Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

Paul D. Langridge; Alejandro Garcia Diaz; Jessica Yu Chan; Iva Greenwald; Gary Struhl

doi:10.1016/j.cub.2022.03.025

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

Paul D. Langridge, Alejandro Garcia Diaz, Jessica Yu Chan, Iva Greenwald, Gary Struhl

Research output: Contribution to journal › Article › peer-review

Abstract

The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. 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 induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.³^,⁴ In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a “pulling force” on Notch that is essential to expose the cleavage site in the NRR.^4–6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,⁶ we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a “leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.¹^,² Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.

Original language	English (US)
Pages (from-to)	2263-2271.e6
Journal	Current Biology
Volume	32
Issue number	10
DOIs	https://doi.org/10.1016/j.cub.2022.03.025
State	Published - May 23 2022
Externally published	Yes

Keywords

ADAM protease
C. elegans
Drosophila
Epsin
NRR
Notch
ectodomain shedding
force sensor
ligand endocytosis
mechano-receptor
negative regulatory region

ASJC Scopus subject areas

Neuroscience(all)
Biochemistry, Genetics and Molecular Biology(all)
Agricultural and Biological Sciences(all)

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10.1016/j.cub.2022.03.025

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@article{8cccd2a9f79144739a137f2eeb57f76f,

title = "Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins",

abstract = "The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. 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 induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a “pulling force” on Notch that is essential to expose the cleavage site in the NRR.4–6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a “leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.",

keywords = "ADAM protease, C. elegans, Drosophila, Epsin, NRR, Notch, ectodomain shedding, force sensor, ligand endocytosis, mechano-receptor, negative regulatory region",

author = "Langridge, {Paul D.} and {Garcia Diaz}, Alejandro and Chan, {Jessica Yu} and Iva Greenwald and Gary Struhl",

note = "Funding Information: We are grateful to Julie Canman, Sophia Hirsch, and Michelle Attner for helpful input into the C. elegans work; Nikola Danev for assistance with the Drosophila constructs; Barth Grant for advice about GPI linkage sequences; Gleniza Gomez and Chunyao Tao for technical assistance; Linlin Zhang for providing Notch sequences; David Fitch for advice on phylogenetic analysis and discussion; and Catherine O{\textquoteright}Keeffe and Andrew Tomlinson for comments on the manuscript. We also thank Zheng Zhou, Sophia Hirsch, and Julie Canman for strains as credited herein, as well as the Caenorhabditis Genetics Center funded by the NIH Office of Research Infrastructure Programs ( P40 OD010440 ). This research was supported by the Institute of General Medicine of the National Institutes of Health ( R35GM131746 to I.G. and R35GM127141 to G.S.) and a Roy and Diana Vagelos Precision Medicine Award (to G.S.). Publisher Copyright: {\textcopyright} 2022 Elsevier Inc.",

year = "2022",

month = may,

day = "23",

doi = "10.1016/j.cub.2022.03.025",

language = "English (US)",

volume = "32",

pages = "2263--2271.e6",

journal = "Current Biology",

issn = "0960-9822",

publisher = "Cell Press",

number = "10",

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T1 - Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

AU - Langridge, Paul D.

AU - Garcia Diaz, Alejandro

AU - Chan, Jessica Yu

AU - Greenwald, Iva

AU - Struhl, Gary

N1 - Funding Information: We are grateful to Julie Canman, Sophia Hirsch, and Michelle Attner for helpful input into the C. elegans work; Nikola Danev for assistance with the Drosophila constructs; Barth Grant for advice about GPI linkage sequences; Gleniza Gomez and Chunyao Tao for technical assistance; Linlin Zhang for providing Notch sequences; David Fitch for advice on phylogenetic analysis and discussion; and Catherine O’Keeffe and Andrew Tomlinson for comments on the manuscript. We also thank Zheng Zhou, Sophia Hirsch, and Julie Canman for strains as credited herein, as well as the Caenorhabditis Genetics Center funded by the NIH Office of Research Infrastructure Programs ( P40 OD010440 ). This research was supported by the Institute of General Medicine of the National Institutes of Health ( R35GM131746 to I.G. and R35GM127141 to G.S.) and a Roy and Diana Vagelos Precision Medicine Award (to G.S.). Publisher Copyright: © 2022 Elsevier Inc.

PY - 2022/5/23

Y1 - 2022/5/23

N2 - The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. 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 induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a “pulling force” on Notch that is essential to expose the cleavage site in the NRR.4–6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a “leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.

AB - The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. 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 induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a “pulling force” on Notch that is essential to expose the cleavage site in the NRR.4–6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a “leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.

KW - ADAM protease

KW - C. elegans

KW - Drosophila

KW - Epsin

KW - NRR

KW - Notch

KW - ectodomain shedding

KW - force sensor

KW - ligand endocytosis

KW - mechano-receptor

KW - negative regulatory region

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DO - 10.1016/j.cub.2022.03.025

M3 - Article

C2 - 35349791

AN - SCOPUS:85130379211

SN - 0960-9822

VL - 32

SP - 2263-2271.e6

JO - Current Biology

JF - Current Biology

IS - 10

ER -

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

Abstract

Keywords

ASJC Scopus subject areas

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