Opposing Roles of Zyxin/LPP ACTA Repeats and the LIM Domain Region in Cell-Cell Adhesion

Abstract

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cellcell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherinactin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cellcell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.Cadherin-mediated cell-cell adhesion integrates individual cells into tissues (1). Strong cell-cell adhesion is required to maintain cell morphology and tissue homeostasis (2). As individual cadherin-cadherin adhesion interactions are weak (3), strong adhesion is thought to require incorporation of cadherin into actin networks (4, 5). The cytoplasmic domain of cadherin binds either ␤-catenin or plakoglobin (6), which in turn binds ␣-catenin (7, 8). ␣-Catenin links cadherin to actin filaments either directly (8) or by binding the actin-binding proteins vinculin (9) or ␣-actinin (10). Cadherin anchors into detergent-insoluble actin networks concomitant with incorporation of ␣-catenin into cadherin complexes (4, 5). However, it remains unclear how binding of cadherin to actin filaments is regulated to coincide with cell-cell adhesion. Although cadherin has been demonstrated to induce dramatic changes in actin organization (11-13) and several actin regulatory programs have been generally implicated in cell-cell adhesion (13, 14), how actin rearrangements and cadherin engagement are coordinated remains poorly understood.Understanding actin assembly mechanisms has been greatly improved by studies of the intracellular pathogen, Listeria monocytogenes (15). Listeria ActA protein hijacks host actin polymerization machinery, directing assembly of actin comet tails that propel the bacterium through the cytosol (16,17). In addition to actin nucleation by the Arp2/3 complex, normal motility requires the activity of VASP family members (18) that are recruited via an array of proline-rich motifs termed ActA repeats (19). It has been proposed that VASP competes with capping protein (CapZ) for available barbed ends of actin filaments, protecting the...

α-Actinin links LPP, but not zyxin, to cadherin-based junctions

“…In both proteins, the LIM domains are separated from the FPPPPP motifs by a central region of over 250 amino acids. In a previous report, zyxin or LPP lacking a single one of these shared domains retained the ability to localize to cell-cell contacts [12]. However, zyxin and LPP are thought to act as scaffold proteins, binding multiple proteins simultaneously and complicating interpretation of how targeting to cellular sites might occur.…”

“…This was particularly important given the observation that zyxin may harbor additional cell-cell junction localization information that is not found in LPP. Zyxin or LPP mutants lacking LIM domains drive accelerated cell-cell junction formation and strengthening [12]. We reasoned that deletion of cell-cell junction targeting information from these mutants would diminish their activity.…”

“…Aggregation assays, as well as their statistical analysis, were performed as described [12]. Briefly, cells were grown in suspension at 2.5×10 5 cells per ml in 20μl drops.…”

“…FPPPPP peptides displace VASP from cell-cell contacts [7], demonstrating that FPPPPP motif containing proteins, such as zyxin and LPP, are required to localize VASP to cell-cell contacts. Zyxin/LPP mutants lacking LIM domains can either accelerate or reduce cell-cell adhesion, an effect that is determined by the presence of intact FPPPPP motifs [12]. Thus, zyxin and LPP likely alter cell-cell adhesion by binding VASP.…”

“…They are recruited to specific cellular sites through interactions with FPPPPP motifs [9]. The focal adhesion protein zyxin and its relative LPP contain arrays of FPPPPP motifs that bind VASP [10][11] and colocalize with VASP at epithelial cell-cell junctions [7,12]. FPPPPP peptides displace VASP from cell-cell contacts [7], demonstrating that FPPPPP motif containing proteins, such as zyxin and LPP, are required to localize VASP to cell-cell contacts.…”