Cis-activation in the Notch signaling pathway

The Notch signaling pathway consists of transmembrane ligands and receptors that can interact both within the same cell (cis) and across cell boundaries (trans). Previous work has shown that cis-interactions act to inhibit productive signaling. Here, by analyzing Notch activation in single cells while controlling cell density and ligand expression level, we show that cis-ligands can also activate Notch receptors. This cis-activation process resembles trans-activation in its ligand level dependence, susceptibility to cis-inhibition, and sensitivity to Fringe modification. Cis-activation occurred for multiple ligand-receptor pairs, in diverse cell types, and affected survival in neural stem cells. Finally, mathematical modeling shows how cis-activation could potentially expand the capabilities of Notch signaling, for example enabling ‘negative’ (repressive) signaling. These results establish cis-activation as an additional mode of signaling in the Notch pathway, and should contribute to a more complete understanding of how Notch signaling functions in developmental, physiological, and biomedical contexts

Cis-activation in the Notch signaling pathway

The Notch signaling pathway consists of transmembrane ligands and receptors that can interact both within the same cell (cis) and across cell boundaries (trans). Previous work has shown that cis-interactions act to inhibit productive signaling. Here, by analyzing Notch activation in single cells while controlling cell density and ligand expression level, we show that cis-ligands can also activate Notch receptors. This cis-activation process resembles trans-activation in its ligand level dependence, susceptibility to cis-inhibition, and sensitivity to Fringe modification. Cis-activation occurred for multiple ligand-receptor pairs, in diverse cell types, and affected survival in neural stem cells. Finally, mathematical modeling shows how cis-activation could potentially expand the capabilities of Notch signaling, for example enabling ‘negative’ (repressive) signaling. These results establish cis-activation as an additional mode of signaling in the Notch pathway, and should contribute to a more complete understanding of how Notch signaling functions in developmental, physiological, and biomedical contexts

Dynamic Ligand Discrimination in the Notch Signaling Pathway

The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes

Cis-interactions between Notch and Delta generate mutually exclusive signalling states

The Notch–Delta signalling pathway allows communication between neighbouring cells during development^1. It has a critical role in the formation of ‘fine-grained’ patterns, generating distinct cell fates among groups of initially equivalent neighbouring cells and sharply delineating neighbouring regions in developing tissues. The Delta ligand has been shown to have two activities: it transactivates Notch in neighbouring cells and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. Here we report the development of a quantitative time-lapse microscopy platform for analysing Notch–Delta signalling dynamics in individual mammalian cells, with the aim of addressing these issues. By controlling both cis- and trans-Delta concentrations, and monitoring the dynamics of a Notch reporter, we measured the combined cis–trans input–output relationship in the Notch–Delta system. The data revealed a striking difference between the responses of Notch to trans- and cis-Delta: whereas the response to trans-Delta is graded, the response to cis-Delta is sharp and occurs at a fixed threshold, independent of trans-Delta. We developed a simple mathematical model that shows how these behaviours emerge from the mutual inactivation of Notch and Delta proteins in the same cell. This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta/low Notch) and receiving (high Notch/low Delta) signalling states. At the multicellular level, this switch can amplify small differences between neighbouring cells even without transcription-mediated feedback. This Notch–Delta signalling switch facilitates the formation of sharp boundaries and lateral-inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviours

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells

Dynamic Ligand Discrimination in the Notch Signaling Pathway

The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes

Synergistic Ca^(2+) Responses by Gα_i- and Gα_q-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβ_γ and Gα_q

Cross-talk between Gα_i- and Gα_q-linked G-protein-coupled receptors yields synergistic Ca^(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca^(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca^(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα_q. Thus, both Gβγ and Gα_q contribute to activation of PLCβ3 in cells for Ca^(2+) synergy. We conclude that Ca^(2+) synergy between Gα_i-coupled and Gα_q-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent

RNAi Methodologies for the Functional Study of Signaling Molecules

RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules

[Revision] Reconstitution of morphogen shuttling circuits

Here we provide raw data, code, and other supplementary material related to the manuscript, "Reconstitution of morphogen shuttling circuits," by Ronghui Zhu et al. This includes computer codes for implementing the shuttling model, maps of constructs used in this manuscript, raw imaging and flow cytometry data, and raw time-lapse images.&nbsp

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q.

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent