# Context-dependent interactions among afadin, ZO-1, and actin filaments

## TL;DR

The study explores how afadin and ZO-1 interact with actin filaments in different cell types, revealing context-dependent regulation of these interactions.

## Contribution

The paper identifies JAM-C as a ZO-1-binding membrane protein and shows how cellular contexts regulate interactions among afadin, ZO-1, and actin.

## Key findings

- Afadin and ZO-1 independently colocalize with actin filaments in EL-derived cells.
- Nectin-2 is found in afadin aggregates but not in ZO-1 aggregates, suggesting a ZO-1-binding membrane protein.
- JAM-C is identified as the membrane protein that binds to ZO-1.

## Abstract

Afadin and ZO-1 are actin-binding scaffold proteins localized at cell-cell junctions. Although these proteins contain multiple protein-binding motifs for various junctional proteins, their binding partners within cells are strictly regulated. Here, we investigated the mutual interactions among afadin, ZO-1, and actin filaments using cells lacking cellular junctions derived from EL and F9 non-epithelial cells. In EL-derived cells, afadin and ZO-1 independently colocalized with various types of actin filaments. In F9-derived cells, afadin and ZO-1 colocalized as aggregates. Gene disruption analyses revealed that afadin and ZO-1 independently form aggregates in the absence of cadherin-catenin complex. Nectin-2, an afadin-binding membrane protein, was detected in afadin aggregates but not in ZO-1 aggregates, suggesting the existence of a membrane protein that binds to ZO-1. We identified this protein as JAM-C. A comparison between α-catenin-deficient and β-catenin-deficient F9 cells suggested that the extracellular domain of E-cadherin interferes with afadin and ZO-1 aggregate formation. Furthermore, gene disruption of nectin-2 suggested that JAM-C-bound ZO-1, rather than unbound ZO-1, preferentially interacts with afadin. Together, these findings indicate that interactions among afadin, ZO-1, and actin filaments are strictly regulated by various cellular contexts.