Journal of Cell Science RESEARCH ARTICLE CLIC4 regulates cell adhesion and b1 integrin trafficking Elisabetta Argenzio ` , Coert Margadant*, Daniela Leyton-Puig, Hans Janssen, Kees Jalink, Arnoud Sonnenberg and Wouter H. Moolenaar ` ABSTRACT Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic–endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell–matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to b1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of b1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of b1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA- dependent integrin trafficking. KEY WORDS: CLIC4, Cell adhesion, Integrin trafficking, Lysophosphatidic acid, Rab35 INTRODUCTION The chloride intracellular channel (CLIC) protein family consists of six members (CLIC1–CLIC6) that are structurally related to the omega class of glutathione S-transferases (GSTs), but appear to have distinct cellular functions (Dulhunty et al., 2001; Harrop et al., 2001; Jiang et al., 2014; Littler et al., 2005; Littler et al., 2010). CLICs are globular proteins that are highly conserved among vertebrates and exist in both soluble and membrane- associated forms. They have been implicated in membrane remodeling, intracellular trafficking, vacuole formation, actin reorganization, ion transport and other processes (for a review, see Jiang et al., 2014; Littler et al., 2010). Gene targeting studies in mice have begun to reveal essential, non-redundant physiological roles of the CLIC proteins, including in platelet and macrophage function (Jiang et al., 2012; Qiu et al., 2010), hearing (Gagnon et al., 2006), angiogenesis and wound healing (Padmakumar et al., 2012; Ulmasov et al., 2009). In Caenorhabditis elegans, disruption of the CLIC homolog EXC-4 dramatically impairs formation and maintenance of the intracellular excretory tube, but vertebrate CLICs cannot rescue the phenotype, indicating that vertebrate and invertebrate CLICs have distinct functions (Berry et al., 2003; Berry and Hobert, 2006). Among the six mammalian CLIC proteins, CLIC4 is the best- studied family member, yet its precise function(s) and regulation are still largely unknown. CLIC4 is ubiquitously expressed and detected in the cytosol as a diffusible protein, but it can also localize to intracellular vesicles, organelles and actin-based structures (Berryman and Goldenring, 2003; Chuang et al., 1999; Chuang et al., 2010) and even to the nucleus (Shukla et al., 2009; Suh et al., 2007). Growing evidence points to a role for CLIC4 in such diverse processes as angiogenesis, differentiation, migration and wound healing. Thus, Clic4 2/2 mice display defective angiogenesis and vacuolization (Ulmasov et al., 2009) as well as spontaneous skin erosions and impaired wound healing, apparently due to defects in cell adhesion (Padmakumar et al., 2012). However, the underlying mechanism is unknown. Cell adhesion is mediated by integrins, heterodimeric transmembrane receptors that link the extracellular matrix to the cytoskeleton. Integrins undergo endocytic–exocytic trafficking, both constitutively and in a stimulus-dependent manner. Integrin endocytic recycling is key to the regulation of cell adhesion, spreading and migration (Bridgewater et al., 2012; Caswell et al., 2009; Margadant et al., 2011; Pellinen and Ivaska, 2006). We previously identified CLIC4 as a new player in a signaling pathway initiated by G-protein-coupled receptor agonists, in particular the lipid mediator lysophosphatidic acid (LPA) (Ponsioen et al., 2009). LPA is a major serum constituent (Eichholtz et al., 1993) and many cellular responses to serum are in fact attributable to LPA receptor stimulation (Moolenaar et al., 2004). Using N1E-115 neuroblastoma cells as a model, we showed that CLIC4 undergoes rapid but transient translocation from the cytosol to distinct regions of the plasma membrane upon LPA or serum stimulation, in a manner strictly dependent on RhoA activity and F-actin integrity (Ponsioen et al., 2009). This observation raises the possibility that CLIC4 functions in a regulated exocytic–endocytic trafficking route; however, very little is known about the rapid agonist-induced recruitment of CLIC4 to the plasma membrane. In the present study, we set out to examine the function and LPA-induced redistribution of CLIC4 in HeLa and MDA-MB- 231 carcinoma cells, focusing on cell adhesion and integrin behavior. We find that CLIC4 is required for integrin-mediated cell adhesion, and that LPA stimulates the recruitment of CLIC4 to b1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 stimulates both the internalization and serum/ LPA-induced recycling of b1 integrin. Furthermore, CLIC4 suppresses Rab35 activity, and antagonizes Rab35-dependent regulation of integrin trafficking. We conclude that CLIC4 is a new player in the agonist-regulated trafficking of b1 integrin. Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands. *Present address: Department of Molecular Cell Biology, Sanquin Research, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands. ` Authors for correspondence (e.argenzio@nki.nl; w.moolenaar@nki.nl) Received 24 January 2014; Accepted 13 October 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5189–5203 doi:10.1242/jcs.150623 5189