Cis-activation in the Notch signaling pathway

  1. Nagarajan Nandagopal
  2. Leah A Santat
  3. Michael B Elowitz  Is a corresponding author
  1. California Institute of Technology, Howard Hughes Medical Institute, United States
5 figures, 2 videos, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
Engineered CHO-K1 N1D1 + Rfng cells show ligand-dependent cis-activation.

(A) Schematic of actual and potential cis- and trans-interaction modes in the Notch pathway. (B) Schematic of the N1D1 + Rfng cell line. CHO-K1 cells were engineered to express a chimeric receptor …

https://doi.org/10.7554/eLife.37880.002
Figure 1—figure supplement 1
Cis-activation assay enables isolation of individual engineered cells.

(A) Schematic of ‘control’ cis-activation assay used to verify that the relative density of cells was low enough to prevent trans-interactions. N1D1 + Rfng cells lacking H2B-Cerulean expression …

https://doi.org/10.7554/eLife.37880.003
Figure 1—figure supplement 2
Cis- and trans-activation share similar features.

(A) Histograms of mCherry fluorescence in cells analyzed in Figure 1E. Cells were categorized as expressing low, medium, or high Dll1 levels (shades of grey). (B) Fold increase in isolated, or …

https://doi.org/10.7554/eLife.37880.004
Figure 1—figure supplement 3
Cis-activation occurs with the wild-type Notch1 receptor and in multiple cell types.

(A) (Left) The N1WTD1+Rfng cell line (schematic). CHO-K1 cells were engineered to express wild-type Notch1 receptor (‘N1WT’, green), an H2B-Citrine reporter (yellow) activated by cleaved NICD …

https://doi.org/10.7554/eLife.37880.005
Figure 1—figure supplement 4
Cis-activation occurs with endogenous ligands and receptors in Caco-2 and NMuMG cells.

(A) Fold increase in mean Notch activation levels in sparsely plated Caco-2 cells, transfected with the 12xCSL-H2B-Citrine reporter construct, with or without 10 uM DAPT. < 24 hr after plating, …

https://doi.org/10.7554/eLife.37880.006
Figure 2 with 2 supplements
Cis-activation is affected by changes in ligand-receptor affinity.

(A) (Top) Cell lines used for analyzing effect of Rfng on cis-activation. (Bottom) Plots showing mean Notch activation (reporter Citrine fluorescence normalized to background fluorescence in …

https://doi.org/10.7554/eLife.37880.009
Figure 2—figure supplement 1
Rfng does not modify the cis-activation behavior of N1D4 cells.

(A) (Top) Cell lines used for analyzing effect of Rfng (purple) on cis-activation, in the context of Notch1 and Dll4. (Bottom) Comparison of mean cis-activation in polyclonal N1D4 cells with …

https://doi.org/10.7554/eLife.37880.010
Figure 2—figure supplement 2
Notch2 lacks cis-inhibition with Dll1 or Dll4.

(A) Mean Notch activation levels, relative to background reporter fluorescence, in polyclonal N2D1 Pop cells plated on surfaces coated with (black) or without (blue) 2.5 ug/ml recombinant human Dll1 …

https://doi.org/10.7554/eLife.37880.011
Figure 3 with 1 supplement
Cis-activation occurs in neural stem cells and regulates survival.

(A) E14.5 mouse cortical neural stem cells (NSCs) were plated sparsely and treated with ± 10 μM DAPT, cultured under growth conditions with low growth factors, and subsequently assayed for …

https://doi.org/10.7554/eLife.37880.012
Figure 3—figure supplement 1
RNAseq analysis of Notch pathway component expression in neural stem cells.

(A) Expression levels of Notch receptors, ligands, and Fringes, measured using RNAseq (see Materials and methods), in neural stem cells cultured in the presence of 0.5 ng/ml EGF and 10 μM DAPT for …

https://doi.org/10.7554/eLife.37880.013
Figure 4 with 1 supplement
Receptor-ligand cell surface interactions are necessary for cis-activation.

(A) Schematics showing how soluble recombinant N1ECD-Fc protein (rN1ECD-Fc) can be used to test whether surface interactions between ligand (red) and receptor (green) are necessary for cis-activation…

https://doi.org/10.7554/eLife.37880.014
Figure 4—figure supplement 1
Surface perturbations affect N1D1+Rfng cis-activation.

(A) (Left) Schematic showing how recombinant N1ECD-Fc protein (rN1ECD-Fc) affects trans-activation between cells expressing ligands (red) and receptors (green). rN1ECD-Fc protein, present in excess, …

https://doi.org/10.7554/eLife.37880.015
Figure 5 with 1 supplement
Mathematical model of cis-activation reveals potential roles in signal processing.

(A) In each cis-activaiton model, Notch (‘N’, green) and Delta (‘D’, blue) interact to produce one or more cis-complexes, which can be active (‘C+’), producing NICD (green arrow) or inhibited (‘C-’, …

https://doi.org/10.7554/eLife.37880.016
Figure 5—source data 1

MATLAB code and parameter sets used for models in Figure 5.

https://doi.org/10.7554/eLife.37880.018
Figure 5—figure supplement 1
1Latin Hypercube Sampling generates evenly distributed parametersScatter plots showing pairwise distributions of parameters tested in Models 1 and 2a-d.

For clarity, each plot only shows 3000 parameter pairs, randomly subsampled from the 10,000 total parameters sets analyzed. Orange dots are subsampled from parameters that produce non-monotonic C+ …

https://doi.org/10.7554/eLife.37880.017

Videos

Video 1
Cis-activation of isolated engineered CHO-K1 cells.

Examples of isolated CHO-K1 N1D1 + Rfng cells activating prior to cell division in the cis-activation assay. (Top row) Blue channel shows fluorescence of the constitutively expressed nuclear …

https://doi.org/10.7554/eLife.37880.007
Video 2
Cis-activation of isolated engineered NMuMG cells.

Examples of isolated NMuMG N1D1 + Rfng cells activating prior to cell division in the cis-activation assay. (Top row) Blue channel shows fluorescence of the constitutively expressed nuclear …

https://doi.org/10.7554/eLife.37880.008

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene
(Mus musculus)
Dll1NCBI ID:
13388
Gene
(Homo sapiens)
Dll1NCBI ID:
28514
Gene
(Homo sapiens)
Dll4NCBI ID:
54567
Gene
(Mus musculus)
R-fringeNCBI ID:
19719
Gene
(Homo sapiens)
Notch1NCBI ID: 4851
Gene
(Homo sapiens)
Notch2NCBI ID: 4853
Cell line
(Cricetulus griseus)
CHO-K1Thermo Fisher
Scientific
(T-REx CHO-K1)
Cat# R71807
RRID:CVCL_D586
Figures 1, 2 and 4;
Figure 1—figure supplements 1, 2 and 3;
Figure 2—figure supplements 1 and 2;
Figure 4—figure supplement 1
Cell line
(Cricetulus
griseus)
N1D1Derived from
CHO-K1
CHO-K1 expressing
pEF-hNECD-Gal4esn
+ pcDNA5-
TO-Dll1-T2A-H2B-mCherry
 + pEV-UAS-H2B-Citrine
Figure 2
Cell line
(Cricetulus
griseus)
N1D1 + RfngDerived from N1D1CHO-K1 expressing
pEF-hNECD-Gal4esn + pcDNA5-
TO-Dll1-T2A-H2B-mCherry +
pEV-UAS-H2B-Citrine + pLenti-
CMV-R-fringe-T2A-Puromycin

Figures 1, 2 and 4;Figure 1—figure supplements 1 and 2; Figure 4—figure supplement 1
Cell line
(Cricetulus
griseus)
N1WTD1 + RfngDerived from CHO-K1CHO-K1 expressing
pcDNA3‐hN1‐mod1 +
pcDNA5-TO-Dll1-mCherry
+ pEV-12xCSL-H2B-Citrine +
piggyBac CMV-R-fringe+pCS-
H2B-Cerulean
Figure 1—figure supplement 3
Cell line
(Mus
musculus)
NMuMGATCCCat# CRL-1636 (Wild-type
cells used to transfect
in piggyBac-12xCSL-
H2B-Citrine)
RRID:CVCL_0075
Figure 1—figure supplement 4
Cell line
(Mus
musculus)
NMuMG + Dll1Derived from NMuMGBase wild-type cell line
expressing
piggyBac-12xCSL-H2B-Citrine
+ piggyBac-TO-Dll1-T2A
-H2B-mCherry-P2A-Hygromycin
Figure 1—figure supplement 4
Cell line
(Mus
musculus)
NMuMG N1D1 + RfngDerived from NMuMGNMuMG ΔN2ΔJ1
expressing piggyBac-
CMV-hNECD-Gal4-ANK
-T2A-H2B-Cerulean +
piggyBac CMV-TO
Dll1-T2A-H2B-mCherry-
P2A-Hygromycin + pEV
-2xHS4-UAS-H2B-Citrine-
T2A-tTS-2xHS4-Blast-T2A-
rTetR-HDAC4-P2A-R-fringe
Figure 1—figure supplement 3
Cell line
(Homo
sapiens)
Caco-2ATCC (Caco-2 C2BBe1)Cat# CRL-2102 (Wild-type
cells used to transfect in
pEV-12xCSL-H2B-Citrine)
RRID:CVCL_1096
Figure 1—figure supplement 4
Cell line
(Cricetulus
griseus)
N1D1 PopDerived from CHO-K1CHO-K1 with pEV-UAS-
H2B-Citrine + pCS-H2B-
Cerulean +
piggyBac-TO-Dll1-T2A-H2
B-mCherry + piggyBac-
CMV-hN1ECD-Gal4 - Cell
population (Pop)
Figure 2
Cell line
(Cricetulus
griseus)
N1D4 PopDerived from CHO-K1CHO-K1 with pEV-UAS-
H2B-Citrine + pCS-H2B-
Cerulean + piggyBac-
TO-Dll4-T2A-H2B-mCherry +
piggyBac-CMV-hN1ECD-Gal4 -
Cell population (Pop)
Figure 2;
Figure 2—figure supplement 1
Cell line
(Cricetulus
griseus)
N2D1 PopDerived from CHO-K1CHO-K1 with pEV-UAS-H2B-
Citrine + pCS-H2B-Cerulean +
piggyBac-TO-Dll1-T2A-H2B
-mCherry + piggyBac-
CMV-hN2ECD-Gal4 - Cell
population (Pop)
Figure 2;
Figure 2—figure supplement 2
Cell line
(Cricetulus
griseus)
N2D4 PopDerived from CHO-K1CHO-K1 with pEV-UAS-
H2B-Citrine + pCS-H2B-
Cerulean + piggyBac
-TO-Dll4-T2A-H2B-mCherry +
piggyBac-CMV-hN2ECD-Gal4 -
Cell population (Pop)
Figure 2;
Figure 2—figure supplement 2
Cell line
(Mus musculus)
NSCEMD MilliporeCat# SCR029 (E14.5 mouse
neural cortical stem cells - NSC)
Figure 3;
Figure 3—figure supplements 1 and 2
Transfected
construct
(recombinant
DNA)
pEV‐UAS‐H2B‐
Citrine
Sprinzak et al., 2010N/AReporter for
Notch1ECD-Gal4 receptor in
CHO cells
Transfected
construct
(recombinant
DNA)
pEV-2xHS4-UAS-
H2B-Citrine-T2A-
tTS-2xHS4-Blast-
T2A-rTetR-HDAC4-
P2A-R-fringe
This paperN/AReporter for Notch1ECD-
Gal4-ANK receptor in
NMuMG cells (tTS was
not relevant for this
work and was inactivated
by 4-epiTc); rTetR-HDAC4
was used to decrease
Delta expression in the
presence of Dox;
Constitutive R-fringe
expression in NMuMG cells
Transfected
construct
(recombinant
DNA)
pEV-12xCSL‐
H2B‐Citrine
Sprinzak et al., 2010N/AReporter for Notch1
wild-type receptor
in CHO and Caco-2 cells
Transfected
construct
(recombinant
DNA)
pEF-hN1ECD
-Gal4
This paperN/ANotch1ECD-Gal4
synthetic receptor
used in CHO clones
Transfected
construct
(recombinant
DNA)
pX330
(CRISPR-Cas9
plasmid system)
Cong et al., 2013N/APlasmid used to insert
RNA guide sequence
for CRISPR knockdown
Transfected
construct
(recombinant
DNA)
piggyBac-12x
CSL-H2B-Citrine
This paperN/ANotchWT reporter
placed into NMuMG
WT cells but not used
in this study
Transfected
construct
(recombinant
DNA)
piggyBac-CMV
-hN1ECD-Gal4
This paperN/ANotch1ECD-Gal4
synthetic receptor
used in CHO populations
Transfected
construct
(recombinant
DNA)
piggyBac-CMV
-hN2ECD-Gal4
This paperN/ANotch2ECD-Gal4
synthetic receptor used
in CHO populations
Transfected
construct
(recombinant
DNA)
pcDNA3‐hN1‐mod1Sprinzak et al., 2010N/AWild-type Notch1
receptor used in
CHO clones
Transfected
construct
(recombinant
DNA)
piggyBac-CMV-
hNECD-Gal4-ANK
-T2A-H2B-Cerulean
This paperN/ANotch1ECD-Gal4-ANK
synthetic receptor
used in NMuMG clones
Transfected
construct
(recombinant
DNA)
pcDNA5-TO-Dll1
-T2A-H2B-mCherry
Nandagopal et al., 2018N/AInducible Delta-like1
ligand used in CHO
clones and populations
Transfected
construct
(recombinant
DNA)
pcDNA5-TO‐
Dll1‐ mCherry
Sprinzak et al., 2010N/AInducible
Delta-like1-mCherry
fusion used in CHO
clones with Notch1WT
receptor
Transfected
construct
(recombinant
DNA)
piggyBac-CMV-TO
Dll1-T2A-H2B-
mCherry-P2A-
Hygromycin
Nandagopal et al., 2018N/AInducible Delta-like1
ligand used in CHO
populations and
NMuMG clones
Transfected
construct
(recombinant
DNA)
piggyBac-CMV-TO-
Dll4-T2A-H2B-mCherry-P2A-Hygromycin
Nandagopal et al., 2018N/AInducible Delta-like4
ligand used in CHO
populations
Transfected
construct
(recombinant
DNA)
pLenti-CMV-R-
fringe-T2A-
Puromycin
This paperN/AConstitutive R-fringe
expression in CHO
N1D1 + Rfng cells
Transfected
construct
(recombinant
DNA)
pCS‐H2B‐CeruleanSprinzak et al., 2010N/ASegmentation color
used in CHO cells
Transfected
construct (siRNA)
Allstar Negative
Control
QiagenSI03650318Control siRNA,
Figure 3C
Transfected
construct (siRNA)
Dll1 siRNAThermo Fisher
Scientific
Cat# 4390771
(ID: s65000)
Dll1 siRNA,
Figure 3C
Antibodyrabbit anti-
mouse Notch2
Cell Signaling
Technologies
Cat# 5732
RRID:AB_10693319
WB (1:1000)
Antibodyrabbit anti-
mouse Jagged1
Cell Signaling
Technologies
Cat# 2620
RRID:AB_659968
WB (1:1000)
Antibodyrabbit anti-
mouse GAPDH
Cell
Signaling
Technologies
Cat# 2118
RRID:AB_561053
WB (1:3000)
Antibodyrabbit anti-
mouse Dll1-ICD
Kindly provided by
Gerry Weinmaster,
UCLA
Antibody 88 cWB (1:2000)
AntibodyECL Rabbit IgG
HRP-linked whole
antibody from
Donkey Secondary
GE Healthcare
Life Sciences
Cat #NA934
RRID:AB_772206
WB (1:2000)
AntibodyAnti-mouse AlexFluor
488 Secondary
Thermo Fisher
Scientific
Cat# A21202
RRID:AB_141607
ICC (1:1000)
OtherSuperSignal West
Pico Chemiluminescent
Substrate
Thermo Fisher
Scientific
Cat# 34580as recommended
per the manufacturer
OtherSuperSignal West
Femto
Chemiluminescent
Substrate
Thermo Fisher
Scientific
Cat# 34095as recommended
per the manufacturer
Recombinant
DNA reagent
Lipofectamine LTX
plasmid transfection
reagent
Thermo Fisher
Scientific
Cat# 15338–100as recommended
per the manufacturer
Recombinant
DNA reagent
ViraPower Lentiviral
Expression System
Thermo Fisher
Scientific
Cat# K497500as recommended
per the manufacturer
Peptide,
recombinant
protein
Recombinant mouse
IgG2A Fc Protein
R and D SystemsCat# 4460 MG-10010 ug/ml
Peptide,
recombinant
protein
Recombinant mouse
Dll1 Fc chimera
R and D SystemsCat# 5026 DL-05010 ug/ml
Peptide,
recombinant
protein
Recombinant
mouse
Notch-1 Fc
chimera
R and D SystemsCat# 5267-TK-05010 ug/ml
Peptide,
recombinant
protein
Recombinant human
Dll1ext-Fc fusion
proteins
Sprinzak et al., 2010Kindly provided
by Irwin Bernstein,
MD at Fred Hutchinson
Cancer Research Center
2.5 ug/ml
Chemical
compound, drug
DAPTSigma AldrichCat# D59421 uM (CHO cells);
10 uM (all other cells)
Chemical
compound, drug
4-epi tetracycline
Hydrochloride
Sigma AldrichCat# 379180–200 ng/ml
Chemical
compound, drug
DoxycyclineTakara Bio USA IncCat# 6313111 ug/ml or 10 ug/ml
Chemical
compound, drug
DexamethasoneSigma AldrichCat# D4902100 ng/ml
Commercial
assay or kit
Miniprep kitQiagenCat# 27106
Commercial
assay or kit
QIAquick PCR
Purification kit
QiagenCat# 28104
Commercial
assay or kit
RNeasy mini kit
for RNA extraction
QiagenCat# 74106
Commercial
assay or kit
iScript cDNA
synthesis kit
Bio-RadCat# 1708890
Commercial
assay or kit
iQ SYBR Green
Supermix
Bio-RadCat# 1708880
Commercial
assay or kit
SsoAdvanced
Universal Probes
Supermix
Bio-RadCat# 172–5282
Commercial
assay or kit
DNA HCR kitMolecular Instruments
Software,
algorithm
Cell segmentation
and tracking
Nandagopal et al., 2018N/AFigure 1,
Figure 1—figure supplement 2
Software,
algorithm
FISH Transcript
detection and
quantification
This paperhttps://github.com/nnandago/elife2018-dot_detection; Nandagopal, 2018aMATLAB code for
visualizing, segmenting,
and detecting transcript
dots in FISH-labeled cells.
Used to generate
Figure 1—figure supplement 2,
Figure 3—figure supplement 1
Software,
algorithm
cis-activation modelThis paperhttps://github.com/nnandago/elife2018-cis_activation_modeling; Nandagopal, 2018bMATLAB code for
modeling steady
state concentrations
of ligand, receptors and
complexes for a
range of parameters.
Used to generate Figure 5,

Figure 5—figure supplement 5–

Additional files

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