Supplementary Materials Supplemental Materials (PDF) JCB_201609095_sm. We conclude that for effective collective migration, the RHOA-GEFs RHOA/C actomyosin pathways should be optimally tuned to bargain between era of motility pushes and limitation of intercellular conversation. Launch Collective cell migration consists of intercellular mechanical conversation through adhesive connections (Tambe et al., 2011; Weber et al., 2012; Zaritsky et al., 2015). In migrating monolayers, such conversation is set up by cells on the monolayer boundary and steadily sent to cells behind the group (Ng et al., 2012; Serra-Picamal et al., 2012; Zaritsky et al., 2014, 2015; Ladoux et al., 2016; Etienne-Manneville and Mayor, 2016). Effective cellCcell conversation requires well balanced control of contractility and cellCcell and cellCmatrix adhesions (Hidalgo-Carcedo et al., 2011; Weber et al., 2012; Cai et al., 2014; Bazellires et al., 2015; Das et al., 2015; Hayer et al., 2016; Notbohm et al., 2016; Plutoni et al., 2016). Coordination between these procedures is governed, among many pathways, by signaling actions from the Rho-family GTPases (Wang et al., 2010; Hidalgo-Carcedo et al., 2011; Timpson et al., 2011; Hall and Omelchenko, 2012; Cai et al., 2014; Omelchenko et al., 2014; Reffay et al., 2014; Plutoni et al., 2016). Rho-family GTPases are and temporally modulated by complicated systems of upstream regulators spatially, OTX008 including 81 activating guanine nucleotide exchange factors (GEFs), 67 deactivating GTPase-activating proteins, and 3 guanine dissociation inhibitors (Jaffe and Hall, 2005; Omelchenko and Hall, 2012). The networks are composed of many-to-one BMP3 and one-to-many interaction motifs; that is, individual GTPases are OTX008 regulated by multiple GEFs, and one GEF often acts upon multiple GTPases. Moreover, some GEFs are effectors of GTPases, leading to nested feedback and feedforward interactions (Schmidt and Hall, 2002; Jaffe and Hall, 2005; Cherfils and Zeghouf, 2013; Hodge and Ridley, 2016). Such pathway design permits an enormous functional specialization of transient signaling events, at specific subcellular locations and with precise kinetics. Our long-term goal is to disentangle these signaling cascades in the context of collective cell migration. Although the roles of GEFs and their interactions with Rho GTPases are widely studied for single-cell migration (Goicoechea et al., 2014; Pascual-Vargas et al., 2017), less is known about how they regulate collective migration (Hidalgo-Carcedo et al., 2011; Omelchenko et al., 2014; Plutoni et al., 2016). Here, we report a comprehensive and validated, image-based GEF screen that identified differential roles of GEFs. By design of quantitative measures that encode the collective dynamics in space and time, we were able to identify a surprising role of RHOA, RHOC, and a group of four upstream GEFs in modulating collective migration via efficient long-range communication. Results and discussion Quantification of monolayer cell migration in space and time Collective cell migration emerges from the individual motility of cells in an interacting group: an action of one cell affects its neighbor and can propagate over time to eventually coordinate distant cells (Zaritsky et al., 2015). To identify molecules implicated in this mechanism, we performed live-cell imaging of the wound-healing response of human bronchial epithelial cells from the 16HBE14o (16HBE) line (Fig. 1 A and Video 1). Cells formed apical junctions and maintained epithelial markers and group cohesiveness before scratching the monolayer, as assessed by the localization of E-cadherin and the tight-junction protein ZO1 at the lateral cellCcell contact areas (Fig. 1 B). Upon scratching, the monolayer transitioned over 2 h from OTX008 a nonmotile phase to an acceleration phase to steady-state wound closure (Fig. 1 C). The acceleration phase was associated with a gradual transition of cells from unorganized local movements to a faster and more organized motility. Cells at the wound edge underwent this changeover 1st, accompanied by a influx of coordinated motility propagating from the wound advantage (Fig. 1 A, insets). The propagation can be regarded as driven by mechanised cellCcell conversation (Matsubayashi et al., 2011; Ng et al., 2012; Serra-Picamal et al., 2012; Zaritsky et al., 2014, 2015; Notbohm et al., 2016). Open up in another window Shape 1. Ramifications of GTPase knockdown on OTX008 collective cell migration. (A) Exemplory case of a wound-healing test. (insets) Speed vectors displaying that front side cells commence to migrate before deeper cells. Pub, 100 m. (B) Immunofluorescent staining of E-cadherin and ZO1 before scratching the monolayer. Pub, 20 m. (C) Monolayer advantage advancement over 500 min. (inset best) Edge advancement during the 1st 125 min. (inset bottom level) Boost of wound-healing price during the 1st 125 min. (D) Building of the kymograph of the wound-healing test: mean acceleration of cells at different ranges.