David Sprinzak

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

The Notch–Delta signalling pathway allows communication between neighbouring cells during development. 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.

Reconstruction of genetic circuits

The complex genetic circuits found in cells are ordinarily studied by analysis of genetic and biochemical perturbations. The inherent modularity of biological components like genes and proteins enables a complementary approach: one can construct and analyse synthetic genetic circuits based on their natural counterparts. Such synthetic circuits can be used as simple in vivo models to explore the relation between the structure and function of a genetic circuit. Here we describe recent progress in this area of synthetic biology, highlighting newly developed genetic components and biological lessons learned from this approach.

An electronic Mach-Zehnder interferometer.

Double-slit electron interferometers fabricated in high mobility two-dimensional electron gases are powerful tools for studying coherent wave-like phenomena in mesoscopic systems. However, they suffer from low visibility of the interference patterns due to the many channels present in each slit, and from poor sensitivity to small currents due to their open geometry. Moreover, these interferometers do not function in high magnetic fields—such as those required to enter the quantum Hall effect regime—as the field destroys the symmetry between left and right slits. Here we report the fabrication and operation of a single-channel, two-path electron interferometer that functions in a high magnetic field. This device is the first electronic analogue of the optical Mach–Zehnder interferometer, and opens the way to measuring interference of quasiparticles with fractional charges. On the basis of measurements of single edge state and closed geometry transport in the quantum Hall effect regime, we find that the interferometer is highly sensitive and exhibits very high visibility (62%). However, the interference pattern decays precipitously with increasing electron temperature or energy. Although the origin of this dephasing is unclear, we show, via shot-noise measurements, that it is not a decoherence process that results from inelastic scattering events.

Phase evolution in a Kondo-correlated system.

We measured the phase evolution of electrons as they traverse a quantum dot (QD) formed in a two-dimensional electron gas that serves as a localized spin. The traversal phase, determined by embedding the QD in a double path electron interferometer and measuring the quantum interference of the electron wave functions manifested by conductance oscillation as a function of a weak magnetic field, evolved by pi radians, a range twice as large as theoretically predicted. As the correlation weakened, a gradual transition to the familiar phase evolution of a QD was observed. The specific phase evolution observed is highly sensitive to the onset of Kondo correlation, possibly serving as an alternative fingerprint of the Kondo effect.

Mutual Inactivation of Notch Receptors and Ligands Facilitates Developmental Patterning

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation. DOI: http://dx.doi.org/10.7554/eLife.02950.001

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

The Notch signaling pathway relies on a proteolytic cascade to release its transcriptionally active intracellular domain, on force to unfold a protective domain and permit proteolysis, on extracellular domain glycosylation to tune the forces exerted by endocytosed ligands, and on a motley crew of nuclear proteins, chromatin modifiers, ubiquitin ligases, and a few kinases to regulate activity and half-life. Herein we provide a review of recent molecular insights into how Notch signals are triggered and how cell shape affects these events, and we use the new insights to illuminate a few perplexing observations.

Charge distribution in a Kondo-correlated quantum dot.

We report on direct measurement of charge and its distribution in a Kondo correlated quantum dot (QD). A noninvasive potential-sensitive detector, in proximity with a QD, reveals that, although the conductance of the QD is significantly enhanced as it enters the Kondo regime, the average charge remains unaffected. This demonstrates the separation between spin and charge degrees of freedom. We find, however, under certain conditions, an abrupt redistribution of charge in the QD, taking place with an onset of Kondo correlation. This suggests a correlation between the spin and charge degrees of freedom.

The cis side of juxtacrine signaling: a new role in the development of the nervous system

Cell-cell communication by juxtacrine signaling plays a key role in the development of the nervous system, from cell fate determination through axonal guidance to synaptogenesis. Interestingly, several juxtacrine signaling systems exhibit an inhibitory interaction between receptors and ligands in the same cell, termed cis inhibition. These include the Notch, semaphorin and ephrin signaling systems. Here we review the role of cis inhibition in these signaling systems in the development of the nervous system. We compare and contrast cis inhibition mechanisms and discuss their potential cellular function as a threshold-generating mechanism. The prevalence of cis inhibition suggests that these interactions and their functional regulatory roles may serve as a general design principle for juxtacrine signaling-mediated processes during and beyond neurodevelopment.

From Notch signaling to fine-grained patterning: Modeling meets experiments

Notch signaling is the canonical signaling pathway between neighboring cells. It plays an important role in fine-grained patterning processes such as the formation of checkerboard-like differentiation patterns and sharp boundaries between developing tissues. While detailed information about many of the genes and proteins involved have been identified, we still lack a quantitative mechanistic understanding of these processes. Here we discuss several recent studies that provide novel insights into Notch-dependent patterning by combining mathematical models with quantitative experimental results. Such approaches allow identification of mechanisms and design principles controlling how patterns are generated in a reproducible and robust manner.