The C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/β-catenin signalling

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/β-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/β-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/β-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/β-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/β-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine

Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

<div><p>Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require <i>de novo</i> RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.”</p></div

U2OS cell competition interactions are short-ranged.

<p>(A) Schematic representation of U2OS transwell cultures, Cells shared culture medium but were separated by a 0.8 μm Durapore membrane. (B) Cp3-IF analysis of apoptosis in transwell cultures. Wt cells induce apoptosis in YFP cells in the insert but not in YFP cells separated by the transwell. (C) Spot-seeding of YFP and H2B-mCherry expressing clone R1. Mixed YFP:R1 spots surrounded by pure YFP cell populations were divided in “inner” (I), “border” (B), and “outer” (O) zones as represented. (D) Time-lapse microscopy tracking of YFP cell fates during 72-hour spot cultures. The number of cell layers separating each YFP cell from its nearest R1 neighbor was recorded, and YFP “B” cells were grouped accordingly: for instance, a YFP cell is labeled “B3” if it comes within 3 cell layers of the nearest R1 cell, while a”B0” YFP cell comes to lie adjacent to an R1 cell at any time during the observation period. The data summarizes the fate of cells present at the beginning of each experiment and their immediate progeny, followed over 72 hours. The percentage of followed cells that underwent cell division is shown at the top; cell death is shown at the middle, and net population size change at the bottom. Increased apoptosis is observed only in inner YFP cells and “B0” border cells that come in direct contact with R1 cells. Data in panel D was derived from 3 independent experiments (Supplemental Movie S1-3), comprising over 5,000 cells counted. *: p<0.05, **: p<0.01 by Student’s <i>t</i>-test; #:p<0.05, ##:p<0.01 by paired <i>t</i>-test.</p

Cell death is triggered by a cell competition-like interaction in clonally-derived mammalian cell lines.

<p>(A) Cell counts showing YFP (“loser” cells) cells first expand, then decline, in the presence of Wt (“winner”) cells but grow unimpeded when cultured alone. Time is measured from cell seeding (t = 0). (B) Cleaved caspase-3 (Cp3) immuno-fluorescence (IF) (red) of U2OS cultures showing increased apoptosis in co-cultured YFP cells (green). Arrows indicate Cp3+ apoptotic YFP cells. Wt cells are counterstained with Hoescht 33342 (blue). (C) Quantification of apoptosis on immune-stained cultures; x-axis, time in days (d) Note that the baseline level of apoptosis increases with cell density by day 6 under all culture conditions. (D) Quantification of cell proliferation in 72-hour U2Os cultures by phospho-histone H3 (PH3) immunofluorescence. (E) Cp3 IF of 72-hour U2OS cultures treated with the caspase-3 inhibitor Z-VAD-FMK. YFP cell counts per microscope field are shown at the bottom. Inhibition of apoptosis by Z-VAD-FM K prevents YFP elimination from Wt:YFP co-cultures. (F) P-H3 IF of U2OS cells cultured for 72 hours in presence of the Cyclin D1 inhibitor purvalanol A as indicated. Quantification of apoptosis is shown below. Purvalanol A treatment inhibits proliferation (top) and rescues YFP elimination (bottom). Images were taken at 100X magnification. Error bars in this and all subsequent figures reflect mean ± SD. *: p<0.05, **: p<0.01 and ***: p<0.001 by Student’s <i>t</i>-test.</p

Cell competition in U2OS cells is mediated by post-transcriptional mechanism.

<p>(A) Microarray RNA analysis experimental design. Cells were grown in mono- or co-cultures for 48h as indicated and sorted by flow cytometry before RNA extraction. (B) Venn diagram distribution of RNAs displaying >2-fold expression change across indicated sample groups. Only six transcripts were found to be differentially expressed in mono-cultured and competing YFP cells, while no transcription changes were observed in response to competition in Wt and R1 cells. (C) Immuno-staining of metabolite-labeled nascent RNA and protein chains in Wt U2OS cells treated with actinomycin D or cycloheximide for 24 hours. Ethynil-uridine (EU) and homopropargyl-glycin (HGP) were added to label nascent RNA and peptide chains 2 h before staining. Complete inhibition of transcription and translation was obtained with actinomycin D and cycloheximide, respectively. (D) Cp3-IF of Wt:YFP 72-hour cultures grown in presence or absence of actinomycin D- and cycloheximide. Cycloheximide completely abolished Wt-induced YFP apoptosis. Actinomycin D treatment resulted in a partial rescue to a degree consistent with its inhibition of cell division (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132437#pone.0132437.s010" target="_blank">S10 Fig</a>). *”p<0.05, **: p<0.01 by Student’s <i>t</i>-test.</p

U2OS cell “fitness” is context-dependent.

<p>(A) Aspect of U2OS G1, R1, and YFP co-cultures in fluorescence microscopy. Cells were plated at a 1:1 ratio as indicated (R1:G1 or R1:YFP) and allowed to grow for 1, 3 or 6 days. (B) Cp3-IF apoptosis quantification. R1 cells behave as “winners” in R1:YFP cultures but as “losers” in the presence of G1 cells, indicating that R1 cells assume “winner” or “loser” status depending on the properties of their co-culture partners. *: p<0.001 by Student’s <i>t</i>-test.</p

Differences in energy metabolism drive cell competition in mammalian cells.

<p>(A) Quantification of apoptosis (Cp3 immunofluorescence) in U2OS cultures grown for 72 hours under normoxic (21% O₂) or hypoxic (1.5% O₂) conditions. Hypoxia inhibits cell competition-induced elimination of YFP and R1 cells in Wt:YFP and Wt:R1 co-cultures. (B) Quantification of Cp3 immunofluorescence in U2OS R1 cells expressing a nonsense (NSC, non-silencing control) shRNA or shRNAs directed against the Hif1a transcription factor. Hif1a knockdown in loser cells does not affect cell competition. qPCR analysis of Hif1a in shRNA-expressing YFP cells is shown on the right. (C) Quantification of Cp3 immunofluorescence in U2OS cultures grown for 72 hours in medium containing standard (4 mM) and reduced (0.4 mM) Glutamin (Gln) concentrations. Withholding Gln arrests cell competition in U2OS cells. (D) Quantification of Cp3 immunofluorescence in U2OS cultures treated with a mitochondrial uncoupling agent (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). Uncoupling respiration from oxidative phosphorylation blocks competition-induced elimination of R1 and YFP cells, indicating that competition is driven by differences in the activity of ATP-generating pathways. (E) Luciferase analysis of intracellular ATP levels in monocultured U2OS cell. YFP, but not R1 cells, display reduced ATP levels in when compared to Wt cells. Hypoxia increases ATP levels YFP cells, suggesting that reduced ATP levels may reflect or underlie YFP cell fitness.*: p<0.001, by one-way ANOVA.</p