Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics

  1. Saskia MA de Man
  2. Gooitzen Zwanenburg  Is a corresponding author
  3. Tanne van der Wal
  4. Mark A Hink  Is a corresponding author
  5. Renée van Amerongen  Is a corresponding author
  1. Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Netherlands
  2. Biosystems Data Analysis, Swammerdam Institute for Life Sciences, University of Amsterdam, Netherlands
  3. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Netherlands
  4. van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Netherlands
19 figures, 6 videos, 8 tables and 3 additional files

Figures

Figure 1 with 2 supplements
Generation of HAP1SGFP2-CTNNB1 cell lines.

(A) Cartoon depicting the current model of the WNT/CTNNB1 pathway. In the absence of WNT ligands (left, ‘OFF’), free cytoplasmic CTNNB1 is captured by the destruction complex consisting of AXIN, …

Figure 1—figure supplement 1
SGFP2-CTNNB1 locus.

(A) Detailed view of CTNNB1 exon two depicting gRNA design relative to the wildtype (top) and repaired (bottom) CTNNB1 allele. Note that the repair template contains the same sequence as the …

Figure 1—figure supplement 2
FACS Gating strategy for haploid HAP1 cells.

(A–C) Single-cell gating based on forward scatter (FSC) and side scatter (SSC). (D) Live cell gating based on DAPI exclusion. (E–F) Haploid cell sorting based on Vibrant live-cell DNA dye. (E) …

Figure 2 with 1 supplement
Functional validation of three independent HAP1SGFP2-CTNNB1 clones.

(A) Western blot, showing CTNNB1 (HAP1WT) and SGFP2-CTNNB1 (HAP1SGFP2-CTNNB1 clones 1, 2, and 3) accumulation in response to CHIR99021 treatment. All panels are from one blot that was cut at the 70 …

Figure 2—figure supplement 1
Verification of the WNT/CTNNB1 responsiveness of HAP1 cells.

(A) Graph depicting AXIN2 qRT-PCR results from HAP1WT cells treated with the indicated range of CHIR99021 (1–10 µM) or DMSO vehicle control (0 µM) for 24 hr. HPRT was used as a reference gene. Error …

Figure 3 with 3 supplements
Live imaging of HAP1SGFP2-CTNNB1.

(A) Representative stills from confocal time-lapse experiments corresponding to Videos 12, showing an increase of SGFP2-CTNNB1 after treatment with 100 ng/ml WNT3A (bottom) relative to a vehicle …

Figure 3—figure supplement 1
Difference analysis of SGFP2-CTNNB1 fluorescence.

(A–B) Plots depicting the relative intensity (left) and the difference in relative intensity to BSA treated cells (right) in the cytoplasm (A) and nucleus (B) after 4 hr of treatment. Circles …

Figure 3—figure supplement 2
Graphs showing quantification of time-lapse microscopy experiments with three independent HAP1SGFP2-CTNNB1 clones.

Stills of this experiment are shown in Figure 3C. Segmentation was performed as described in Figure 4. Arrow indicates the moment of starting the different treatments (BSA in red or 100 ng/ml WNT3A …

Figure 3—figure supplement 3
Unnormalized nuclear and cytoplasmic intensity measurements.

Graphs showing the unnormalized fluorescence intensities quantified for a single biological replicate shown in Figure 3. The vertical black lines indicate the moment of starting the different …

Figure 4 with 1 supplement
Two diffusion-component fit-model for SGFP2-CTNNB1 FCS measurements.

(A) Schematic representation of the point FCS technique, depicting the confocal volume with fluorescent particles diffusing in and out. Particles in FCS are defined by their coherent movement; …

Figure 4—figure supplement 1
Quantification of SGFP2-CTNNB1 fast component using an unfixed two diffusion component model.

Graph depicting the first (i.e. fast) diffusion coefficient in SGFP2-CTNNB1 cells (treated with BSA or WNT3A, data as in Figures 4 and 5) in a two diffusion component model where both the first and …

Figure 5 with 2 supplements
Abundance and mobility of SGFP2-CTNNB1 molecules in living cells after 4 hr WNT3A treatment or control.

Details on sample size and statistics can be found in Supplementary file 1. (A) Graph depicting the total concentration of SGFP2-CTNNB1 particles (monomeric plus complexed) as measured with FCS. (B) …

Figure 5—figure supplement 1
Quantification of SGFP2-CTNNB1 particles, fluorescence and fluorescence lifetime.

Details on sample size and statistics can be found in Supplementary file 1. (A) Graph depicting the total number of SGFP2-CTNNB1 particles as measured with N and B. (B) Graph depicting the average …

Figure 5—figure supplement 2
Number and Brightness analysis.

(A) A series of images are acquired over time (t) (B-C) Representation of the confocal volume in a single pixel of the image in (A) representing monomers with a brightness of 1 (B) and trimers with …

Computational model of WNT/CTNNB1 based on FCS concentrations for free and complexed CTNNB1 (Tables 12).

(A) Schematic overview of the model. DC=destruction complex, DC* = DVL-inactivated DC, CB=cytoplasmic CTNNB1, CB*=phosphorylated CB, NB=nuclear CTNNB1, TCF=TCF/LEF transcription factors, …

Figure 7 with 1 supplement
Cytoplasmic complex characteristics in absence of SGFP2-CTNNB1 N-terminal phosphorylation.

The S45F mutant was introduced using CRISPR (see Figure 7—figure supplement 1) and CHIR treated and control cells were measured after 24 hr. Details on sample size and statistics can be found in Supp…

Figure 7—figure supplement 1
Generation and characterization of a S45F mutant cell line (HAP1SGFP2-CTNNB1(S45F)).

(A–C) FACS graphs showing the selection strategy for HAP1SGFP2-CTNNB1(S45F) cells. HAP1SGFP2-CTNNB1 clone one was transfected with CRISPR constructs similar to Figure 1C, see Materials and methods …

Figure 8 with 2 supplements
In silico and experimental perturbation of WNT signaling.

Details on experimental sample size and statistics can be found in Supplementary file 1. (A) Representative confocal images of HAP1-SGFP2WT (WT, top) and HAP1-SGFP2S45F (S45F, bottom) cells acquired …

Figure 8—figure supplement 1
Live imaging of HAP1SGFP2-CTNNB1 upon CHIR99021 stimulation.

(A) Representative stills from confocal time-lapse experiments corresponding to Video 3, showing an increase of SGFP2-CTNNB1 after treatment with 8 µM CHIR99021. Scale bar = 20 μm. (B–D) …

Figure 8—figure supplement 2
additional biophysical properties of SGFP2-CTNNB1S45F and SGFP2-CTNNB1 under CHIR99021 stimulation.

The S45F mutant was introduced using CRISPR (see Figure 7—figure supplement 1) and CHIR-treated and control cells were measured after 24 hr. Details on sample size and statistics can be found in Supp…

Author response image 1
Boxplots and individual datapoints from a single biological experiment (that is part of Figure 3 previously Figure 4) at 0 and 4 hours after treatment.

The upper panels (raw) show the unnormalized intensity and the bottom panels (normalized) the normalized data. Note that although the axis is very different (0-750 compared to 0-5), the spread in …

Author response image 2
Cells were divided into 3 size categories (small: <500 pixels, middle: 500-1500 pixels, large: >1500 pixels).

Graphs show the normalized cytoplasmic intensity (top), normalized nuclear intensity (middle) and the nuclear/cytoplasmic ratio (bottom) for cells treated for 4 hours with BSA (control) 100 ng/ml …

Author response image 3
These images show a double-tagged line that has endogenously tagged SGFP2-CTNNB1 (left panel, green) and endogenously tagged mScI-GSK3B (middle panel, magenta).
Author response image 4
Figure displaying the relation between the particle number (x-axis) and the normalized brightness (y-axis).

The number of particles is up to 3fold higher than on average in our endogenously tagged SGFP2CTNNB1 cell lines. However, we see no apparent effect of these higher particle numbers on the normalized …

Author response image 5
Waterwheel model of CTNNB1 turnover.
Author response image 6
Comparison of old model without steep (A,C,E) and new model with steep (B,D,F).

In the old model the change in k6/k7 and k9/k8 is immediate at the addition of WNT3A. This results in a sudden loss of cytoplasmic CTNNB1 (A) and gain in the nucleus (C) as is also apparent from the …

Author response image 7
These images show a double-tagged line that has endogenously tagged SGFP2CTNNB1 (left panel, green) and endogenously tagged mScI-GSK3B (middle panel, magenta).
Author response image 8
Autocorrelation curves from the same experiment with a small offset of 0.

002 (A) as original example in Figure 5G (now Figure 4G) or without offset (B).

Author response image 9
Comparison of analysis of short and long FCS traces.

Autocorrelation curves were made from nine 10s portion of the measurement (trace), or a single 90s trace from the same FCS measurement of SGFP2-CTNNB1 and subsequently fitted with the same fitting …

Author response image 10
SGFP2-CTNNB1 parameters fitted by a two-diffusion component model where T1 is free (A-C) or fixed at 14.9 um2/s (D-F).

(A and D) Concentration of SGFP2-CTNNB1 (B and E) Fraction of slow SGFP2-CTNNB1 (C and F) Speed of slow SGFP2-CTNNB1.

Author response image 11
Differences between EGFP and SGFP2.

(A) protein alignment of EGFP (top) and SGFP2 (bottom) showing the 4 amino acid difference. (B) Independent measurements in an additional mammalian cell line of monomeric EGFP and SGFP2 brightness. …

Videos

Video 1
Representative video of confocal time-lapse experiments, showing SGFP2-CTNNB1 (left, green), SiR-DNA staining (middle, magenta), and transmission image (right, gray) after treatment with vehicle control (BSA).

Time of addition is at 00:00:00 (indicated at the top left). Scale bar in the lower right represents 20μm.

Video 2
Representative video of confocal time-lapse experiments, showing SGFP2-CTNNB1 (left, green), SiR-DNA staining (middle, magenta), and transmission image (right, gray) after treatment with 100 ng/ml WNT3A.

Time of addition is at 00:00:00 (indicated at the top left). Scale bar in the lower right represents 20μm.

Video 3
Representative video of confocal time-lapse experiments, showing SGFP2-CTNNB1 (left, green), SiR-DNA staining (middle, magenta), and transmission image (right, gray) after treatment 8 µM CHIR99021.

Time of addition is at 00:00:00 (indicated at the top left). Scale bar in the lower right represents 20μm.

Video 4
Video showing the quantification of the normalized intensity of SGFP2-CTNNB1 in the cytoplasm of time-lapse microscopy series (from Figure 4 and Videos 13) at each time point showing all individual cells from three biological experiments.

Time of addition of the indicated substances is at 00:00:00 (indicated at the top left). The left graph represents the raw data (colored dots, each dot is one cell, n=155–400 cells for each …

Video 5
Video showing the quantification of of the normalized intensity of SGFP2-CTNNB1 in the nucleus of time-lapse microscopy series (from Figure 4 and Videos 13) at each time point showing all individual cells from three biological experiments.

Time of addition of the indicated substances is at 00:00:00 (indicated at the top left). The left graph represents the raw data (colored dots, each dot is one cell, n=155–400 cells for each …

Video 6
Video showing the quantification of the nuclear-cytoplasmic ratio of SGFP2-CTNNB1, calculated from raw intensity values underlying Videos 4 and 5.

At each time point showing all individual cells from three biological experiments. Time of addition of the indicated substances is at 00:00:00 (indicated at the top left). The left graph represents …

Tables

Table 1
Total number of SGFP2-CTNNB1 molecules in the confocal volume and corresponding calculated concentrations obtained from FCS measurements in n=3 independent experiments.

The concentration is calculated from the number of molecules in the confocal volume and the calibrated confocal volume (see Materials and methods). The number of molecules is consistent with those …

Number of moleculesConcentration (nM)
CompartmentTreatmentNMedian95% CIMedian95% CI
CytoplasmBSA218070–116180127–218
WNT3A219585–122221144–250
NucleusBSA216353–7212291–158
WNT3A18135127–150240217–325
Table 2
Number of SGFP2-CTNNB1 molecules in the confocal volume and corresponding calculated concentration of SGFP2-CTNNB1 molecules with the fast or slow diffusion coefficient obtained from FCS measurements in n=3 independent experiments.

The concentration is calculated from the number of molecules and the calibrated confocal volume (see Materials and methods).

Fast SGFP2-CTNNB1Slow SGFP2-CTNNB1
Number of moleculesConcentration (nM)Number of moleculesConcentration (nM)
CompartmentTreatmentnMedian95% CIMedian95% CIMedian95% CIMedian95% CI
CytoplasmBSA215140–639166–1392920–375738–76
WNT3A216047–8014576–1683530–416857–76
NucleusBSA214841–668778–119132–22224–40
WNT3A189681–101170147–2144737–498664–104
Table 3
Brightness of SGFP2-compared relative to EGFP-monomer and -dimer controls in n=2 independent experiments. N is the number of analyzed cells.

p-Values were calculated using PlotsOfDifferences that uses a randomization test (Goedhart, 2019).Note that only the EGFP-dimer is significantly different to the EGFP-monomer control, while …

FluorophoreCompartmentTreatmentNMedian95 CI medianp-value to matched control (EGFP monomer in the nucleus or cytoplasm)
EGFP-monomerCytoplasmNA1510.79–1.341.000
EGFP-dimerCytoplasmNA141.41.29–1.600.011*
SGFP2-CTNNB1CytoplasmBSA690.920.83–1.000.738
SGFP2-CTNNB1Cytoplasm100 ng/ml WNT3A461.010.93–1.110.919
EGFP-monomerNucleusNA1510.91–1.071.000
EGFP-dimerNucleusNA141.621.44–1.69<0.001*
SGFP2-CTNNB1NucleusBSA690.870.78–0.960.192
SGFP2-CTNNB1Nucleus100 ng/ml WNT3A461.050.95–1.150.578
Table 4
Variables Minimal Model of WNT signaling.
Model nameVariableCompoundValues obtained fromWNT OFF (nM)WNT ON (nM)
CBx1Free cytoplasmic CTNNB1FCS data this report91145
DCx2Free destruction complexModel equations82.452
CB*-DCx3DC-bound phosphorylated CTNNB1FCS data this report*62.5*62.5*
DC*x4Inactivated destruction complexModel equations0**30.5**
NBx5Free nuclear CTNNB1FCS data this report87170
TCFx6Free TCFModel equations8117
NB-TCFx7TCF-bound nuclear CTNNB1FCS data this report22.286
TCF0TCF0Total TCFx7 and Tan et al., 2012 - Figure 11103103
  1. *Under the assumption that k3 does not change, the levels of CB*-DC remain equal. Since there was no significant difference between the concentration of slow SGFP2-CTNNB1 in the absence or presence of WNT3A (57 nM versus 68 nM, not significant, Table 2) the average of both medians (62.5 nM) was used. ** In WNT OFF, w=0, and no inactivated destruction complex is formed. In WNT ON, w=1, which induces the formation of inactivated destruction complex at the expense of free destruction complex (see Equations 7a and 9 in the model description in Materials and methods).

Table 5
Equilibrium conditions for the Minimal Model of WNT signaling.

All rates are multiplied with factor R=20, so that the equilibrium is reached at 4.5 hr according to Figure 4C–D.

Rate constantBiological processValues based onWnt offWnt on
bnMmin−1CTNNB1 synthesisv12 from Lee0.4230.423
k2k1nMBinding to and phosphorylation by the destruction complex of cytoplasmic CTNNB1K8 from Lee120120
k3min−1Dissociation and degradation of phosphorylated CTNNB1 from the destruction complexDeduced from b and x30.00680.0068
k5k4nMInactivation of the destruction complex by activated DVLFitted to x1 and x7N.A.*1.7
k6k7Ratio between nuclear import and export of CTNNB1Deduced from x1 and x50.961.17
k9k8nMDissociation of nuclear CTNNB1 from TCFDeduced from x5, TCF0, x732033.6
  1. *In WNT OFF, w=0, and no inactive destruction complex is formed. Only in WNT ON, w=1, which induces the formation of inactivated destruction complex at the expense of free destruction complex (see Equations 7a and 9 in the model description in Materials and methods).

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HAP1Whitehead InstituteCellosaurus: CVCL_Y019kind gift from Thijn Brummelkamp (NKI)
Cell line (Homo sapiens)HAP1SGFP2-CTNNB1This paper
Cell line (Homo sapiens)HAP1SGFP2-CTNNB1(S45F)This paper
Transfected construct (Homo sapiens)pSpCas9(BB)−2A-Puro (PX459) V2.0Ran et al., 2013RRID:Addgene_62988
Transfected construct (Homo sapiens)MegaTopflashHu et al., 2007kind gift from Christophe Fuerer and Roel Nusse, Stanford University
Transfected construct (Homo sapiens)CMV RenillaPromegaE2261
Transfected construct (Homo sapiens)pSGFP2-C1Kremers et al., 2007RRID:Addgene_22881kind gift from Dorus Gadella
Transfected construct (Homo sapiens)pmScarlet-i_C1Bindels et al., 2017RRID:Addgene_85044kind gift from Dorus Gadella
Transfected construct (Homo sapiens)pSYFP2-C1Kremers et al., 2006RRID:Addgene_22878kind gift from Dorus Gadella
Transfected construct (Homo sapiens)mTurquoise2-C1Goedhart et al., 2012RRID:Addgene_54842kind gift from Dorus Gadella
Transfected construct (Homo sapiens)pEGFPClontech
Transfected construct (Homo sapiens)pEGFP2Pack et al., 2006kind gift from Masataka Kinjo
Transfected construct (Homo sapiens)pEGFP3Pack et al., 2006kind gift from Masataka Kinjo
Transfected construct (Homo sapiens)pBluescript II KS(+)Stratagene
Transfected construct (Homo sapiens)pX459-CTNNB1-ATGThis paperRRID:Addgene_153429
Transfected construct (Homo sapiens)pX459-CTNNB1-S45This paperRRID:Addgene_164587
Transfected construct (Homo sapiens)pRepair-SGFP2-CTNNB1This paperRRID:Addgene_153430
Recombinant DNA reagentpRepair-mScI-CTNNB1This paperRRID:Addgene_153431
Recombinant DNA reagentpRepair-SYFP2-CTNNB1This paperRRID:Addgene_153432
Recombinant DNA reagentpRepair-mTq2-CTNNB1This paperRRID:Addgene_153433
Chemical compound, drugCHIR99021Biovision1677–56 mM stock in DMSO
Peptide, recombinant proteinRecombinant Mouse Wnt-3aR and D systems1324-WN-00210 µg/ml stock solution in 0.1% BSA in PBS
Chemical compound, drugDapiInvitrogenD1306
Chemical compound, drugVybrant DyeCycle Violet StainInvitrogenV35003
Chemical compound, drugVybrant DyeCycle Ruby StainInvitrogenV10273
AntibodyNon-phosphorylated (Active) β-catenin clone D13A1 (Rabbit monoclonal)Cell Signaling8814S
RRID:AB_11127203
WB (1:1000)
AntibodyTotal β-catenin clone 14 (mouse monoclonal)BD610153
RRID:AB_397554
WB (1:2000)
Antibodyα-Tubulin clone DM1A (mouse monoclonal)Sigma-AldrichT9026
RRID:AB_477593
WB (1:1000)
AntibodyGFP Antibody (Rabbit polyclonal)InvitrogenA-6455
RRID:AB_221570
WB (1:1000)
AntibodyIRDye 680LT Goat anti-Rabbit IgGLI-COR926–68021
RRID:AB_10706309
WB (1:20,000)
AntibodyIRDye 800CW Donkey anti-Mouse IgGLI-COR926–32212
RRID:AB_621847
WB (1:20,000)
Chemical compound, drugSiR-DNASpirochromeSC007
Chemical compound, drugAlexa Fluor 488 NHS EsterMolecular probesA20000
Software, algorithmFlowJo
Software, algorithmCellProfiler pipelineThis paperavailable at https://osf.io/6pmwf/
Software, algorithmFIJI/ImageJ
Software, algorithmFFS Dataprocessor version 2.3SSTC
Software, algorithmptu converterCrosby et al., 2013
Software, algorithmImageJ macro scriptmodified from Crosby et al., 2013available at https://osf.io/ys5qw/
Software, algorithmPlotsOfDifferencesGoedhart, 2019https://huygens.science.uva.nl/PlotsOfDifferences/
Software, algorithmRStudio
Software, algorithmR scriptThis paperavailable at https://osf.io/sxakf/
Software, algorithmR shiny appThis paperWNT_minimal_model_v2.4.Rapp available at https://wntlab.shinyapps.io/WNT_minimal_model/, source script available at https://osf.io/27ya6/
Table 6
primers/oligonucleotides used in this study.
primersequence
RVA24CAAGTTTGTTGTAGGATATGCCC
RVA25CGATGTCAATAGGACTCCAGA
RVA124AGTGTGAGGTCCACGGAAA
RVA125CCGTCATGGACATGGAAT
RVA555GCCAAACGCTGGACATTAGT
RVA558AGACCATGAGGTCTGCGTTT
RVA561CACCGTTGCCTTTACCACTCAGAGA
RVA562AAACTCTCTGAGTGGTAAAGGCAAC
RVA567CACCGTGAGTAGCCATTGTCCACGC
RVA568AAACGCGTGGACAATGGCTACTCAC
RVA581tgctcaccatggtggGATTTTCAAAACAGTTGTATGGTATACTTC
RVA582actgttttgaaaatcCCACCATGGTGAGCAAGGGC
RVA583agtagccattgtccaCTTGTACAGCTCGTCCATGCCG
RVA584gacgagctgtacaagTGGACAATGGCTACTCAAGGTTTG
RVA618atacgactcactatagggcgaattggagctGATGCAGTTTTTTTCAATATTGC
RVA619ttctagagcggccgccaccgcggtggagctCTCTCTTTTCTTCACCACAACATTTTATTTAAAC
RVA622AAGAGAGAGCTCCACCGCGGTGGCGGCCG
RVA623TGCATCAGCTCCAATTCGCCCTATAGTGAGTCG
RVA1616tgtccacgctgGATTTTCAAAACAGTTGTATGG
RVA1617atacaactgttttgaaaatccagcgtggacaATGGTGAGCAAGGGCGAG
RVA1618cacaaaccttgagtagccatCTTGTACAGCTCGTCCATGC
RVA1619ATGGCTACTCAAGGTTTGTGTCATTAAATC
RVA2540CTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTTCCTGTCCGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGT
Author response table 1
This table describes different scenarios of how different brightness species would impact the average apparent brightness.
Scenario 1actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB115050-
complex containing 1 SGFP2-CTNNB115050-
1
Scenario 2actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB115020-
complex containing 2 SGFP2-CTNNB125080-
1.67
Scenario 3actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB118050-
complex containing 2 SGFP2-CTNNB122050-
1.33
Scenario 4actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB115010-
complex containing 3 SGFP2-CTNNB135090-
2.5
Scenario 5actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB119050-
complex containing 3 SGFP2-CTNNB131050-
1.5
Scenario 6actual brightnessactual fraction (%)apparant fraction (%)average apparent brightness
monomer SGFP2-CTNNB119950-
complex containing 10 SGFP2-CTNNB110150-
1.83

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