Alan S. Fanning

PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane

PDZ domains have recently emerged as central organizers of protein complexes at the plasma membrane. Although new information about PDZ domains appears at a bewildering rate, we will attempt to distill a few general concepts that have emerged about their biology. These include the structural basis for specificity of their binding interactions and ideas about how they organize both small local protein complexes used for signal transduction (transducisomes) and larger two-dimensional complexes like cell junctions and plasma membrane domains.

Protein modules as organizers of membrane structure.

Investigations conducted over the past 18 months have shed new light on how modular protein-binding domains, in particular PDZ domains, co-ordinate the assembly of functional plasma membrane domains. Members of the MAGUK (membrane-associated guanylate kinase) protein family, like PSD-95, use multiple domains to cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses, cellular junctions, and polarized membrane domains. Other PDZ proteins, like the Drosophila protein INAD and the epithelial Na(+)/H(+) regulatory factor (NHERF), organize cellular signaling by localizing transmembrane and cytosolic components to specific membrane domains and assembling these components into functional complexes. The organization of these proteins into discreet structures has functional consequences for downstream signaling.

Protein-protein interactions: PDZ domain networks.

PDZ domains can dimerize or bind to the carboxyl termini of unrelated proteins. Crystallographic studies demonstrate the structural basis for these interactions, which contribute to the ability of PDZ domains to create networks associated with the plasma membrane.

ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.

Zonula Occludens-1 and -2 Are Cytosolic Scaffolds That Regulate the Assembly of Cellular Junctions

The integrity of the tight junction barrier in epithelial and endothelial cells is critical to human health, but we still lack a detailed mechanistic knowledge of how the barrier is formed during development or responds to pathological and pharmacological insults. This limits our understanding of barrier dysfunction in disease and slows the development of therapeutic strategies. Recent studies confirm the long‐maintained but previously unsupported view that the zonula occludens (ZO) proteins ZO‐1 and ZO‐2 are critical determinants of barrier formation. However, ZO proteins can also be components of adherens junctions, and recent studies suggest that ZO proteins may also promote the assembly and function of these junctions during epithelial morphogenesis. We review these studies and outline several recent observations that suggest that one role of ZO proteins is to regulate cytoskeletal dynamics at cell junctions. Finally, we propose a model by which the functional activities of ZO proteins in the adherens junction and tight junction are differentiated by a novel regulatory motif known as the U6 or acidic motif.

Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1

Zonula occludens (ZO)‐1 is a member of the MAGUK (membrane‐associated guanylate kinase homologs) family of membrane‐associated signaling molecules that binds directly to both cytosolic and transmembrane components of the tight junction and is believed to organize these proteins within the apical junctional complex. It also binds directly to F‐actin, although the functional relevance of this interaction is unknown. To address this issue, we have used VSVG‐ tagged transgenes to dissect ZO‐1 and have identified a 220 amino acid region of ZO‐1 that is necessary for its association with F‐actin in MDCK cell pull‐down assays. A GST fusion expressing this region can bind directly to F‐actin in vitro, whereas a GFP fusion expressing this domain decorates actin stress fibers when expressed in MDCK cells. These results indicate that this actin‐binding region (ABR) is both necessary and sufficient for binding to F‐actin in vitro and in vivo. VSVG‐tagged transgenes that lack the ABR still accumulate at both early and late cell‐cell contacts in MDCK cells, suggesting that the ABR is not required for tight junction localization. However, accumulation of constructs lacking the ABR is markedly reduced at tight junctions in confluent cells, suggesting that the ABR does play an important role in the localization of ZO‐1 at junctions. Furthermore, the ABR is required for localization to a novel actin‐rich pool of ZO‐1 that accumulates in puncta at the free edge of cells before initiation of cell‐cell contact. We conclude that direct interactions between ZO‐1 and F‐actin play a role in several different steps of junction assembly.

Tricellulin Is a Tight-Junction Protein Necessary for Hearing

The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear.

The structure and regulation of tight junctions.

Tight junctions create a paracellular barrier between both epithelial and endothelial cells. Recent advances have helped define their molecular composition and regulation. Studies in cultured cell lines provide new insights into how assembly and barrier properties may be controlled by signal transduction cascades.

Cortactin is necessary for E-cadherin–mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation.

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

ETOC: Our study reveals that ZO proteins in fully polarized cells regulate the assembly and contractility of the perijunctional actomyosin ring associated with the adherens junction.