation in this manuscript was to review the literature for single molecule studies of transmembrane proteins and junctional mechanotransduction, and the effects of shear force and cyclic stretch on junctional proteins. Thus, I contributed to the chapters about mechanotransduction by cytoplasmic proteins of cell-cell junctions and by transmembrane junctional proteins.

Biol. Cell (2017)109,139–161 DOI: 10.1111/boc.201600075

Review

The role of apical cell–cell junctions and associated cytoskeleton

in mechanotransduction

Sophie Sluysmans², Ekaterina Vasileva², Domenica Spadaro, Jimit Shah, Florian Rouaud and Sandra Citi¹ Department of Cell Biology, Institute of Genomics and Genetics of Geneva (iGE3), University of Geneva, Geneva, Switzerland

Tissues of multicellular organisms are characterised by several types of specialised cell–cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.

Introduction

Multicellular organisms rely on specialised struc-tures and molecules to ensure (i) the physical co-hesion and specific recognition between individual cells, to form tissues and organs, (ii) the transmis-sion of information and signals from and to cells and (iii) the separation between different tissue compart-ments, to allow polarised absorption and secretion.

Cell–cell junctions play a particularly important role in fulfilling these functions. There are several differ-ent morphological and functional classes of cell–cell junctions, and junctions have undergone consider-able structural and molecular remodelling through-out evolution. Here, we focus on cadherin-based and barrier-forming junctions of vertebrate organisms:

we summarise basic concepts about their structural and functional connection to the cytoskeleton, and

1To whom correspondence should be addressed (email:

sandra.citi@unige.ch)

2These authors contributed equally to this work.

Key words:Actin, Adhesion, Cytoskeleton, Junctions, Microtubule.

Abbreviations: AJ, adherens junctions; CAM, cell adhesion molecule; JAM, junctional adhesion molecule; KO, ; MDCK, Madin Darby Canine Kidney; PAMR, perijunctional actomyosin ring; TJ, tight junctions; ZA,zonula adhaerens; ZO, zonula occludens.

highlight recent studies implying junctional pro-teins in mechanosensing and mechanotransduction.

Mechanotransduction consists in the ability of cellu-lar structures to sense tension, adapt to it and transmit downstream signals. This process was first described at actin-based junctions between cells and the extra-cellular matrix (focal contacts) (Riveline et al., 2001;

Galbraith et al., 2002). However, mechanosensing at cell–substrate junctions will not be discussed here (see (Chen et al., 2015) and (Hytonen and Wehrle-Haller, 2016) for recent reviews): we will focus on the role of apical cell–cell junctions and associated cy-toskeleton, and primarily on the junctional proteins, which have been shown to interact directly with the cytoskeleton.

In vertebrate epithelial cells, the apical junctional complex comprises tight junctions (TJ) and, immedi-ately below, cadherin-basedzonulae adhaerentes(ZA).

Both TJ and ZA are ‘zonular’, that is, they form a con-tinuous circumferential belt around the apex of the cells, which distinguishes them from other forms of specialised cell–cell contacts, such as lateral contacts (puncta adhaerentia), desmosomes,fasciae adhaerentesof cardiac myocytes and other junctions that do not fit

S. Sluysmans and others

these schemes (Farquhar and Palade, 1963; Franke, 2009; Franke et al., 2009).

The canonical functions of TJ are (i) to form a semipermeable paracellular barrier between the api-cal and basal extracellular compartments (Diamond, 1977), and (ii) to topologically define the physi-cal separation between apiphysi-cal and basal domains of the plasma membrane, thus maintaining apico-basal epithelial cell polarity (fence function). The barrier function is carried out through the formation of pores and channels by 4-pass transmembrane proteins of the claudin family. Additional TJ membrane proteins comprise MARVEL proteins, including tricellulin, which is concentrated at tricellular TJ (Furuse et al., 2014; Choi et al., 2016), and angulins (Masuda et al., 2011). The establishment of polarity is regulated by members of the Par protein complex, which crosstalk with RhoGTPases to control cytoskeleton organisa-tion (Iden and Collard, 2008). The non-canonical functions of TJ are related to the regulation of a num-ber of signalling pathways, via their interaction with transcription factors, with regulators of the activities of Rho GTPases, and others (for reviews, see (Furuse, 2010; Citi et al., 2014; Tamura and Tsukita, 2014;

Van Itallie and Anderson, 2014; Zihni et al., 2016)).

Adherens junctions (AJ) are critically important for cell–cell adhesion and sorting, and tissue morpho-genesis and integrity. They are constituted by two main protein complexes, associated with: (i) mem-bers of the calcium-dependent cadherin family, and (ii) members of the calcium-independent, Ig-like-cell adhesion molecule (CAM) nectin family (Gumbiner, 2005; Niessen and Gottardi, 2008; Franke, 2009;

Takeichi, 2014; Mandai et al., 2015). Cytoplasmic proteins associated with cadherins comprise p120-catenin, β-catenin, α-catenin, α-actinin, vinculin and EPLIN, whereas nectins form complexes with afadin and ponsin (Mandai et al., 2015). The ZA is a specialised, circumferential form of AJ, present in polarised epithelia, but cadherin and nectin com-plexes are also present along lateral contacts of epithe-lial cells, and in non-epitheepithe-lial cell types (Gumbiner, 2005; Niessen and Gottardi, 2008; Franke, 2009;

Takeichi, 2014; Mandai et al., 2015). Desmosomes (Godsel et al., 2004; Garrod and Chidgey, 2008) can be viewed as a specialised subclass of AJ, since they contain transmembrane proteins (desmogleins and desmocollins), which belong to the broader cad-herin superfamily, but are not calcium dependent.

Desmosomes are particularly important for resistance of epithelial tissues to mechanical stress, and are the only epithelial junctions associated with intermedi-ate filaments. Desmosomes will not be extensively discussed here, except for their association with the microtubule cytoskeleton, since little is known about their implication in epithelial mechanotransduction processes.

Cell–cell junctions have been described both in epithelial and endothelial cells. However, endothe-lial junctions are different from epitheendothe-lial junctions with regards to their molecular organisation and cy-toskeletal anchoring (reviewed in (Dejana, 2004; Gi-annotta et al., 2013; Tietz and Engelhardt, 2015)).

For example, endothelial cells lack desmosomes, and their TJ and AJ are spatially closely associated. Fur-thermore, mechanosensing in endothelial cells occurs in the context of physiological exposure to the shear stress of blood flow. These concepts will be expanded in the section “Mechanosensing by transmembrane junctional proteins”.

Actin cytoskeleton: Evidence for its association with cell–cell junctions, and implication in junction-dependent epithelial and endothelial physiology The actin and microtubule cytoskeletons are essen-tial to organise and maintain cell shape and polar-isation, and support a wide range of fundamental cellular processes, such as vesicle transport, cytoplas-mic organisation, chromosome segregation, plasma membrane specialisation and cell migration (Musch, 2004; Pollard and Cooper, 2009). Together with in-termediate filaments, they are also fundamental to establish and support cell–cell adhesion and tissue integrity, which is required for tissue and organs physiology, both during development and in adult tissues. Actin and myosin coat the cytoplasmic faces of the plasma membrane to provide structural rigidity and mechanical resistance to the cell cortex (Heuser and Cooke, 1983; Citi and Kendrick-Jones, 1991;

Bretscher et al., 2002). Actomyosin contractility is involved in a large number of cellular functions, be-sides its involvement at cell–cell junctions, and de-pends not only on actin and myosin, but also on a variety of regulatory and scaffolding proteins (Zaidel-Bar et al., 2015), which will not be discussed in detail here.

Junctions, cytoskeleton and mechanotransduction

Review

Actin filaments (F-actin) are 4–7 nm wide, polarised polymers of globular monomeric actin (G-actin, 42 kDa). Actin polymerisation generates either branched or bundled filaments, and produces motility, for example the migration of cells, by sup-porting the spreading of lamellipodia. Myosins are actin-associated motors, and exist in multiple iso-forms (Hodge and Cope, 2000), typically moving towards the plus end of the actin filament. Nonmus-cle myosin-II (conventional myosin, with two heavy chains, two essential and two regulatory light chains) is the major actor of contractility at apical cell–cell junctions. Furthermore, specific isoforms of nonmus-cle myosin-II (A, B, C) and β-actin and γ-actin are implicated in non-redundant functions at cell–

cell junctions, in a cell-type-specific manner (Ivanov et al., 2007; Baranwal et al., 2012; Ebrahim et al., 2013). Regulation of nonmuscle myosin-II contrac-tility is crucial for mechanotransduction. The major regulatory switch for nonmuscle myosin II is myosin light chain kinase-dependent phosphorylation of the regulatory light chains, which controls myosin fil-ament assembly, conformation, and actin-activated Mg²⁺-ATP-ase activity (Tan et al., 1992; Vicente-Manzanares et al., 2009). Myosin light chain phos-phorylation is enhanced by activation of the RhoA-ROCK pathway, primarily through inhibition of the myosin light chain phosphatase (Vicente-Manzanares et al., 2009). At apical junctions myosin-II is organ-ised into a sarcomeric-like belt, where 400–600 nm-long bipolar filaments are aligned end-to-end within one cell, and are in register with filaments in adjacent cells (Ebrahim et al., 2013).

The architecture of the actin cytoskeleton at cell–

cell junctions was explored by electron microscopy in the early 1980s. In epithelial chick hair cells, quick-freeze, deep-etch and rotary shadowing tech-niques revealed the presence of two distinct but in-terconnected populations of actin filaments: (i) one at the level of the ZA, organised as a ring and com-posed of filaments that run in parallel to the plasma membrane, (ii) the other just beneath the TJ mem-brane, organised as a meshwork of filaments (Hi-rokawa and Tilney, 1982; Hi(Hi-rokawa et al., 1983) (Figure 1). Electron microscopy and immunofluores-cence studies also showed that actin and myosin have a specialised organisation at sites such as the brush border (microvilli and terminal web) of intestinal epithelial cells (Mooseker, 1985; Drenckhahn and

Dermietzel, 1988). The thick circumferential belt of F-actin detected at the cytoplasmic face of the ZA is sometimes referred to as perijunctional acto-myosin ring (PAMR) (Turner, 2000). Its ability to undergo a circumferential contraction was first iden-tified in intestinal brush borders (Burgess, 1982), and has subsequently been shown to control not only TJ barrier function (Cunningham and Turner, 2012), but also tissue shape changes and remodelling during morphohenesis (Lecuit and Yap, 2015). The distribu-tion of actin and myosin in the PAMR was recently studied by structured illumination microscopy, re-vealing a continuous cortical actin ring, with un-derlying sarcomeric-alike nonmuscle myosin bipolar filaments (Ebrahim et al., 2013; Van Itallie and An-derson, 2014; Choi et al., 2016). The identity of the junctional and cytoskeletal proteins associated with actomyosin in the PAMR have been described in sev-eral excellent reviews (Franke, 2009; Brieher and Yap, 2013; Takeichi, 2014).

The orientation of actin filaments with respect to the junction-associated plasma membrane de-pends on the type of cadherin-based contact (re-viewed in (Takeichi, 2014)). In linear zonular junc-tions (ZA), filaments run parallel to the membrane and form a thick, circumferential bundle, i.e. a PAMR (Figure 1). Higher tension at this site is associated with increased junctional accumulation of vinculin and EPLIN, as discussed in more detail in the sec-tion “The machinery linking the actin cytoskeleton to AJ”. In contrast, at ‘punctate’ AJ, such as those that form at the tips of E-cadherin-based contacting filopodia, filaments run perpendicular to the plane of the plasma membrane. Punctate AJ (‘puncta adhaeren-tia’) distributed along the lateral contacts of polarised epithelial cells contain actin filaments with mixed orientations, which are not bundled and do not accu-mulate vinculin and EPLIN, suggesting lower ten-sion (Takeichi, 2014). However, at tricellular junc-tions actomyosin bundles distributed along neigh-bouring bicellular junctions separate into fan-like ar-rays and anchor end-on at discrete puncta, where vin-culin staining is enhanced (Choi et al., 2016). Thus, there are different modes of organisation of actin fil-aments at cadherin-based junctions (see also (Zhang et al., 2005; Wu et al., 2014)) and actin filaments can subjected to different degrees of tension, regard-less of their orientation with respect to the plasma membrane.

S. Sluysmans and others

Figure 1 The organization of actin filaments and their anchoring to tight junctions (TJ) andzonula adhaerens(ZA) in polarized epithelial cells

Actin filaments and associated myosin-II bipolar filaments (not shown for the sake of clarity) are distributed throughout the cortex, and are clustered at the apical junctional complex in the form of a circumferential actomyosin zonular belt (PAMR), and at cell–

substratum adhesions on the basal part of the cells. Magnified insets show simplified schemes of the machineries that link the actin cytoskeleton to either TJ (top) or ZA (bottom). At TJ, ZO-1 and ZO-2 form the submembrane scaffold for TJ transmembrane proteins by binding to them through their N-terminal PDZ domains, and connect to F-actin filaments, through their C-terminal domain. The C-terminal domain of ZO-1 also interacts with the globular head domain of cingulin and paracingulin. Cingulin is localised in the same region of the cytoplasmic plaque as myosin, that is farther away from the membrane than ZO-1. At the ZA, actin filaments are organised in bundles parallel to the membrane, and are connected to nectins and cadherins via afadin and α-catenin–vinculin complexes. EPLIN is characteristically associated with actin filaments at the ZA, but not at punctate AJ and at lateral contacts of epithelial cells.

The structural associations between microfilaments and TJ have historically been more difficult to vi-sualise, compared to those with ZA, where the actomyosin bundle is significantly thicker. Trans-mission electron microscopy of detergent-extracted cells demonstrated that TJ-associated actin fila-ments, decorated by the actin-binding region of myosin (subfragment-1, S1), occur predominantly at sites of intercellular membrane apposition, and appear to insert directly into the submembrane TJ space (Madara and Pappenheimer, 1987; Madara et al., 1988). Following the discovery of specific TJ-associated proteins, it was shown that these proteins co-localise with the PAMR by confocal fluorescence microscopy (Turner, 2000). However, structured il-lumination microscopy experiments, which provide

an improved spatial resolution, indicate that the non-muscle myosin sarcomeres are centred at midpoint in the apical junctional complex, likely forming a dis-tinct structure that overlaps partially with both TJ and AJ (Ebrahim et al., 2013), and might topologi-cally correspond to the localisation of the ZA proteins afadin and PLEKHA7 (Pulimeno et al., 2010). Thus, it is not clear whether the barrier-forming units of TJ, for example claudins and zonula occludens (ZO) pro-tein scaffolds (see section “The machinery linking the actin cytoskeleton to TJ”), are anchored exclusively to the thick bundles of actin filaments (PAMR), or might be anchored more apically to differently or-ganised actomyosin filaments.

The role of the actin cytoskeleton in the organ-isation and function of cell–cell junctions was first

Junctions, cytoskeleton and mechanotransduction

Review

demonstrated using drugs that inhibit actin poly-merisation. For instance, treatment of cultured cells or animal models with cytochalasins, which inhibit actin polymerisation, disrupted the barrier function of epithelial and endothelial TJ (Bentzel et al., 1980;

Meza et al., 1980; Cereijido et al., 1981; Shasby et al., 1982; Madara et al., 1986), altered the struc-ture of TJ in vivo (Rassat et al., 1982), and inhib-ited the nectin-dependent adhesive properties of cul-tured cells (Friedlander et al., 1989). These drugs induced the clumping, aggregation and disorgani-sation of fibrillar structures, and their detachment from the membrane, suggesting contraction of acto-myosin filaments (Meza et al., 1980; Madara et al., 1986; Madara et al., 1987; Madara et al., 1988).

Following the development of antibodies recognis-ing specific markers, it became possible to investi-gate the functions of the actomyosin cytoskeleton in orchestrating the spatial distribution of junctional and membrane proteins. For example, it was shown that cytochalasins disrupt the polarised distribution of Na⁺K⁺-ATPase in mouse trophectoderm (Watson et al., 1990), the localisation of cadherin and desmo-somal proteins during junction assembly (Pasdar and Li, 1993), and the circumferential distribution of TJ proteins in cultured cells (Citi et al., 1994; Steven-son and Begg, 1994). It is important to note that TJ and the ZA/AJ are ontologically related, molecularly interconnected and spatially very close. As a conse-quence, it is very difficult to determine whether any TJ phenotype is induced by actin-active drugs that act directly on TJ components, or indirectly through the action on the nearby ZA-associated PAMR.

Following up on initial studies using cytochalasins, a growing number of experimental, pharmacologi-cal and physiologipharmacologi-cal agents (reviewed in (Fanning, 2001)), including phalloidin, latrunculin (Shen and Turner, 2005), glucose, cytokines, kinase agonists and antagonists such as PMA and H-7 (Citi et al., 1994) and the ROCK kinase inhibitor Y27632 (Walsh et al., 2001), were shown to affect TJ molecular in-tegrity and paracellular permeability, through their actions on the organisation of the actin cytoskeleton and the contractility of the PAMR. Furthermore, a number of pathogens, includingClostridium difficile, Vibrio cholerae, Bacteroides fragilis and Escherichia coli induce toxin-dependent and other pathogenic effects on intestinal epithelia by disrupting the integrity of their apical domain the organisation of the

acto-myosin cytoskeleton, thus affecting TJ barrier func-tion (reviewed in (Berkes et al., 2003)).

Experimental modulation of extracellular calcium was used to highlight the existence of junction-associated contractility. Extracellular calcium is critical for the integrity of cadherin-based junc-tions, since calcium binding to the conserved se-quences in the extracellular EC cadherin repeats (LDRE, DxNDN and DxD (Overduin et al., 1995)) is necessary for homophylic adhesion, via trans-dimerisation of cadherin ectodomains on neighbour-ing cells (Pokutta et al., 1994). Thus, removal of extracellular calcium in cultured cells leads to junc-tion disrupjunc-tion, endocytosis of the cadherin complex and indirect disruption of neighbouring TJ (Karten-beck et al., 1991). Junction disruption and rapid loss of TJ barrier function are suppressed by inhibitors of protein kinases, which act by blocking the contractil-ity of the actomyosin cytoskeleton (Citi, 1992; Citi et al., 1994). Thus, cadherin-mediated adhesion is

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