Epithelial Cell Polarity From the Outside Looking In

Abstract

Epithelial cell polarity may be regulated by protein sorting in the Golgi and delivery to different membrane domains, a view from the inside looking out. But from the outside looking in, cell adhesion may be required first to establish sites for delivery, retention, and separation of membrane proteins, and delivery of presorted proteins from the Golgi subsequently reinforces and maintains different membrane domains.

In addition to distinctive organizations of membrane proteins, cytoskeleton, and organelles, polarized epithelial cells have a higher-ordered organization involving cell-cell and cell-extracellular matrix contacts that orients cells into a monolayer that separates different biological compartments in the body (Fig. 1A). The cell surface bounded by these contacts (the basolateral domain; Fig. 1A, shown in red) and facing the inside (serosa) of the organism is structurally and functionally distinct from the unbounded surface (the apical domain; Fig. 1A, shown in blue) facing a free space (lumen) that is usually continuous with the outside of the organism (2). The tight junction (zonula occludens), located at the boundary between the apical and basolateral membrane domains (Fig. 1A), acts as a selective permeability barrier to the paracellular space (gate function) and as an intramembranous fence to prevent free mixing of membrane domain-specific proteins and lipids (fence function). Functional differences between these plasma membrane domains are required to regulate vectorial transport of ions and solutes across the epithelium (2)

FIGURE 1.

A: tubular epithelium in which a closed monolayer of epithelial cells surrounds a fluid-filled lumen and ions and solutes are vectorially transported across the epithelium (green arrow) from the apical (blue) to the basolateral (red) plasma membrane domain (absorption). B: cut-through cyst of Madin-Darby canine kidney cells formed in suspension culture, in which cells secrete and accumulate an extracellular matrix (ECM) (laminin, type IV collagen) in the lumen. Under these conditions, a monolayer of polarized cells is formed in which the basolateral membrane (red) faces the ECM and lumen and the apical membrane (blue) faces the growth medium facing the outside of the cyst. Note that this polarity is opposite to that of epithelial monolayers as shown in A due to the experimentally induced orientation of the ECM. C: random distribution of apical (blue) and basolateral (red) membrane proteins in a single epithelial cell in suspension culture (left) and following formation of cell-cell adhesion (right), which generates a bounded surface (red) and unbounded/free surface (blue) that will develop into the basolateral and apical membrane domains, respectively.

How are different distributions of apical and basolateral membrane proteins generated? Consider the observation that fibroblasts have the same capacity as polarized epithelial cells to sort apical and basolateral membrane proteins in the Golgi complex (15), and yet the degree of cell surface organization of plasma membrane domains is considerably less in fibroblasts than in polarized epithelial cells. We can ask, therefore, what biological process distinguishes fibroblasts from polarized epithelial cells such that membrane proteins in the latter organize into different membrane domains? A good candidate is specialized cell-cell adhesion. Thus understanding how different membrane domains are organized in polarized epithelial cells requires knowledge of how cells adhere to each other and how the resulting bounded and unbounded cell surfaces are converted into different membrane domains by localized assembly and targeted delivery of specific proteins (14).

Observations on roles of cell adhesion in organizing polarized epithelial cells

One approach to investigate mechanisms involved in polarized cell organization is to start with a nonpolarized state and observe how polarity is established. Single Madin-Darby canine kidney (MDCK) cells in suspension culture have a random, mixed distribution of “apical” and “basolateral” membrane proteins on the cell surface (Fig. 1B). However, adhesion between cells in suspension is sufficient to cause endogenous apical membrane proteins to localize to the free surface domain facing the culture medium and to cause basolateral proteins to localize to the cell-cell contacting membranes (Fig. 1B; Ref. 13). Interestingly, the tight junction protein zonula occludens-1 is localized all along the cell-cell contacts similar to E-cadherin, although it would be expected at the boundary of the apical (outside surface) and lateral (cell-cell contact) membrane domains (13). However, coincident with the accumulation of an endogenously secreted extracellular matrix (type IV collagen, laminin) in the central luminal space of the cyst, the location of the tight junction becomes restricted to the apicolateral membrane boundary (13). Thus cell-cell adhesion is sufficient to generate a bounded (basolateral domain) and an unbounded/free (apical domain) surface, but the apicobasal axis of polarity is established in these cell aggregates only after an extracellular matrix accumulates on one side of the cells. Further discussion will now focus on the role of cell-cell adhesion and how it leads to development of the basolateral (bounded) membrane domain.

Mechanisms of epithelial cell-cell adhesion

Epithelial cell-cell adhesion is mediated by a variety of membrane proteins, including classic cadherins, claudins/occludin, nectin, and desmosomal cadherins. Classic cadherins are required to initiate cell-cell contacts, and other adhesion protein complexes subsequently assemble specific junctions required for controlling paracellular diffusion (tight junction) and maintaining the structural continuum of the epithelium (desmosomes) (5, 6).

Cadherins are single-membrane-spanning proteins with a divergent extracellular domain of five repeats and a conserved cytoplasmic domain (5). Binding between extracellular domains, which requires Ca²⁺ for protein conformation, is thought to involve multiple cis-dimers of cadherin, which then form trans-oligomers between cadherins on opposing cell surfaces (5). Binding between cadherin extracellular domains is weak, but strong cell-cell adhesion develops during lateral clustering of cadherins by proteins that link cadherin to the actin cytoskeleton (5); β-catenin binds to cadherin cytoplasmic domain and to α-catenin, which is linked directly or indirectly through vinculin/α-actinin to the actin cytoskeleton (Fig. 2). Little is known about how these protein complexes assemble in cells, how the cadherin/catenin complex binds and organizes the actin cytoskeleton, or how other proteins identified at cell-cell contacts [e.g., actin-related protein 2/3, vasodilator-stimulated phosphoprotein (6)] modify cadherin/catenin function and actin organization.

FIGURE 2.

Cadherin cell-cell adhesion proteins (left) are linked through scaffolding proteins α- and β-catenin to the actin cytoskeleton (membrane cytoskeleton) and thereby to additional scaffolding proteins (ankyrin, fodrin) that bind to a different class of membrane proteins.

High-resolution, live cell imaging of normal MDCK epithelial cells showed that functional E-cadherin/green fluorescent protein and associated catenins are immediately recruited to initial cell-cell contacts where they become progressively immobilized into puncta, more of which are added as the contact expands laterally (1). Actin filaments reorganize underneath cell-cell contacts and appear to associate with E-cadherin puncta (1), but it is not known if cytoplasmic actin filaments are captured end-on or at the side or if they are nucleated from cadherin/catenin complexes. Members of the RhoA family of small GTPases control membrane and actin cytoskeleton dynamics during cell-cell adhesion (7).

Steps in converting cell-cell adhesion into formation of a new membrane subdomain

How does cadherin-mediated cell-cell adhesion lead to the formation of a new membrane domain? Two mechanisms will be discussed. First, changes in the actin cytoskeleton at sites of cell-cell adhesion induce formation of the membrane cytoskeleton and the recruitment of a subset of membrane proteins. Second, cell-cell adhesion results in recruitment of specific docking sites for vesicles containing basolateral membrane proteins presorted in the Golgi complex.

Localized assembly of the membrane-associated cytoskele-ton and recruitment of membrane proteins

E-cadherin-mediated cell-cell contact between MDCK cells leads to formation of the (baso-) lateral membrane domain. Some membrane proteins (e.g., Na⁺-K⁺-ATPase) become localized to cell-cell contacts through integration into the actin membrane cytoskeleton, although most proteins must be delivered there in transport vesicles from the Golgi complex or endosomes (14).

Changes in the local organization of the actin cytoskeleton as a consequence of cell-cell adhesion also coincide with the recruitment of actin-associated proteins, which for the membrane-associated cytoskeleton include the scaffolding proteins anykrin and fodrin (spectrin). Fodrin is a long, rod-shaped protein that assembles into a protein scaffold along with actin, protein 4.1, adducin, and other proteins (3). The fodrin scaffold is associated with the cadherin/catenin complex (Fig. 2). Fodrin also binds to ankyrin, which in turn binds with high affinity to integral membrane proteins, including Na⁺-K⁺-ATPase and Cl⁻/HCO₃⁻ exchanger (Fig. 2; Ref. 3). These inter-actions result in localized assembly of the fodrin-based scaffold, which may play a direct role in the early formation of a basolateral membrane domain at sites of cell-cell contact by directing the retention and accumulation of specific proteins that have affinity for the fodrin lattice (Fig. 2; Ref. 14).

Specifying delivery of exocytic transport vesicles to (basolateral membrane) sites of cell-cell adhesion

What mechanisms are involved in the docking and fusion of post-Golgi transport vesicles on their arrival at the plasma membrane at sites of cell-cell contact? Early studies showed involvement of the soluble NSF attachment protein receptor (SNARE) complex, in which binding between cognate vesicle (v-) SNAREs and target (t-) membrane SNAREs specifies vesicle delivery (10) and showed that different t-SNAREs are localized to apical (syntaxin3) and basolateral (syntaxin4) membranes (9). However, several other proteins have been reported to be involved in localized delivery of vesicles (14). In budding yeast, vesicle delivery to the growing plasma membrane of the daughter cell bud involves a large protein complex termed the exocyst-Sec6/8 complex located at the bud tip (Fig. 3; Ref. 11). Genetic disruption of this complex results in the accumulation of vesicles under the tip of the daughter cell plasma membrane. The epithelial Sec6/8 complex has subunit proteins, physical characteristics, and functions similar to those of the yeast exocyst complex (14).

FIGURE 3.

Relative distributions of the Sec6/8 complex and t-SNAREs on lateral membrane domain of polarized epithelial cells (left) and at the tip of the daughter cell bud in budding yeast. In both cases, the distribution and activity of the Sec6/8 complex is directly associated with localized plasma membrane growth.

During E-cadherin-mediated cell-cell adhesion in MDCK cells, Sec6/8 complex is recruited rapidly (t_1/2 ~ 6 h), efficiently (>80%), and specifically to sites of cell-cell contact. As cells develop polarity, Sec6/8 complex gradually localizes to the apex of the lateral membrane (Fig. 3). Mechanisms involved in specifying recruitment of Sec6/8 complex to sites of cell-cell adhesion (in polarized epithelial cells) are also unknown. Addition of Sec6/8 antibodies to permeabilized MDCK cells inhibited delivery of a basolateral but not an apical membrane protein, strongly indicating that, as in budding yeast, Sec6/8 complex regulates delivery of a subset (in the case of epithelial cells, basolateral membrane proteins) of vesicles to a localized site on the plasma membrane (Fig. 3; Ref. 4).

It is unknown how Sec6/8 complex regulates vesicle delivery to the plasma membrane: does it play indirect roles by concentrating transport vesicles before docking or as a pre-docking receptor for t-SNAREs, or does it play a more direct role with t-SNAREs in the process of vesicle fusion with the plasma membrane? However, it should be noted that recruitment of Sec6/8 complex to the forming basolateral membrane domain coincides with increased efficiency of basolateral transport vesicle delivery to the plasma membrane and that on cell-cell adhesion in MDCK cells the lateral membrane, but neither the apical nor basal membrane, increases about sixfold in surface area, presumably as a result of increased vesicle, and hence membrane, delivery (Fig. 3; Ref. 12).

Sorting and delivery of proteins to the basolateral membrane domain

How are basolateral membrane proteins presorted for specific delivery to the forming basolateral membrane domain? Sorting of exocytic membrane proteins occurs predominantly in the trans-Golgi network in both polarized epithelia and “nonpolarized” fibroblasts (15) and is mediated by well-defined sorting signals that are recognized by less-well-understood sorting machinery (8, 9). Basolateral sorting signals frequently contain a critical tyrosine residue within the amino acid sequence NPXY or YXXØ (where X is any amino acid and Ø is an amino acid with a bulky hydrophobic group) that are predicted to form a structure known as a “tight β-turn.” In some cases, a dihydrophobic motif serves to target the protein basolaterally. In addition, a growing number of basolateral sorting signals bear little or no sequence similarity to these motifs. Both YXXØ- and dihydrophobic-based sorting signals selectively bind the medium (μ) chains of clathrin adaptor protein complexes AP-1 and AP-2, and dihydrophobic signals also bind β-chains (8, 9). Presumably, interacting basolateral membrane proteins are selectively clustered into vesicles by adaptor/clathrin coats.

Summary

At a minimum, two biological processes are required so that epithelial cells develop and maintain a structural and functional basolateral membrane domain: cell-cell adhesion and protein sorting in the exocytic pathway (Fig. 4). Cell-cell adhesion is required to establish a “landmark” on the cell surface, which defines a domain of the plasma membrane that is different from the rest of the plasma membrane not in contact with another cell. Cell adhesion causes a reorganization of the actin (and microtubule) cytoskeleton that leads to assembly of the actin-based membrane cytoskeleton, which can restrict a small class of membrane proteins to the site of cell-cell adhesion (and the forming basolateral membrane domains), and a targeting patch containing Sec6/8 complex, which specifies the delivery of a subset of transport vesicles containing pre-sorted basolateral membrane proteins (Fig. 4). Presorting of basolateral membrane proteins occurs in the trans-Golgi network and is mediated by intrinsic sorting signals in the cytoplasmic domain that are recognized by the clathrin-adaptor complex and clustered into transport vesicles for delivery along microtubules, and possibly actin filaments, to the targeting patch assembled at cell-cell contacts. Of these processes, cell-cell adhesion appears to be required, and in its absence, as in fibroblasts, sorting of proteins in the Golgi complex is not sufficient to generate polarized distributions of membrane proteins.

FIGURE 4.

The “outside-looking-in model” for how cell-cell adhesion leads to activation of cadherin and a signaling complex, the assembly of the membrane-cytoskeleton, and a targeting patch at sites of cell-cell adhesion. Subsequently, vesicle-containing basolateral membrane proteins that have been presorted in the Golgi complex are delivered to the targeting patch.