Increasing β-catenin/Wnt3A activity levels drive mechanical strain-induced cell cycle progression through mitosis
- Stanford University, United States
Figures
Figure 1 with 2 supplements
Mechanical strain is sufficient to drive cell cycle re-entry, but not entry into mitosis.
(A) Distribution of mAG-Geminin in Fucci MDCK monolayers 0 hr or 24 hr after the application of No Strain or High Strain (~8.5%) using the biaxial live cell stretcher. Scale bar: 150 μm. (B) …
-
Figure 1—source data 1
Data used to construct graphs in Figure 1 and Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.19799.004
Figure 1—figure supplement 1
Design and calibration of bi-axial live cell stretcher.
Schematic representation of the side view (A) and top view (B) of the biaxial live cell stretcher. Representative image (C) and quantification (D) of displacement of immobilized fluorescent …
Figure 1—figure supplement 2
Mitotic events and distance to nearest neighbor in fixed MDCK monolayers after mechanical strain.
(A) Nuclear stain (Hoescht) of fixed MDCK monolayers 2–24 hr after application of mechanical strain using the ISA. (B) Quantification of average distance to nearest neighbor (B) or number of mitotic …
Figure 2
Mechanical strain does not induce significant DNA damage or activation of DNA repair.
Distribution of phospho-γΗ2A.X (A), p53 (C), and p53BP1 (E) in MDCK monolayers 24 hr after No Strain or High Strain (15%) using the ISA, or in actively cycling cells treated with DMSO or MMS. …
-
Figure 2—source data 1
Data used to construct graphs in Figure 2.
- https://doi.org/10.7554/eLife.19799.010
Figure 3 with 5 supplements
Mechanical strain induces a Src-dependent increase in Y654 phosphorylated β-catenin and β-catenin transcriptional activity.
Distribution of TOPdGFP at 8 hr (A), β-catenin at 8 hr (A, insets), EdU at 24 hr (C), and pY654 β-catenin at 8 hr (E) in MDCK monolayers after No Strain or High Strain (15%) applied by the ISA, …
-
Figure 3—source data 1
Data used to construct graphs in Figure 3 and Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.19799.012
Figure 3—figure supplement 1
Distribution of β-catenin in monolayers treated with either DMSO or the Src Inhibitor SU6656 8 hr after the application of No Strain or High Strain (15%) using the ISA.
Scale bars: 25 μm.
Figure 3—figure supplement 2
The Src Inhibitor PP2 blocks strain-induced increases in Y654 phosphorylated β-catenin and β-catenin transcriptional activity.
Distribution of TOPdGFP at 8 hr (A), β-catenin at 8 hr (A, insets), EdU at 24 hr (C), and pY654 β-catenin at 8 hr (E) in MDCK Monolayers after No Strain or High Strain (15%) applied by the ISA, …
Figure 3—figure supplement 3
Distribution of β-catenin in monolayers treated with either DMSO or the Src Inhibitor PP2 8 hr after the application of No Strain or High Strain (15%) using the ISA.
Scale bars: 25 μm.
Figure 3—figure supplement 4
EGFR inhibition reduces an increase in pY654 β-catenin following mechanical strain, but does not affect β-catenin transcriptional activity or cell cycle re-entry.
Distribution of TOPdGFP at 8 hr (A), β-catenin at 8 hr (A, insets), EdU at 24 hr (C), and pY654 β-catenin at 8 hr (E) in MDCK monolayers after No Strain or High Strain (15%) applied by the ISA and …
Figure 3—figure supplement 5
Distribution of β-catenin in monolayers treated with either DMSO or the EGFR Inhibitor PD153035 8 hr after the application of No Strain or High Strain (15%) using the ISA.
Scale bars: 25 μm.
Figure 4 with 1 supplement
Inhibition of Casein Kinase I (CKI) increases β-catenin transcriptional activity in MDCK quiescent monolayers, independent of mechanical strain.
Distribution of TOPdGFP at 8 hr (A), β-catenin at 8 hr (A, insets), EdU at 24 hr (C), and pY654 β-catenin at 8 hr (E) in MDCK Monolayers after No Strain or High Strain (15%) applied by the ISA and …
-
Figure 4—source data 1
Data used to construct graphs in Figure 4 and Figure 4—figure supplement 1.
- https://doi.org/10.7554/eLife.19799.019
Figure 4—figure supplement 1
Distribution of β-catenin in monolayers treated with either DMSO or the CKI Inhibitor D4476 8 hr after the application of No Strain or High Strain (15%) using the ISA.
Scale bars: 25 μm.
Figure 5
Inhibition of β-catenin degradation promotes progression from G1 into S/G2 following mechanical strain.
(A) Distribution of mAG-Geminin in Fucci MDCK monolayers treated with DMSO, D4476, or D4476 and iCRT3 0 hr or 24 hr after the application of No Strain or High Strain (~8.5%) using the biaxial live …
-
Figure 5—source data 1
Data used to construct graphs in Figure 5.
- https://doi.org/10.7554/eLife.19799.022
Figure 6
Inhibition of β-catenin degradation promotes mitotic entry following mechanical strain.
(A) Distribution of mitotic events (white arrow heads) in Fucci MDCK monolayers 12 hr or 24 hr after the application of No Strain or High Strain (~8.5%) using the biaxial live cell stretcher and …
-
Figure 6—source data 1
Data used to construct graphs in Figure 6.
- https://doi.org/10.7554/eLife.19799.026
Figure 7 with 1 supplement
Mechanical strain induced increase in the progression from G1 into S/G2 in the presence of Wnt3A.
(A) Distribution of mAG-Geminin in Fucci MDCK monolayers treated with control or Wnt3A-conditioned media 0 hr or 24 hr after the application of No Strain or High Strain (~8.5%) using the biaxial …
-
Figure 7—source data 1
Data used to construct graphs in Figure 7 and Figure 7—figure supplement 1.
- https://doi.org/10.7554/eLife.19799.030
Figure 7—figure supplement 1
Wnt3A induces increased β-catenin transcriptional activity, independent of mechanical strain.
Distribution of TOPdGFP at 8 hr (A) and pY654 β-catenin at 8 hr (C) in MDCK monolayers after No Strain or High Strain (15%) using the ISA, treated with either control or Wnt3A-conditioned media. …
Figure 8
Mechanical strain induced increased mitotic entry in the presence of Wnt3A.
(A) Distribution of mitotic events (white arrow heads) in Fucci MDCK monolayers 12 hr or 24 hr after the application of No Strain or High Strain (~8.5%) using the biaxial live cell stretcher in the …
-
Figure 8—source data 1
Data used to construct graphs in Figure 8.
- https://doi.org/10.7554/eLife.19799.037
Videos
Video 1
Representative movie of a Fucci-MDCK monolayer treated with DMSO after no strain.
https://doi.org/10.7554/eLife.19799.007
Video 2
Representative movie of a Fucci-MDCK monolayer treated with DMSO after mechanical strain.
https://doi.org/10.7554/eLife.19799.008
Video 3
Representative movie of a Fucci-MDCK monolayer treated with D4476 after no strain.
https://doi.org/10.7554/eLife.19799.023
Video 4
Representative movie of an FUCCI-MDCK monolayer treated with D4476 after mechanical strain.
https://doi.org/10.7554/eLife.19799.024
Video 5
Representative movie of a Fucci-MDCK monolayer treated with D4476 and iCRT3 after no strain.
https://doi.org/10.7554/eLife.19799.027
Video 6
Representative movie of a Fucci-MDCK monolayer treated with D4476 and iCRT3 after mechanical strain.
https://doi.org/10.7554/eLife.19799.028
Video 7
Representative movie of a Fucci-MDCK monolayer treated with control-conditioned media after no strain.
https://doi.org/10.7554/eLife.19799.032
Video 8
Representative movie of a Fucci-MDCK monolayer treated with control-conditioned media after mechanical strain.
https://doi.org/10.7554/eLife.19799.033
Video 9
Representative movie of a Fucci-MDCK monolayer treated with Wnt3A-conditioned media after no strain.
https://doi.org/10.7554/eLife.19799.034
Video 10
