A Cell-based Assay to Investigate Non-muscle Myosin II Contractility <em>via</em> the Folded-gastrulation Signaling Pathway in <em>Drosophila</em> S2R+ Cells

Abstract

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a Gα12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

TheDrosophila melanogasterRab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function

“…During Drosophila gastrulation, activation of the Fog pathway leads to signaling cascade converging on Rho1 activation and subsequent apical constriction generated by NMII contractility (Parks and Wieschaus, 1991;Costa et al, 1994;Barrett et al, 1997;Peters and Rogers, 2013;Manning and Rogers, 2014;Vasquez et al, 2014;Kerridge et al, 2016;Xie et al, 2016;Jha et al, 2018). This pathway may be recapitulated in Drosophila S2R+ cells; when perfused with exogenous recombinant Fog-Myc (from S2: Fog-Myc cells), S2R+ cells undergo a stereotypical contraction coincident with phase-dark and phase-light ruffles visible by phasecontrast microscopy (Manning et al, 2013;Manning and Rogers, 2014;Peters and Rogers, 2013;Peters et al, 2018) (Figure 5, A and B). We treated S2R+ cells with RNAi targeting RN-tre, Rho, and con-trol, applied concentrated Fog-Myc media or control media, and counted the cells that underwent constriction (Figure 5, A-F).…”

“…This was expressed as a fraction, with the number of phosphomyosin-positive cells over the total number of cells counted. Manning et al, 2013;Peters and Rogers, 2013;Peters et al, 2018. a Lowercase letters prime to the vector, uppercase letters prime to the insertion sequence.…”

“…For a detailed description of Fog media harvesting, see Peters et al (2018). CuSO 4 (1 mM) was added to S2: pMTFog-Myc cells were grown to 100% confluency and incubated at 25°C for 48-72 h. Cells were pelleted at 2734 × g for 10 min at 4°C.…”

“…For a detailed description of the assay protocol, see Peters et al (2018). Cells were treated with appropriate dsRNA for 7 d and induced with 1 mM CuSO 4 on day 5 where appropriate.…”