Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.
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.
Studies of embryogenesis conducted with genetic model organisms have proved invaluable to our understanding of how cells are assembled into tissues. Studies of Drosophila melanogaster, in particular, have led to the identification of key genes and biological principles that direct the morphogenesis and development of organisms from fertilization to adulthood1,2,3. In parallel to the genetic research conducted with Drosophila, cultured cell lines derived from fruit fly tissues have also emerged as a powerful system to address a wide range of molecular and cell biological questions4,5,6. Drosophila tissue culture cells have minimal requirements for maintenance, as they are cultured at room temperature and without CO2. As such, they are amenable to high-resolution imaging, and they have a high degree of susceptibility to gene inhibition by RNAi6,7. Many groups have used Drosophila tissue culture cells as a tool to discover genes involved in defining cell shape, cytoskeletal dynamics, viability, phagocytosis, and signal transduction pathways4,8,9,10,11. When employed as a model alongside the whole animal, cultured Drosophila cell lines offer a set of very complementary approaches that accelerate the identification of molecules important for the development and provide a framework in which to determine their mechanistic roles12. Here, we describe a cell-based assay to study the signaling pathways that trigger apical constriction via the folded-gastrulation (Fog) pathway13,14. This cell-based contractility assay allows researchers to investigate both the Fog signaling pathway and the molecular mechanisms that regulate non-muscle myosin II contractility.
Gastrulation of the early Drosophila embryo has been studied for many years as a genetic model for the epithelial morphogenesis and the cellular transition from epithelial to mesenchymal cell identity. One of the key events of gastrulation is the morphogenesis of a subset of epithelial cells along the embryonic ventral midline from columnar to pyramidal in shape15,16,17,18. This simple cell shape change results in the internalization of the presumptive mesodermal cells and is driven by the motor activity of non-muscle myosin II constricting the actin network16,19,20. Decades of genetic research has identified the molecular components of this pathway and has sequentially placed them in the following order: 1) Fog is secreted from the apical domain of the epithelial cells at the ventral midline; 2) Fog binds to the G-protein-coupled co-receptors, Mist and Smog, and signals through a heterotrimeric G-protein complex containing the Gα12/13 subunit Concertina (Cta), which is chaperoned by the non-canonical GEF Ric-8; 3) Cta activates a guanine nucleotide exchange factor, RhoGEF2, which in turn activates the small G-protein Rho1; 4) Rho1 activates Rho kinase (Rok); 5) Rok activates non-muscle myosin II contractility at the apical domain through phosphorylation of the regulatory light chain (RLC), thus producing apical constriction (Figure 1)15,21,22,23,24,25,26,27,28,29. Mutations in several of these components interfere with the normal gastrulation and other morphogenetic movements, including the formation of the wing disc and salivary glands, indicating that this pathway is used at several stages of Drosophila embryogenesis30,31,32. The Fog pathway is one of the best-studied models for epithelial shaping and has provided important insights into how tissue-level morphogenesis is regulated from gene transcription to cytoskeleton-driven cell movements14,15,21.
We developed a cell-based contractility assay that recapitulates many of the cellular responses downstream of Fog that have been observed in developing fly embryos17. We engineered a stable S2 cell line that expresses Fog tagged at its C-terminus with myc under the control of an inducible metallothionine promoter that can be harvested upon the addition of copper sulfate (CuSO4) to the medium. When Fog-conditioned media is applied ectopically to S2 Receptors+ (S2R+) cells, which are a subline of S2 cells distinguished by their differential expression of receptors such as Frizzled and integrin subunits, the cells undergo a reorganization of the cytoskeleton highly reminiscent of apical constriction12,17,27,33. These changes can be observed by phase-contrast microscopy in which Fog treatment leads to the appearance of phase-dark ruffles indicative of a radial increase in non-muscle myosin II contractility, or by fluorescence microscopy where Fog treatment leads to the formation of non-muscle myosin II rings in cells expressing EGFP-tagged RLC34. These rings contain a myosin-phosphorylated regulatory light chain (pRLC) visible via immunostaining23,34,35. This Fog-induced response required Cta, RhoGEF2, Rho, and Rok; thus, using recombinant Fog-Myc and S2R+ cells, we have established a means to investigate Fog-induced constriction in a tissue-culture-based system24,25,34.
1. Maintenance of Drosophila Tissue Culture Cells
2. RNAi Treatment of Drosophila S2R+ Cells
NOTE: Refer to Rogers and Rogers6 for detailed instructions on how to produce dsRNA suitable for insect culture.
3. Fog Production and Harvest
4. Preparation of Concanavalin-A-coated Glass-bottomed Dishes and the Plating of S2R+ Cells
5. Efficacy Test of the Fog-conditioned Medium, and the Cellular Contractility Assay
6. Fixing and Staining of Constricted S2R+ Cells
7. Imaging and Quantification of the Cellular Contractility Assay
S2 and S2:Fog-Myc cells were grown to near confluent conditions in a 150-cm2 tissue culture flask and were treated with 25 – 75 µM CuSO4 over a period of 24 – 48 hours to induce expression of Fog-Myc (Figure 2). Samples from S2 cells (Figure 2A) and S2:Fog-Myc cells (Figure 2B) were removed and Fog-Myc production was monitored by western blot. As expected, we failed to detect Fog-Myc in media harvested from S2 cells under any conditions. However, we did detect Fog in media harvested from the S2:Fog-Myc stable cell line as early as 24 hours after the induction with 75 µM CuSO4. S2R+ cells attached to con-A-coated glass-bottomed dishes expressing an EGFP-tagged regulatory light chain (GFP-RLC) of non-muscle myosin II (Figures 3A and 3B). Live-cell imaging was performed on an inverted microscope equipped with a 100X/1.4 NA objective lens during the perfusion of Fog-conditioned media. Approximately 5 minutes post-treatment with Fog-Myc, the RLC formed rings indicative of non-muscle myosin II constriction. These rings can be observed through immunostaining S2R+ cells with an antibody raised against a synthetic phosphopeptide corresponding to residues surrounding Ser19 of human myosin RLC (Figures 4A and 4B). This antibody cross-reacts with Drosophila RLC.
Here we have a representative example of the cellular contractility assay following a 7-day treatment of cells with control RNAi and dsRNA against Rho1 (Figures 5A – 5E). S2R+ cells were plated on con-A-coated glass-bottomed dishes and treated with Fog-conditioned (+Fog) media or control media (-Fog). The number of contracted cells were then quantified following fixation. The phase-dense ruffles indicative of constriction was prominent in control depleted cells treated with Fog-conditioned media (Figure 5B). In Figure 5C, Rho-depleted cells treated with S2-conditioned medium are a typical example of smooth-edged, fried-egg-like morphology. Note that Rho plays a role in the Fog signaling pathway, as well as in cytokinesis; thus, Rho-depleted cells often fail to properly divide, leading to an increase in cell size and ploidy. Treatment of control depleted cells with Fog-conditioned media typical leads to 30% – 50% of cells undergoing constriction, whereas we failed to observe substantial constriction in Rho-depleted cells.
Figure 1:The putative Fog signaling pathway. In the absence of Fog, the Gα12/13 subunit, Concertina (Cta), along with its binding partners Gϒ and Gβ, are inactive and associated with the co-receptors Mist and Smog. Ric-8 chaperones Cta and regulates its subcellular localization. Upon Fog binding, Cta disassociates from Gϒ, and Gβ recruits RhoGEF2 to the cell membrane where it catalyzes the exchange of GDP for GTP in the small GTPase Rho1. GTP-bound Rho1, now active, activates Rho Kinase (Rok), which phosphorylates the RLC of non-muscle myosin, leading to its activation and subsequent cellular contractility. Please click here to view a larger version of this figure.
Figure 2: Time-course of Fog-Myc production. These panels show cell culture medium harvested from a near 100% confluent flask of (A) control S2 cells or (B) S2:Fog-Myc stable cells. The cells were treated with 25 – 75 µM CuSO4 over a period of 24 – 48 hours. The cell culture medium was collected and prepared for SDS-PAGE and western blotting. Fog-Myc was detected by an anti-Myc antibody. Please click here to view a larger version of this figure.
Figure 3: Live-cell imaging of Fog-induced constriction. Stable S2R+ cell lines expressing EGFP-tagged RLC of non-muscle myosin II were imaged by swept-field confocal microscopy during the perfusion of Fog-conditioned media. Time is shown in minutes and seconds. (A) This panel shows an image of a focal plane close to the cell-coverslip interface at time 0:00 before the perfusion of Fog-Myc. (B) This panel shows a time series (0:30 – 10:00) of images taken at a focal plane near the top of the cell. The white arrows indicate the reorganization of non-muscle myosin II as indicated by EGFP-RLC into contractile rings during constriction. The scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 4: Phosphorylated-RLC localizes to contractile rings following induction of constriction by Fog-Myc. S2R+ cells were fixed and stained for actin using phalloidin (red), phosphorylated RLC using a phosphoserine-19 antibody (green), and DAPI (DNA, blue), following treatment with (A) control or (B) Fog-Myc-conditioned media. Note the reorganization of phosphorylated RLC into rings upon the addition of Fog-Myc. Please click here to view a larger version of this figure.
Figure 5: Representative quantification of the Fog-induced cellular contractility assay. S2R+ cells were treated with (A and B) control RNAi or (C and D) dsRNA against the small GTPase Rho1 for 7 days. Following this time, the cells were plated on con-A-coated glass-bottomed dishes and were treated with (A and C) control media or (B and D) Fog-Myc-conditioned media. The scale bar represents 10 µm. (E) A scatter plot indicates the mean and 95% confidence intervals of the fraction of contracted cells per condition. Individual points represent the fraction of contracted cells per field of view at 40X magnification (10 – 120 cells per field) and the numbers in parentheses indicate the total number of cells counted for both contracted and non-contracted cells over three separate RNAi experiments. Asterisks denote a statistically significant difference between RNAi- and Fog-treated conditions as determined by one-way ANOVA (p < 0.0001) with Tukey's multiple comparison post hoc analysis. Note that there was no statistically significant difference (n.s.) between Rho1 RNAi-treated samples treated with control or with Fog-Myc-conditioned media. Please click here to view a larger version of this figure.
Here, we present a detailed protocol for a cell-based contractility assay using a Drosophila tissue culture cell line (S2R+ cells), which undergoes non-muscle myosin II constriction as a response to Fog signaling. This assay is useful for investigating the Fog pathway, as well as mechanisms that regulate non-muscle myosin II contractility.
Cell-culturing Considerations:
The conditions under which S2R+ cells are maintained is critical to achieve reliable data from this assay. When planning to perform the contractility assay, it best to maintain S2R+ cells in Shield and Sang M3 insect medium. While S2R+ cells can thrive in other insect cell culture media (e.g., SF900 or Schneider's insect medium), they often lose their responsiveness to Fog. S2R+ cells that have a high passage number, generally more than 20 – 25 passages, begin to lose sensitivity to RNAi. It is best to use early-passage cells for all experiments. Another important aspect to RNAi depletion experiments is choosing appropriate controls. Common negative controls include dsRNA targeting EGFP or pBlueScript, neither of which have homology to the fly genome. Targeting proteins in the Fog pathway (Rho, Rok, etc.), which, when depleted, prevent the activation of non-muscle myosin contractility, are also useful controls. S2:Fog-Myc cells can be maintained in an alternative cell culture medium such as SF900 supplemented with 100 units/mL of penicillin, 100 µg/mL streptomycin, and 0.25 amphotericin B. Note that FBS is not required when culturing cells in SF900. Cell density is also a critical component to all aspects of Drosophila tissue culture cells. Unlike mammalian tissue culture cells, Drosophila-derived tissue culture cells thrive best under higher cell densities (densities no lower than 5 x 105 cells/mL). However, when performing this assay, it is critical that cells are not plated on con-A-coated glass-bottomed dishes above 80% confluence, as the quantification of contracted versus non-contracted cells will become extremely tedious.
Critical Aspects and Alternative Approaches of the Cellular Contractility Assay:
The production of Fog, the ligand that triggers non-muscle myosin II contractility, is a key component of this assay. The fog gene encodes a protein of 730 amino acids with a predicted molecular weight of ~78 kDa26. Hydropathy analysis revealed a stretch of 12 hydrophobic residues at Fog's amino-terminus that could function as a secretion signal sequence. In addition, the coding sequence also contains multiple sites for potential N- and O-linked glycosylation, further suggesting Fog is a secreted protein26. In support of this, Fog was localized to secretory vesicles in presumptive epidermal cells undergoing apical constriction16. Fog-Myc expression was induced upon addition of copper sulfate to the medium, and antibodies against Fog or Myc recognized a 150-kD protein from culture medium harvested from induced cultures but not from induced media from S2 cells lacking the Fog-Myc construct. This molecular weight was higher than the predicted 80-kD molecular weight of Fog and suggests that the secretory machinery of S2 cells may glycosylate the protein prior to exocytosis. Due to the potential variability in Fog purification, it is advisable to use the same batch of Fog-conditioned media for all experiments. Making up large batches of concentrated Fog-Myc media will help in maintaining consistency throughout rounds of experiments.
The success of this assay also depends on confidently identifying cells that have undergone constriction. While constricted cells can be observed by fluorescence microscopy, by staining for actin or non-muscle myosin II, the most reliable way to identify and quantify constricted cells is through phase-contrast or DIC microscopy. Accurate counts can be achieved using 20X – 40X magnification on most standard light microscopes. Although the protocol written here uses glass-bottomed dishes, the assay can also be performed using standard 1.5 glass coverslips coated with con A. The addition of Fog can be done in 35 mm of tissue culture on a coverslip placed on parafilm, in order to limit the amount of Fog used for each assay. The quality of fixation is a critical component of the assay, as poorly fixed cells can lead to false positives. Using fresh fixation solution and making sure the cells never dry once fixed will lead to more reliable results. Finally, it is important to count a large number of cells. Typically, only 30% – 50% of untreated or control RNAi-treated cells constrict following the perfusion of Fog. However, there is a basal level of constriction that occurs in S2R+ cells, so a large number of cells is needed to ensure any change in the fraction of constricted cells is due to treatments. Furthermore, the depletion of some proteins involved in the regulation of non-muscle myosin II contractility may lead to hyper-contractility, even in the absence of Fog. The data presented here is from over 3,600 cells that were counted in three successive RNAi treatments using the same batch of Fog-conditioned media.
Applications of the Cellular Contractility Assay:
This contractility assay, when coupled with RNAi screening methods, offers a powerful system to study cell signaling, morphogenesis, and cellular contractility. Previously, it has been used to identify one of two Fog co-receptors21. A targeted screen of 138 G-protein-coupled receptors (GPCRs) is depleted by RNAi in S2R+ cells, and its ability to respond to Fog was assayed as described in the protocol presented here. Of the 138 GPCRs, a single, previously uncharacterized gene, now known as Mist, was uncovered. Further investigation into the function of Mist demonstrated that not only was it required for Fog-induced cellular contractility in S2R+ cells, but it was also essential for gastrulation in developing Drosophila21. Furthermore, this assay was used to demonstrate that Ric-8, a non-canonical GEF, is also a component in the Fog signaling pathway27. A series of epistasis RNAi experiments coupled with the contractility assay demonstrated that Ric-8 interacts with the Gα12/13 subunit Cta and functions to localize it within the cell, which is critical to Fog-induced cellular contractility31.
Drosophila tissue culture is well suited to novices, as the cells are easily maintained at room temperature, do not require CO2 or buffered cell culture medium, and are robust as long as proper cell culture densities are maintained. The cellular contractility assay was successfully performed by the laboratory section of an undergraduate cell biology course, where the students had little to no experience with culturing cells or microscopy. The cellular contractility assay presented here represents a powerful, cell-based tool that can be employed in gene discovery, or to interrogate the Fog signaling pathway, helping us to better understand developmental processes, such as apical constriction and non-muscle myosin II contractility, in general.
The authors have nothing to disclose.
The authors would like to thank members of the Rogers Lab and the Applewhite lab, Greta Glover, and the students in the Reed College’s spring 2018 Cellular Biology course, who have contributed to the development of this protocol. Furthermore, the authors would like to thank members of the Ritz lab for carefully reading and editing this manuscript. Research reported in this publication was supported by the National Science Foundation under award number 716964 to D.A.A. and A.R. and the Reed College Biology Department.
S2R+ cells | Drosophila Genomics Resorce Center | 150 | cell culture line, undergoes apical constriciton |
S2:Fog-myc cells | Drosophila Genomics Resorce Center | 218 | cell culture line, produces Fog-myc under pMT promoter |
S2R+ :Sqh-EGFP cells | Drosophila Genomics Resorce Center | 196 | cell culture line, stably expresses Sqh(RLC of non-muscle myosin II) tagged with EGFP |
Shield and Sang M3 | Sigma-Aldrich | S3652 | cell culture medium |
SF900 insect medium | Thermofisher Scientific | 12658019 | alternative cell cutlure medium |
Schneider’s insect medium | Thermofisher Scientific | 21720024 | alternative cell cutlure medium |
Fetal Bovine Serum | Thermofisher Scientific | 16000044 | media supplement |
Antibiotic-Antimycotic (100X) | Thermofisher Scientific | 15240112 | antimicrobial/antifugal media supplement |
Concanavalin A | MP Biomedicals | 150710 | used to coat dishes and coverslip for cellular adhesion |
Glass bottom plates | Maktek | P35G-1.5-10 | used for assay and for fixing and staining cells |
Glass cover slips | Corning | 2940-225 | alternative to glass bottom dishes |
Protein concentrators | Millipore | UFC903008 | 30,000 molecular weight cutoff, used to concentrate media |
25 cm2, 75 cm2, 150 cm2 tissue culture flasks | Genessee | 25-204,25-206,25-208,25-210 | used to culture cells |
6-well and 12-well tissue culture dishes | Genessee | 25-105, 25-106 | used to culture cells |
Flourescent mounting medium | Dako | S3023 | to preserve fixed cells |
32% Paraformaldehyde | EMS | 15714-S | used to fix cells |
Pipes | EMS | 19240 | used in PEM buffer for cell fixation |
Anti-Myc Antibody | Drosophila Hybirdoma Bank | cat# 9E10 | used to dectect Fog-Myc |
PhosphoSerine-19 antibody | Cell Signaling | 3671 | antibody against phosphorylated regulatory light chain (RLC) serine 19 |
488-Phalloidin, 568-Phalloidin | Thermofisher Scientific | A12379, A12380 | to stain for f-actin |
Hawk-VT multi-point array scanning confocal system | VisiTech International | Used for live cell imaging of S2R+:Sqh cells | |
Eclipse TE300 Inverted microscope | Nikon | Used for live cell imaging of S2R+:Sqh cells |