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Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1¹

ALAN S. FANNING^*2, THOMAS Y. MA and JAMES MELVIN ANDERSON^*

^* Department of Cell and Molecular Physiology, The University of North Carolina at Chapel Hill, North Carolina, USA; and
Department of Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA

²Correspondence: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, CB# 7545, Medical Sciences Research Bldg., Chapel Hill, NC 27599-7545, USA. E-mail: alan_fanning{at}med.unc.edu

SPECIFIC AIMS

The assembly and regulation of the tight junction in epithelial cells is believed to be regulated by cytosolic proteins such as ZO-1, a member of the membrane-associated guanylate kinase homologue (MAGUK) family of signaling and scaffolding proteins. Like other MAGUKs, ZO-1 interacts with transmembrane components of the tight junction as well as the actin cytoskeleton. The aim of this study was to determine the structural basis for the interaction between ZO-1 and F-actin and to examine how it might be involved in the assembly of tight junctions in epithelial cells.

PRINCIPAL FINDINGS

1. F-actin binds directly to a 220 residue region between amino acids (aa) 1151–1371 of ZO-1
Using epitope-tagged ZO-1 transgenes with successive carboxyl-terminal truncations, we identified a 220 aa between residues 1151–1371 that was required for cosedimentation of ZO-1 with F-actin in a crude cell lysate pull-down assay. We designated this segment as the actin binding region (ABR). Using standard cosedimentation assays, a purified GST fusion protein encoding the 220 aa ABR was demonstrated to bind directly to F-actin in vitro. The GST-ABR fusion bound saturably to F-actin in vitro (Fig. 1 A) and could be competed off of F-actin with a maltose binding protein fusion with the ABR, further confirming the specificity of the interaction of the ABR with ZO-1.

View larger version (29K): [in this window] [in a new window]   Figure 1. GST-ABR binds saturably and specifically to F-actin. A) Saturation binding curve with GST-ABR. Increasing concentrations of the GST-ABR fusion protein were mixed with F-actin, sedimented, and analyzed by Western blot with an antisera against the VSVG epitope tag. B) Densitometric plot of the data in panel A illustrating that the binding of GST-ABR clearly saturates above 75 µM. C) Competition binding assay. The GST-ABR fusion protein (10 µM) was mixed with F-actin (5.5 µM) and increasing concentrations of a maltose binding protein fusion encoding the ABR (MBP-ABR). After sedimentation, the pellet fractions were resolved by SDS-PAGE and analyzed by Western blot with an antisera against a carboxyl-terminal VSV-G tag. The relative positions of GST-ABR (arrowhead) and MBP-ABR (asterisk) are indicated at left. Note that increasing concentrations of the MBP-ABR fusion result in displacement of the GST-ABR fusion from F-actin.

2. The 220 aa ABR interacts with F-actin in vivo
To examine the role of the ABR in vivo, we generated green and yellow fluorescent proteins (GFP and YFP, respectively) that encoded the ABR and the amino- and carboxyl-terminal halves of ZO-1 and introduced these constructs into cultured MDCK cells by transfection. We observed that the carboxyl-terminal half of ZO-1 (GFP ZC), which encodes aa 1033–1736, and the smaller construct encoding amino acids 1151–1371 (GFP ABR) both colocalize with F-actin stress fibers and other filamentous actin structures in transfected cells. In contrast, the amino-terminal half (aa 1–876) localizes at the tight junction. Taken with the findings described above, these results indicate that the ABR is necessary and sufficient for interaction with F-actin both in vitro and in vivo.

3. The ABR promotes localization to the tight junction
To determine whether interactions with F-actin are required for tight junction localization of ZO-1, we examined the subcellular distribution of epitope-tagged carboxyl-terminal deletions in MDCK cells (Fig. 2 ). We found that deletion constructs that lacked the ABR (z1151) were still localized to the tight junction, similar to constructs encoding the full-length molecule (ZO1myc) or a carboxyl-terminal deletion that still contained the ABR (z1371). However, there were distinct qualitative and quantitative differences in the localization of constructs like z1151, which lacked the ABR. We observed large gaps in the circumferential pattern of junctional staining normally observed with the endogenous protein or with constructs that contain the ABR. There was also an increased amount of cytosolic staining of z1151 relative to other transgene products. These observations suggest that the ABR, although not necessarily required for junction localization, enhances the efficiency of localization or the retention and stabilization of ZO-1 at the tight junction.

View larger version (87K): [in this window] [in a new window]   Figure 2. The actin binding region (ABR) promotes efficiency and accuracy of localization to the tight junction. Stably transfected MDCK lines were plated at confluent density, cultured for 10 days, and treated with sodium butyrate for 20 h to induce transgene expression. Cells were then fixed and stained with antiserum against the VSVG epitope tag and an antiserum against the endogenous ZO-1 (R40.76) that does not recognize the VSVG-tagged human ZO-1 transgene. All three constructs colocalize at the tight junction with the endogenous ZO-1. However, the transgene lacking the ABR, z1151, is often undetectable at some junctions in expressing cells. This is especially evident where expressing cells contact nonexpressing cells (arrows). Bar = 10 µm.

4. The ABR is required for localization of ZO-1 to a novel pool of ZO-1 at the free edge of cell monolayers
ZO-1 has been documented within several distinct pools as the assembly of junctions progresses from initial spot-like filopodial contacts to the circumferential junction complex characteristic of polarized epithelial cells. To determine whether direct interactions with F-actin are required for the assembly of ZO-1 into these different pools, we examined the subcellular distribution of the epitope-tagged ZO-1 transgenes in scrape-wounded cell monolayers and subconfluent islands of MDCK epithelial cells. We observed that ZO-1 was not required for the assembly of ZO-1 into the early cadherin-based filopodial cell–cell contacts. However, it was required for assembly into a series of periodically spaced spots along the free cell border in which ZO-1 has not been described. This pool appears as spots spaced at intervals of 2 µm along the free cell border. It colocalizes with a pool of F-actin that is associated with, but distinct from, the circumferential stress fibers present at the free edge of cell monolayers. These observations suggest that interactions with F-actin may play a direct role in the localization of ZO-1 to other cellular structures.

CONCLUSIONS

The actin cytoskeleton has long been recognized as a possible regulator of tight junction assembly and permeability. Electron microscopy has indicated that F-actin is intimately associated with tight junctions; more recent studies have indicated that many cytosolic components of the tight junction are either directly or indirectly associated with F-actin. Furthermore, pathological and pharmacological agents that disrupt cytoskeletal structure can have a dramatic effect on junction structure and function. However, the molecular mechanisms by which the cytoskeleton might regulate tight junctions are poorly understood.

We have proposed that the tight junction protein ZO-1 is a key link between the cytoskeleton and components of the paracellular seal. ZO-1 is a member of a large family of membrane-associated signaling molecules, many of which can act as molecular scaffolds that organize transmembrane proteins into discrete plasma membrane domains, like epithelial cell–cell junctions and neuronal synapses. We hypothesized that ZO-1 organizes transmembrane proteins of the tight junction like claudin or occludin into a single complex and that direct interactions between ZO-1 and F-actin either promote localization and/or stabilization of this complex within the cortical cytoskeleton of the lateral plasma membrane.

In the present study, we have identified the F-actin binding site within the unique carboxyl terminus of ZO-1 and demonstrated that this 220 aa region is both necessary and sufficient for interaction with F-actin in vitro and in vivo. Although this domain is not necessary for localization to the tight junction, it does promote the localization or stabilization of ZO-1 within cell–cell contacts. That it is not necessary in our assays is not surprising, since ZO-1 has multiple binding sites for other tight junction proteins that could recruit it to tight junctions. In fact, we had previously demonstrated that sequences in the amino-terminal half of the protein are sufficient to localize ZO-1 to the tight junction. However, the observation that epitope-tagged transgene products lacking the ABR have a significantly altered distribution at the tight junction suggests that F-actin binding is important for the proper assembly of ZO-1 into the tight junction (see Fig. 3 ).

View larger version (19K): [in this window] [in a new window]   Figure 3. Direct interactions between ZO-1 and F-actin promote the localization of ZO-1 to the tight junction and actin-rich puncta at the free edge of wounded cells. F-actin (dotted lines) is distributed throughout epithelial cells and organized into distinct structures such as the perijunctional actomyosin ring (PAMR) at the apical aspect of polarized cells or the circumferential stress fibers that line the free edge of a wounded monolayer. Actin filaments are also intimately associated with the cell–cell contact sites at the tight junction. ZO-1 is produced in the cytosol (1); localization of ZO-1 to puncta at the free edge of cell monolayers (2) requires direct interaction with F-actin. Localization of ZO-1 to the tight junction (3) is enhanced by direct actin binding and does not necessarily require this interaction. It is possible (?) that the pool of ZO-1 at the free edge serves as a precursor to junction assembly.

The actin binding region is required for localization of ZO-1 to a previously unappreciated pool of ZO-1 and F-actin observed at the free edge of wounded or subconfluent monolayers of MDCK cells. The functional relevance of this pool is unclear and is under investigation. Both the ZO-1 and F-actin in this pool have a punctuate distribution and appear to be closely associated with circumferential ring of stress fibers that characteristically line the free edge of wounded cell monolayers. One hypothesis under investigation is that this is a precursor pool of junction components placed near the free edge in preparation for assembly into nascent cell–cell contacts. This might position ZO-1 close to sites of initial actin-rich filopodial cell–cell contacts that form when opposing cells are brought together by the closing wound margin. Alternatively, this pool may reflect a previously unappreciated step in the recruitment of tight junction proteins into the apical junctional complex (see Fig. 3 ). Regardless, it suggests that direct interactions between ZO-1 may have several distinct roles during assembly of cell–cell contacts.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0121fje; to cite this article, use FASEB J. (September 19, 2002) 10.1096/fj.02-0121fje

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