Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states
- Howard Hughes Medical Institute, California Institute of Technology, United States
- California Institute of Technology, United States
- Baylor College of Medicine, United States
- Tel Aviv University, Israel
Figures
Figure 1
Cis interactions between receptors and ligands lead to exclusive sending and receiving signaling states.
(A) In the blue shaded region, receptor expression exceeds ligand expression (as indicated schematically above plot), so that mutual cis interactions leave mainly free receptors, allowing the cell …
Figure 2
The availability assay labels receptors and ligands that can participate in trans signaling.
(A and B) Stable CHO-K1 cell lines constitutively express a Notch1-Gal4 chimeric receptor and a tetracycline-inducible Dll1 (A) or Jag1 (B) ligand fused to cerulean fluorescent protein. (C and D) In …
Figure 3 with 4 supplements
Dll1 and Jag1 exhibit mutual cis-inhibition with Notch1.
(A and B) Single cell data show decreasing receptor availability and increasing ligand availability with increasing ligand expression. Circles denote the medians of data points in logarithmically …
Figure 3—figure supplement 1
Calibration of availability assay reagents.
(A) The working concentration of Dll1ext-Fc was determined by incubating the parental hN1(ΔICD)-Gal4esn cells with increasing concentrations of soluble Dll1ext-Fc, and staining with secondary …
Figure 3—figure supplement 2
Receptor and ligand availability correlate with signal receiving and sending ability, respectively.
(A) To validate the Notch availability assay, we tested cells expressing hN1(ΔICD)-Gal4esn and observed high levels of availability fluorescence, while CHO-K1 cells lacking ectopic Notch expression …
Figure 3—figure supplement 3
Availability protocol and analysis pipeline.
(A) Test cells were induced for 48 hr with varying concentrations of 4-epiTc. On the day of the experiment, the cells are dissociated with trypsin, split at low density and allowed to reattach to …
Figure 3—figure supplement 4
Untagged DSL ligands show similar effects to their fluorescently tagged counterparts.
(A) We compared the abilities of tagged and untagged DSL ligands to reduce Notch availability in cis. We transiently transfected the Dll1-cerulean plasmid (Figure 2A) or the Jag1-cerulean plasmid (Fi…
Figure 4
Fringe proteins show distinct effects on Jag1-Notch1 and Dll1-Notch1 cis interactions.
(A) Available Notch1 levels for the Notch1+Dll1 cell line without Fringe (red) or with Lfng (magenta), Mfng (orange), or Rfng (blue). Lines are fits to model (‘Materials and methods’). Addition of …
Figure 5
Lfng relieves Jag1-Notch1 but not Dll1-Notch1 cis-inhibition.
(A) Schematic design of time-lapse experiment to analyze cis-interactions. Before the video, cells are pre-induced to express high levels of ligand and then seeded on plates coated with Dll1-Fc. …
Figure 6 with 1 supplement
Delta and Serrate have distinct cis-inhibition phenotypes, and decreasing Fringe activity affects each of these phenotypes differently.
(A) A wild-type fly wing. The five wing veins are marked. Inset is a close-up of the area marked by the rectangle. (B) Loss of one copy of Notch leads to mild wing margin loss and wing vein …
Figure 6—figure supplement 1
Genomic Delta and Serrate transgenes behave similarly to endogenous copies of Notch ligands in Drosophila.
(A and B) Schematic of genomic regions harboring Delta (A) or Serrate (B), their neighboring genes and their corresponding rescue transgenes. (C and D) Addition of two extra copies of Delta (C) or Se…
Figure 7
Fringe modulation of cis interactions generates a set of distinct Notch signaling states.
(A) Lfng or Mfng modification of Notch1 enhances trans activation from Dll1 and weakens trans activation from Jag1 (left). Lfng or Mfng modification of Notch1 enhances cis interactions with Dll1 and …
Figure 8
A model for Notch signaling states during dorsal-ventral boundary formation in the Drosophila wing disc.
(A) A schematic of the Drosophila wing imaginal disc during the third larval instar. During this stage of development, a sharp stripe of Notch signaling occurs at the interface of the dorsal and …
Videos
Video 1
Dilution video assay with a cell line expressing Notch1+Jag1.
Reporter activation shows a delay, as cell divisions are required to dilute out cis-Jag1. Frame rate is 10 fps, with a frame taken ever 20 min.
Video 2
Dilution video assay with a cell line expressing Notch1+Jag1+Lfng.
Reporter activation is immediate despite high cis-Jag1 levels. Frame rate is 10 fps, with a frame taken ever 20 min.
Additional files
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Supplementary file 1
(A) Cell lines used in this work. (B) Plasmids used in this work.
- https://doi.org/10.7554/eLife.02950.018
