Biology subjects

Langridge, P. D.

Publications and source records attributed to Langridge, P. D..

3 recordsLinked to original sources

bioRxiv2024

An in vivo screen for proteolytic switches that can mediate Notch activation

Notch proteins are single pass transmembrane receptors activated by sequential extracellular and intramembrane cleavages that release their cytosolic domains to function as transcription factors in the nucleus. Upon binding, Delta/Serrate/LAG-2 (DSL) ligands activate Notch by exerting a "pulling" force across the intercellular ligand/receptor bridge. This pulling force is generated by Epsin-mediated endocytosis of ligand into the signal-sending cell, and results in the extracellular cleavage of the force-sensing Negative Regulatory Region (NRR) of the receptor by an ADAM10 protease [Kuzbanian (Kuz) in Drosophila]. Here, we have used chimeric Notch and DSL proteins to screen for other domains that can function as ligand-dependent proteolytic switches in place of the NRR in the developing Drosophila wing. While most of the tested domains are either refractory to cleavage or constitutively cleaved, we identify several that mediate Notch activation in response to ligand. These NRR analogues derive from widely divergent source proteins and have strikingly different predicted structures. Yet, almost all depend on force exerted by Epsin-mediated ligand endocytosis and cleavage catalyzed by Kuz. We posit that the sequence space of protein domains that can serve as force-sensing proteolytic switches in Notch activation is unexpectedly large, a conclusion that has implications for the mechanism of target recognition by Kuz/ADAM10 proteases and is consistent with a more general role for force dependent ADAM10 proteolysis in other cell contact-dependent signaling mechanisms. Our results also validate the screen for increasing the repertoire of proteolytic switches available for synthetic Notch (synNotch) therapies and tissue engineering. One sentence summaryA scalable in vivo screen for Epsin and ADAM10 dependent proteolytic switches that can mediate Notch activation in response to force.

cell biology↗

bioRxiv2023

Emergent spatiotemporal patterning of synthetic Notch signal transduction in vivo

Cell-cell communication through direct contact, or juxtacrine signaling, is important in development, disease, and many areas of physiology. Synthetic forms of juxtacrine signaling can be precisely controlled and operate orthogonally to native processes, making them a powerful reductionist tool with which to address fundamental questions in cell-cell communication in vivo. Here we investigate how cell-cell contact length and tissue growth dynamics affect juxtacrine signal responses through implementing a custom synthetic gene circuit in Drosophila wing imaginal discs alongside mathematical modeling to determine synthetic Notch (synNotch) activation patterns. We find that the area of contact between cells largely determines the extent of syn-Notch activation, leading to the prediction that the shape of the interface between signal-sending and signal-receiving cells will impact the magnitude of the synNotch response. Notably, synNotch outputs form a graded spatial profile that extends several cell diameters from the signal source, providing evidence that the response to juxtacrine signals can persist in cells as they proliferate away from source cells, or that cells remain able to communicate directly over several cell diameters. Our model suggests the former mechanism may be sufficient, since it predicts graded outputs without diffusion or long-range cell-cell communication. Overall, we identify that cell-cell contact area together with output synthesis and decay rates likely govern the pattern of synNotch outputs in both space and time during tissue growth, insights that may have broader implications for juxtacrine signaling in general.

biophysics↗

bioRxiv2021

The C. elegans Notch proteins LIN-12 and GLP-1 are tuned to lower force thresholds for activation than Drosophila Notch

The conserved transmembrane receptor Notch mediates cell fate decisions in all animals. In the absence of ligand, a Negative Regulatory Region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site [1, 2]; ligand binding makes the cleavage site accessible, leading to shedding of the Notch ectodomain as the first step of signal transduction [3, 4]. In Drosophila and vertebrates, the ligands are all single-pass transmembrane Delta/Serrate/LAG-2 (DSL) proteins; the endocytic adaptor Epsin binds to the ubiquitinated intracellular domain, and the resulting Clathrin-mediated endocytosis exerts a "pulling force" that exposes the cleavage site in the NRR [4-6]. However, in C. elegans, the presence of natural secreted DSL proteins [7] and other observations suggested that Epsin-mediated endocytosis may not be required to activate the Notch proteins LIN-12 and GLP-1. Here, we confirm that neither Epsin nor the cytosolic domains of DSL proteins are required for Notch signaling in C. elegans. Furthermore, we provide evidence that the NRRs of LIN-12 and GLP-1 are tuned to a lower force level than the NRR of Drosophila Notch. Finally, we show that adding a Leucine "plug" that occludes the cleavage site in vertebrate and Drosophila Notch proteins but is absent in the C. elegans Notch proteins [1, 2] renders the LIN-12 and GLP-1 NRRs dependent on Epsin-mediated ligand endocytosis, indicating that greater force is now required to expose the cleavage site. Thus, the NRRs of LIN-12 and GLP-1 appear to be tuned to a lower force threshold, accounting for the different requirements for signaling in C. elegans.

developmental biology↗