Abstract
BackgroundNotch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.ResultsTo test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems—as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture.ConclusionsTogether these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.
Highlights
Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition
Myosin II activity is required for robust Notch signaling Myosin II motors contribute to the generation of actindependent pulling forces to drive a wide range of developmental processes [21,22,23]
In order to determine whether actomyosin contractility is required for lateral inhibition signaling during notum pattern formation, we asked how decreasing actomyosin tension affects the activity of a transcriptional reporter of Notch signaling, NsfGFP (Fig. 1a, b) [24]
Summary
Myosin II activity is required for robust Notch signaling Myosin II motors contribute to the generation of actindependent pulling forces to drive a wide range of developmental processes [21,22,23]. These appeared to be a combination of the two phenotypes observed in the single mutants, i.e., there was an increase in the variability of bristle spacing together with an enhanced number of GFP-positive cell clusters relative to the expression of either RNAi alone (Fig. 5a, b; Additional file 5: Figure S3F) These data support the idea that Delta ligand endocytosis and myosin II activity act in distinct ways to impact Notch signaling in vivo via lateral and protrusion-mediated signaling, respectively.
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