2.2. Cell adhesion structures

2.2.1.1. Cadherin-based adherens junctions

In epithelial cells cadherin-based cell-cell contact is a specialised region of the plasma membrane, where cadherin molecules of the adjacent cells interact in a calcium-dependent manner. Actin filaments are associated to this structure through catenins located at the undercoat of the adherens junction (Ozawa et al. 1990, reviewed by Geiger & Ginsberg 1991, Rimm et al. 1995, reviewed by Takeichi 1995). Cadherins are subdivided into classic cadherins (E; epithelial, P; placental, N; neuronal cadherins) found in adherens junctions, and desmosomal cadherins, found in desmosomes (reviewed by Yap et al. 1997). Classical cadherins have an extracellular part consisting of five distinct domains (EC1-5) and a conserved cytoplasmic domain. The extracellular part interacts homotypically with cadherins of the neighbouring cells, and the conserved cytoplasmic tail associates with intracellular proteins involved in the formation of the junctional structure. E-cadherin mediates the assembly of adherens junctions, but it also affects the formation of desmosomes and tight junctions (Gumbiner et al. 1988, Wheelock & Jensen 1992).

The carboxy-terminal part of E-cadherin cytoplasmic domain binds to β -catenin or to γ -catenin (plakoglobin) which, in turn, form the linkage to α-catenin (Fig. 2). Furthermore, through a site near its transmembrane domain, E-cadherin binds directly to a special catenin, called p120ctn (p120cas, Reynolds et al. 1994, Yap et al. 1998, Thoreson et al. 2000) that was originally characterised as a substrate of v-Src kinase (Reynolds et al. 1992). In the complex of E-cadherin, α-catenin is the only catenin that forms either direct or indirect linkage to actin cytoskeleton through α-actinin, vinculin, ZO-1, spectrin and a number of other molecules associated to cadherin complex (reviewed by Yamada & Geiger 1997).

The function of cadherins is not only limited to formation of protein complexes inside the cells and linkage of the cells together, but they also regulate the signalling events during differentiation, proliferation and migration (reviewed by Knudsen et al. 1998). Cadherins contribute either to the organisation of signalling components or, by formation of close cell-cell contact, they affect the signalling mechanisms indirectly (reviewed by Fagotto & Gumbiner 1996). Cell-cell contacts are also involved in the long-term signalling events that regulate cell growth and gene expression. During differentiation the cells express the characteristic, unique cadherins and in several cases cadherins determine the differentiated phenotype of the cells. The presence of many signalling molecules, such as Src family tyrosine kinases, receptor kinases and phosphatases in the adherens junctions of the epithelial cells (Tsukita et al. 1991, reviewed by Woods & Bryant 1993, Brady-Kalnay et al. 1995) may be important for the regulation of the function of cadherin as well as for transduction of the signals from cadherin or adherens junction into the cell. In tissues, the establishment of strong adhesion or contractility is dependent on the assembly of adherens junction. On the other hand, in epithelial cells, the apico-lateral belt of adherens junction strengthens the adhesion by linking the actin cytoskeleton to sites of strong adhesion. The absence of E-cadherin weakens the intercellular adhesion by affecting other junctional proteins, and loss of its expression or function is associated with tumour cell invasion of epithelial cancers (reviewed by Birchmeier & Behrens 1994).

β -catenin is a key component of cell-cell adhesion linking cadherin receptors to the cytoskeleton (Fig. 2). Moreover, it is also part of the Wnt/Wingless signalling pathway that controls numerous events in development, including differentiation, proliferation and morphogenesis (reviewed by Wodarz & Nusse 1998). β -catenin uses this pathway for transmission of signals from cell-adhesion components or Wnt protein to the nucleus. In the presence of Wnt signals unphosphorylated β -catenin regulates gene expression through its association with transcription factors, LEF-1 (lymphocyte-enhancer factor-1) and TCFs (reviewed by Behrens et al. 1996, reviewed by Seidensticker & Behrens 2000). In the absence of Wnts β -catenin is phosphorylated and degraded in proteasomes. In tumours degradation of β -catenin is blocked due to mutation of β -catenin or tumour suppressor gene APC (adenomatous polyposis coli). This leads to formation of TCF/β -catenin complexes and activation of oncogenes (reviewed by Seidensticker & Behrens 2000).

2.2.1.2. Tight junctions

In epithelial and endothelial cells, tight junctions are the most apical intercellular junctions that function as selective (semipermeable) diffusion barriers between individual cells. They maintain the different composition of proteins and lipids between the apical and basolateral plasma membrane domains (“fence” function). Furthermore, the tight junctions regulate the growth and differentiation of the cells. (Reviewed by Balda & Matter 1998, reviewed by Tsukita et al. 1999.)

The tight junction is identified as a belt-like region in which two lipid-apposing membranes lie close together (tight junction strands). Tight junction strands of the adjacent cells form tightly connected pairs. The proteins involved in the formation of tight junctions are divided into two categories: 1) integral membrane proteins, such as occludin, claudin and junctional adhesion molecule, JAM and 2) peripheral membrane proteins (cytoplasmic plaque proteins), MAGUK (membrane-associated guanylate kinase) homologue proteins, such as ZO-1, 2, 3, cingulin, symplekin, 19B1, and AF-6. Moreover, various signalling proteins (protein kinases, heterotrimeric G-proteins and small GTP-binding proteins) are either localised at the cytoplasmic plaque domain of tight junction, or they have a central role in the assembly or function of junction (reviewed by Tsukita et al. 2001). ZO-1 was the first tight junctional protein identified (Stevenson et al. 1986). In epithelial cells ZO-1 is a critical protein in the initial steps during the formation of cell-cell junctions, because it accumulates together with cadherin to tips of cellular protrusions of the adjacent cells forming spot-like junctions (Yonemura et al. 1995). As polarisation of the cells proceeds, occludin is gradually carried to spot-like junctions of ZO-1 to form a belt-like tight junction, whereas E-cadherin is released from ZO-1 junctions in order to form adherens junction (Ando-Akatsuka et al. 1999). In non-epithelial cells ZO-1 has also been found in other subcellular sites, e.g. in the nucleus of growing cells (Gottardi et al. 1996) and in adherens junctions of cells which lack tight junctions, such as fibroblasts (Howarth et al. 1992, Itoh et al. 1993). These variations in the distribution of ZO-1 might be a consequence of its ability to bind both occludin (Furuse et al. 1994) and α-catenin (Itoh et al. 1997), or the presence of forms of the protein of varying degrees of solubility.

In tight junctions of the epithelial cells, ZO-1 interacts with ZO-2, ZO-3, occludin and claudin (reviewed by Tsukita et al. 1999). Other molecules also associate directly with ZO-1, such as AF-6 (afadin), a putative target for Ras (Yamamoto et al. 1997), gap junctional protein connexin-43 (Toyofuku et al. 1998, Giepmans & Moolenaar 1998) and spectrin (Itoh et al. 1991). Occludin and claudin are linked to actin cytoskeleton through the C-terminal half of ZO-1 (Itoh et al. 1997). This part of ZO-1 contains various alternatively spliced domains, of which the α-domain corresponds to the plasticity of tight junctions (Balda & Anderson 1993, Sheth et al. 1997). Occludin and claudin have four transmembrane domains with N- and C-termini in the cytoplasm and two extracellular loops (reviewed by Tsukita et al. 1999). The extracellular domains and at least one of the transmembrane domains of occludin are responsible for regulation of paracellular permeability of tight junctions (McCarthy et al. 1996). Occludin is not only the structural and functional component of tight junction, but it also affects the phenotype of the epithelial cells during transformation (Li & Mrsny 2000). In Ras-transformed MDCK cells occludin is localised to cytoplasm together with claudin-1 and ZO-1, but the proteins are recruited to membrane if activation of mitogen-activated protein kinase (MAPK) is blocked (Chen et al. 2000). Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte transmigration (reviewed by Fanning et al. 1999). At tight junction JAM is linked to strands of claudin through ZO-1. JAM regulates the polarity of epithelial cells through its association with and recruitment of ASIP (atypical protein kinase C-specific interacting protein)/PAR3-atypical PKC complex to tight junction (Ebnet et al. 2001). In man, the claudin superfamily consists of at least 18 members, which are involved on paracellular transport as structural and functional components of tight junction. Claudins are directly associated with ZO-1, 2 and 3 and indirectly with AF-6 and cingulin (Itoh et al. 1999, reviewed by Tsukita et al. 1999). The tight junction of choroids plexus epithelium has a unique molecular composition of claudins composed of claudin-1, 2 and 11 that have a unique regulatory role in barrier function of blood-cerebrospinal fluid (Wolburg et al. 2001).

Nectin, afadin and ponsin are the components of a recently found cell-cell adhesion system at adherens junctions that are likely involved in the formation of cadherin– and claudin-based junctions (Mandai et al. 1997, Asakura et al. 1999, Ikeda et al. 1999). Nectin is a Ca2+-independent immunoglobulin-like adhesion molecule (Takahashi et al. 1999). The nectin family consists of at least three members, nectin 1, 2 and 3. Most nectins interact with the PDZ domain of afadin through their conserved motif of C-terminus (Takahashi et al. 1999, Satoh-Horikawa et al. 2000). A family of afadins consists of at least two members, larger (l) –and smaller (s)-afadin, of which l-afadin is ubiquitously expressed in epithelial cells, whereas s-afadin is found in neural tissues. Both afadins have one PDZ domain, but s-afadin lacks the F-actin binding domain and there are also variations in the proline-rich region of the proteins (Mandai et al. 1997). Human s-afadin is identical to AF-6, the protein that forms a complex with ZO-1 at tight junction of epithelial cells (Yamamoto et al. 1997). L-Afadin is linked to cadherin-based adherens junction via ponsin, which in turn associates through its SH3 domain to the proline-rich region of vinculin (Mandai et al. 1999). The nectin-afadin-ponsin complex is important in the formation of primordial spot-like junctions of epithelial cells, but it is not sensitive to disruption of existent cell-cell contacts induced by low Ca²⁺ concentration (Asakura et al. 1999). Thus, the nectin-afadin-ponsin complex serves as an independent adhesion system at epithelium.

2.2.1.3. Desmosomes and gap junctions

Desmosomes are specialised junctional structures that form a tight connection between all the epithelial cells and cardiac myocytes (reviewed by Schwartz et al. 1990). Within the cells, they are linked to cytokeratins, a class of intermediate filaments. The complex desmosomal structure consists of several transmembrane adhesive glycoproteins and cytoplasmic plaque proteins (reviewed by Garrod 1993). The glycoproteins, such as desmogleins and desmocollins, belong to the cadherin superfamily and the others, such as desmoplakins link the intermediate filaments to the membrane. Plakoglobin, a member of the cytoplasmic desmosomal plaque proteins, is directly bound to desmosomal cadherins; desmoglein-1 (Korman et al. 1989, Mathur et al. 1994, Troyanovsky et al. 1994) and desmocollin-1 (Troyanovsky et al. 1994). It is also found at adherens junction of the epithelial cells associated to classical cadherin (Cowin et al. 1986, Peifer et al. 1992) and α-catenin (Huber et al. 1997).

Gap junctions are intercellular structures that make possible the passive diffusion of the ions and small molecules in aqueous intercellular channels (connexons) between the cytoplasms of the neighbouring cells (reviewed by Kumar & Gilula 1996). Most cells of the normal tissues, except skeletal muscle cells, erythrocytes and circulating lymphocytes, generally communicate via these junctions. Gap junctions are specialised regions of the cell membrane in which each gap junction pore is formed by a juxtaposition of two hemichannels in neighbouring cells. These interact to span the plasma membranes of two adjacent cells joining the cytoplasms of the cells. The hemichannels are composed of connexins, highly related transmembrane proteins consisting of at least 13 members (reviewed by Beyer et al. 1990, reviewed by Saez et al. 1993, reviewed by Goodenough et al. 1996). To date, cadherins and catenins are excluded from the gap junction plaques but cadherin-catenin cell adhesion system may be involved in the formation of gap junctions (Fujimoto et al. 1997). The gap junctions are not only communicating channels but they also promote the fusions of placental cytotrophoblasts (Cronier et al. 1997). In fibroblasts proteins such as connexin 43 affect cell growth independently of gap junction formation (Moorby & Patel 2001). Inhibition of gap junction either with heptan-1-ol treatment or culturing cells at low density has no effect on connexin 43 to control cell growth.

2.2.2.1. Focal adhesions

Most cultured and stationary cells adhere tightly to the underlying growth substratum through distinct regions of their plasma membrane called cell-matrix junctions, known as focal adhesion plaques, focal contacts, or focal adhesions, FAs (Abercrombie et al. 1971, reviewed by Burridge et al. 1988). At these sites, transmembrane receptors, or integrins, interact with extracellular matrix (ECM) proteins e.g. fibronectin, collagens, laminins and vitronectin. On the cytoplasmic side of focal adhesions, integrins together with cytoskeletal proteins link the large bundles of microfilaments, stress fibres, to these structures. Thus, the focal adhesions are a structural connection between the ECM and actin cytoskeleton (Fig. 3). The focal adhesions of the cultured cells are typically 2-10 µm long and 0.25-0.5 µm wide.

Integrin ligation is the first step in the formation of the focal adhesions. Thereafter, integrin makes the linkage with actin cytoskeleton via vinculin, talin, α-actinin, paxillin and p125 focal-adhesion kinase, FAK (reviewed by Yamada & Miyamoto 1995) and other components. Various regulatory proteins, such as calcium-dependent protease; calpain II, protein kinase C, FAK and Src family tyrosine kinases control the assembly of focal adhesion (Beckerle et al. 1987, Jaken et al. 1989, reviewed by Burridge & Chrzanowska-Wodnicka 1996, Kaplan et al. 1994, reviewed by Zamir & Geiger 2001). In many cultured cells, a number of proteins in focal adhesions are highly tyrosine phosphorylated (Burridge et al. 1992), and the formation of focal adhesions can be prevented by inhibitors of tyrosine kinases, such as herbimycin (Burridge et al. 1992) or by serum starvation, or it can be stimulated by inhibition of tyrosine phosphatases with vanadyl hydroperoxide (Barry & Critchley 1994, Chrzanowska-Wodnicka & Burridge 1994, Retta et al. 1996). In living, migrating fibroblasts microtubules and small GTPases of Rho family control the turnover of contact sites (Ridley & Hall 1992, reviewed by Small et al. 1999b).

2.2.2.2. Hemidesmosomes

Hemidesmosomes are multimeric protein complexes that attach epithelial cells to their underlying matrix and serve as cell surface anchorage sites for the keratin cytoskeleton. They are morphologically similar to desmosomes and localised to the basal surface of some epithelial cells (reviewed by Schwartz et al. 1990, reviewed by Garrod 1993). This kind of structures are typically described in epidermis of the skin. In spite of the morphological similarities (cytoplasmic plaques and connection to cytokeratin filaments) with desmosomes, the protein composition is distinct. Hemidesmosomes contain α6β 4 integrin that is important for hemidesmosomal attachment. The plaque protein BP230 (BPAG1, bullous pemphigoid antigen 1) is sequencially related to desmoplakin (Green et al. 1999), and antigen BP180 (BPAG2) is a collagen-like transmembrane protein. Keratin bundles associate to the hemidesmosome plaque via complex of BP230, BP180 and α6β 4 integrin (Hopkinson & Jones 2000).