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(2010). as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ Etidronate (Didronel) complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cellCcell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. Introduction Cell-to-cell adhesion is one of the hallmarks of the epithelium, which is found disrupted in many cancers during malignant transformation (Niessen et al., 2011). The connection between adherens junction (AJ) complexes and the actin cytoskeleton has long been appreciated, but how actin is assembled and regulated during de novo cellCcell contact formation under physiological conditions such as 3D environments is not well understood (Weis and Nelson, 2006; Ratheesh and Yap, 2012). Actin remodeling is essential not Etidronate (Didronel) only for AJ formation and expansion, but also during junctional maintenance when actin turnover at cellCcell contacts reaches a steady state (Ivanov et al., 2005; Cavey and Lecuit, 2009). Thus, actin nucleation and polymerization appear highly specialized and dynamically regulated at the AJ. The Arp2/3 complex is involved in the regulation of junctional actin polymerization (Kovacs et al., 2011; Tang and Brieher, 2012); however, evidence for a role of formins, the largest group of actin nucleators, remains poorly understood (Michael and Yap, 2013). Formins are multidomain proteins, controlled through intramolecular interaction of their C-terminal, Diaphanous autoregulatory domain (DAD) to the N terminus (NT). The current model of activation involves binding of active RhoGTPases to Etidronate (Didronel) the RhoGTP binding domain (GBD), thereby releasing autoinhibition (Baarlink et al., 2010; Breitsprecher and Goode, 2013). Formin-1, which lacks an apparent GBD, was shown to modulate AJs in mouse keratinocytes (Kobielak et al., 2004; Campellone and Welch, 2010), whereas RhoA/Dia1/myosin II activity was proposed to strengthen AJs in a monolayer cell culture (Carramusa et al., 2007). However, a role for formins-mediated actin dynamics in the de novo formation of AJs in human epithelial cells in real time has not been addressed. Moreover, how experimental approaches to studying monolayer cells grown on rigid surfaces can be translated into more physiological 3D environments remains unclear (Baker and Chen, 2012; Kutys et al., 2013). A suitable model system is human MCF10A breast Etidronate (Didronel) epithelial cells cultured in 3D Matrigel, which resembles components of a basement membrane (Debnath and Brugge, 2005). Here we report that formin-like 2 (FMNL2) controls junctional actin assembly and turnover during initial AJ formation as well as epithelialization in 3D environments. FMNL2 associates with components of the AJ complex in a regulated fashion in which Rac1 promotes rapid and dynamic localization of FMNL2 and subsequent actin assembly at newly forming cellCcell contacts. Results and discussion FMNL2 localizes to newly formed cellCcell contacts MCF10A cells develop into a two-cell stage within the first 24 h when seeded into Matrigel. Within 2 wk they grow into larger spheroids, and lumen formation occurs via apoptosis of the inner cells (Debnath and Brugge, 2005; Fig. 1 A). Open in a separate window Figure 1. FMNL2 localizes to AJs in a 3D model for nascent cellCcell adhesion formation. (A) Confocal images of MCF10A cells in 3D stained for F-actin after 1, 4, or 14 d. (B) 3D reconstructions of MCF10A cells expressing LifeAct-mCherry and E-CadherinCGFP during cellCcell contact formation. Merged images show Rabbit Polyclonal to OR4A15 magnification (bar, 2 m). (C) MCF10A cells grown in Matrigel and labeled as indicated. The asterisk marks the junctional area. (D) Expression of formins in MCF10A cells assessed by qPCR. (E) MCF10A cells grown in Matrigel were labeled as indicated. FMNL2 localizes to the AJ (asterisk). (F) MCF10A cells expressing FMNL2-GFP were labeled for E-Cadherin. (G) 3D reconstructions of a time series of MCF10A cells expressing LifeAct-mCherry and FMNL2-GFP. Merged images magnify the AJ area (bar, 2 m). (H) Representative images of MCF10A cells expressing GFP or FMNL2DAD-GFP stained for F-actin. Arrows illustrate line scans used for quantifications. (I) Corresponding line scan profiles to H. (J) Quantification of F-actin line scan profiles (GFP, = 47; FMNL2DAD-GFP, = 66). *, P 0.05. Error bars indicate SEM. To analyze de novo junctional actin formation, we aimed at visualizing actin.