Search

Notch Ligand Binding Assay Using Flow Cytometry   

Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
eLIFE
Apr 2017

Abstract

Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.

Keywords: Notch ligand binding assay, DLL1, DLL4, JAG1, JAG2, Fc-tag, Flow cytometry

Background

Cell proliferation, differentiation, and apoptosis are well known to be regulated by Notch signaling. Aberrant changes in Notch signaling are related to diverse disorders, giving rise to a range of developmental and adult diseases (Bray, 2016). The canonical Notch signaling pathway in mammals is initiated by the binding of Notch ligands Delta or Jagged to the extracellular domain of Notch receptors (NECD), expressed on opposing cells. Receptor-ligand binding initiates two sequential proteolytic cleavages, resulting in the release of the Notch intracellular domain (NICD). Released NICD complexes with the transcriptional repressor CSL (CBF-1/Suppressor-of-hairless/Lag-1), also termed recombination signal binding protein for immunoglobulin kappa J region (RBPjk), and the co-activator Mastermind (MAML), activate Notch target genes. The binding of Notch receptors to different ligands results in distinct consequences (Benedito et al., 2009; Bray, 2016). For example, the maintenance of hematopoietic stem cells is regulated by low strength JAG1-mediated Notch signaling, whereas arterial cell fate is determined by high strength DLL4-mediated Notch signaling (Gama-Norton et al., 2015). The addition of N-acetylglucosamine (GlcNAc) to the O-fucose on epidermal growth factor (EGF) repeats of the NECD by a Fringe glycosyltransferase generally enhances signaling by Notch receptors induced by Delta-like ligands DLL1 and DLL4, while reducing signaling induced by Jagged ligands JAG1 and JAG2 (Bruckner et al., 2000; Moloney et al., 2000; Yang et al., 2005; Kovall et al., 2017). Recent structural studies have revealed molecular interactions between O-glycans on a Notch1 fragment including EGF repeats 8-13, and soluble ligands DLL4 (Luca et al., 2015) and JAG1 (Luca et al., 2017). Notch ligand EGF repeats are also modified with O-glycans but mutant ligands lacking O-glycans largely remain functional (Muller et al., 2014; Serth et al., 2015). The protocol described below is a method of determining the relative binding of soluble Notch ligand ECDs to the ECD of endogenous or introduced Notch receptors (Figure 1).


Figure 1. Diagram of the Notch ligand binding assay. Notch receptors expressed on the cell surface have an extracellular domain (NECD) comprised of 29-36 N-terminal EGF like repeats followed by 3 Lin-12 Notch repeats. Cell surface expression is confirmed using anti-NECD antibodies. NECD is non-covalently attached to the intracellular domain (NICD) which gets released from the cell membrane and translocates to the nucleus upon proteolysis following ligand binding. Notch ligands Delta-like (DLL1 and DLL4) and Jagged (JAG1 and JAG2) extracellular domains (ECD) comprise a Module at the N-terminus of Notch Ligand (MNNL) motif, followed by a Delta-Serrate-LAG2 (DSL) domain, followed by 6-16 EGF repeats. For this assay, the C-terminus of Notch ligand ECD is linked to a human Fc-tag which is recognized by a fluorescently-labeled secondary antibody (PE-Ab). Ligand binding buffer must contain calcium for Notch ligand binding to occur. Chelation of calcium is used as a control for the specificity of ligand binding.

Copyright Varshney and Stanley. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Varshney, S. and Stanley, P. (2017). Notch Ligand Binding Assay Using Flow Cytometry. Bio-protocol 7(23): e2637. DOI: 10.21769/BioProtoc.2637.
  2. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.