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TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

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Stiffness of NMuMG cells measured by passive microrheology. (A) Mean-squared displacement (MSD) of 2 μm, FN-coated beads as a function of time scale between 0.1 and 10 s for NMuMG cells with or without TGF-β treatment; each curve is the ensemble average of cell populations: untreated (n = 1050) and TGF-β treated (n = 1010). MSD curves show a slope of <1 (illustrated by the black guide line), indicating subdiffusive viscoelastic response of beads anchored to the cortical actin cytoskeleton through integrin receptors (Wirtz, 2009). Data were taken using a high-throughput microscopy system described previously (Spero et al., 2008). Error bars represent SEM; the MSD of beads attached to untreated and TGF-β treated NMuMG were statistically significant at *p < 0.001 for all time scales. (B) Effective stiffness, G′ (Pa), of NMuMG cells with or without TGF-β treatment at the 1-s time scale. Using the MSD trajectories computed in Supplemental Figure S1B, we calculated the complex, frequency-dependent shear modulus, G*(ω), using the generalized Stokes–Einstein relation, and the elastic contribution is shown at the 1-s time scale.
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Figure 3: Stiffness of NMuMG cells measured by passive microrheology. (A) Mean-squared displacement (MSD) of 2 μm, FN-coated beads as a function of time scale between 0.1 and 10 s for NMuMG cells with or without TGF-β treatment; each curve is the ensemble average of cell populations: untreated (n = 1050) and TGF-β treated (n = 1010). MSD curves show a slope of <1 (illustrated by the black guide line), indicating subdiffusive viscoelastic response of beads anchored to the cortical actin cytoskeleton through integrin receptors (Wirtz, 2009). Data were taken using a high-throughput microscopy system described previously (Spero et al., 2008). Error bars represent SEM; the MSD of beads attached to untreated and TGF-β treated NMuMG were statistically significant at *p < 0.001 for all time scales. (B) Effective stiffness, G′ (Pa), of NMuMG cells with or without TGF-β treatment at the 1-s time scale. Using the MSD trajectories computed in Supplemental Figure S1B, we calculated the complex, frequency-dependent shear modulus, G*(ω), using the generalized Stokes–Einstein relation, and the elastic contribution is shown at the 1-s time scale.

Mentions: To determine whether the decrease in stiffness observed after EMT was a result of the force magnitude applied during the magnetic tweezers (active microrheology) assay, we performed an external passive microrheology assay to measure the basal stiffness of NMuMG cells before and after TGF-β–initiated EMT. Consistent with observations with the magnetic tweezers assay, measurement of the thermal motion of FN-coated beads revealed a threefold decrease in cell stiffness upon TGF-β treatment (Figure 3, A and B).


TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

Stiffness of NMuMG cells measured by passive microrheology. (A) Mean-squared displacement (MSD) of 2 μm, FN-coated beads as a function of time scale between 0.1 and 10 s for NMuMG cells with or without TGF-β treatment; each curve is the ensemble average of cell populations: untreated (n = 1050) and TGF-β treated (n = 1010). MSD curves show a slope of <1 (illustrated by the black guide line), indicating subdiffusive viscoelastic response of beads anchored to the cortical actin cytoskeleton through integrin receptors (Wirtz, 2009). Data were taken using a high-throughput microscopy system described previously (Spero et al., 2008). Error bars represent SEM; the MSD of beads attached to untreated and TGF-β treated NMuMG were statistically significant at *p < 0.001 for all time scales. (B) Effective stiffness, G′ (Pa), of NMuMG cells with or without TGF-β treatment at the 1-s time scale. Using the MSD trajectories computed in Supplemental Figure S1B, we calculated the complex, frequency-dependent shear modulus, G*(ω), using the generalized Stokes–Einstein relation, and the elastic contribution is shown at the 1-s time scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Stiffness of NMuMG cells measured by passive microrheology. (A) Mean-squared displacement (MSD) of 2 μm, FN-coated beads as a function of time scale between 0.1 and 10 s for NMuMG cells with or without TGF-β treatment; each curve is the ensemble average of cell populations: untreated (n = 1050) and TGF-β treated (n = 1010). MSD curves show a slope of <1 (illustrated by the black guide line), indicating subdiffusive viscoelastic response of beads anchored to the cortical actin cytoskeleton through integrin receptors (Wirtz, 2009). Data were taken using a high-throughput microscopy system described previously (Spero et al., 2008). Error bars represent SEM; the MSD of beads attached to untreated and TGF-β treated NMuMG were statistically significant at *p < 0.001 for all time scales. (B) Effective stiffness, G′ (Pa), of NMuMG cells with or without TGF-β treatment at the 1-s time scale. Using the MSD trajectories computed in Supplemental Figure S1B, we calculated the complex, frequency-dependent shear modulus, G*(ω), using the generalized Stokes–Einstein relation, and the elastic contribution is shown at the 1-s time scale.
Mentions: To determine whether the decrease in stiffness observed after EMT was a result of the force magnitude applied during the magnetic tweezers (active microrheology) assay, we performed an external passive microrheology assay to measure the basal stiffness of NMuMG cells before and after TGF-β–initiated EMT. Consistent with observations with the magnetic tweezers assay, measurement of the thermal motion of FN-coated beads revealed a threefold decrease in cell stiffness upon TGF-β treatment (Figure 3, A and B).

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

Show MeSH
Related in: MedlinePlus