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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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Modeling of cell stiffness and stiffness response. (A) Schematic of the magnetic tweezers experiment: a 50-pN force was applied for 5 s, followed by a 10-s relaxation time, for a total of eight pulls. The time-dependent displacement for a typical bead is shown below the force regimen. (B) The time-dependent compliance (black data points) is calculated from the displacement of a bead and the applied force. The Jeffrey model (inset) is a mechanical circuit that models the elastic (or stiffness, G) and viscous (η­1 and η2) responses for a viscoelastic liquid during force application. The Jeffrey model (blue line) was used to quantify the stiffness of the cell as measured during force application. In experiments, the cell stiffness was defined to be the stiffness obtained in modeling the compliance of the cell during the first pulse of force. (C, D) Compliance signatures for representative examples of (C) cell stiffening and (D) cell softening, where time progression is encoded by increasing intensity of red, such that black is the compliance during the first pulse of force. To examine the stiffness of cells in response to force, we used the Jeffrey model to quantify the stiffness for each pulse of force during the 2-min experiment. The obtained stiffness measurements were normalized to the stiffness derived from the first pulse of force (G1) to give relative force response fractions G­2/G1 through G­8/G1. The full, nonoverlapping compliance signatures are provided in Supplemental Figure S1, C and D.
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Figure 1: Modeling of cell stiffness and stiffness response. (A) Schematic of the magnetic tweezers experiment: a 50-pN force was applied for 5 s, followed by a 10-s relaxation time, for a total of eight pulls. The time-dependent displacement for a typical bead is shown below the force regimen. (B) The time-dependent compliance (black data points) is calculated from the displacement of a bead and the applied force. The Jeffrey model (inset) is a mechanical circuit that models the elastic (or stiffness, G) and viscous (η­1 and η2) responses for a viscoelastic liquid during force application. The Jeffrey model (blue line) was used to quantify the stiffness of the cell as measured during force application. In experiments, the cell stiffness was defined to be the stiffness obtained in modeling the compliance of the cell during the first pulse of force. (C, D) Compliance signatures for representative examples of (C) cell stiffening and (D) cell softening, where time progression is encoded by increasing intensity of red, such that black is the compliance during the first pulse of force. To examine the stiffness of cells in response to force, we used the Jeffrey model to quantify the stiffness for each pulse of force during the 2-min experiment. The obtained stiffness measurements were normalized to the stiffness derived from the first pulse of force (G1) to give relative force response fractions G­2/G1 through G­8/G1. The full, nonoverlapping compliance signatures are provided in Supplemental Figure S1, C and D.

Mentions: To determine the effect of EMT on cell stiffness and stiffening response, we induced EMT in normal murine mammary gland (NMuMG) epithelial cells, a well-established TGF-β–induced EMT model (Piek et al., 1999; Yu et al., 2002; Xie et al., 2004). A magnetic tweezers system (Fisher et al., 2006; O'Brien et al., 2008) was then used to apply force via integrins (Matthews et al., 2006; Guilluy et al., 2011) to the cytoskeleton through externally attached paramagnetic beads coated with fibronectin (FN). The viscoelastic response of a cell was observed by monitoring the displacement of a bound bead over time during force application (Figure 1A). To quantify the mechanical phenotype in terms of stiffness and stiffening response, we calculated the time-dependent compliance of the cell and fitted it to a Jeffrey model for viscoelastic liquids (Figure 1B; Larson 1999). The spring constant obtained during the first pulse of force provided a measure of stiffness, and by normalizing the spring constants of subsequent force pulses to the first, we obtained the stiffness-response to force, or stiffening response. Two classifications of mechanical response were observed: a stiffening response (Figure 1C) and a softening response (Figure 1D). TGF-β–induced EMT was verified by monitoring reduced E-cadherin levels (see later discussion of Figure 4A) and actin reorganization (insets, Figure 2, 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)

Modeling of cell stiffness and stiffness response. (A) Schematic of the magnetic tweezers experiment: a 50-pN force was applied for 5 s, followed by a 10-s relaxation time, for a total of eight pulls. The time-dependent displacement for a typical bead is shown below the force regimen. (B) The time-dependent compliance (black data points) is calculated from the displacement of a bead and the applied force. The Jeffrey model (inset) is a mechanical circuit that models the elastic (or stiffness, G) and viscous (η­1 and η2) responses for a viscoelastic liquid during force application. The Jeffrey model (blue line) was used to quantify the stiffness of the cell as measured during force application. In experiments, the cell stiffness was defined to be the stiffness obtained in modeling the compliance of the cell during the first pulse of force. (C, D) Compliance signatures for representative examples of (C) cell stiffening and (D) cell softening, where time progression is encoded by increasing intensity of red, such that black is the compliance during the first pulse of force. To examine the stiffness of cells in response to force, we used the Jeffrey model to quantify the stiffness for each pulse of force during the 2-min experiment. The obtained stiffness measurements were normalized to the stiffness derived from the first pulse of force (G1) to give relative force response fractions G­2/G1 through G­8/G1. The full, nonoverlapping compliance signatures are provided in Supplemental Figure S1, C and D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Modeling of cell stiffness and stiffness response. (A) Schematic of the magnetic tweezers experiment: a 50-pN force was applied for 5 s, followed by a 10-s relaxation time, for a total of eight pulls. The time-dependent displacement for a typical bead is shown below the force regimen. (B) The time-dependent compliance (black data points) is calculated from the displacement of a bead and the applied force. The Jeffrey model (inset) is a mechanical circuit that models the elastic (or stiffness, G) and viscous (η­1 and η2) responses for a viscoelastic liquid during force application. The Jeffrey model (blue line) was used to quantify the stiffness of the cell as measured during force application. In experiments, the cell stiffness was defined to be the stiffness obtained in modeling the compliance of the cell during the first pulse of force. (C, D) Compliance signatures for representative examples of (C) cell stiffening and (D) cell softening, where time progression is encoded by increasing intensity of red, such that black is the compliance during the first pulse of force. To examine the stiffness of cells in response to force, we used the Jeffrey model to quantify the stiffness for each pulse of force during the 2-min experiment. The obtained stiffness measurements were normalized to the stiffness derived from the first pulse of force (G1) to give relative force response fractions G­2/G1 through G­8/G1. The full, nonoverlapping compliance signatures are provided in Supplemental Figure S1, C and D.
Mentions: To determine the effect of EMT on cell stiffness and stiffening response, we induced EMT in normal murine mammary gland (NMuMG) epithelial cells, a well-established TGF-β–induced EMT model (Piek et al., 1999; Yu et al., 2002; Xie et al., 2004). A magnetic tweezers system (Fisher et al., 2006; O'Brien et al., 2008) was then used to apply force via integrins (Matthews et al., 2006; Guilluy et al., 2011) to the cytoskeleton through externally attached paramagnetic beads coated with fibronectin (FN). The viscoelastic response of a cell was observed by monitoring the displacement of a bound bead over time during force application (Figure 1A). To quantify the mechanical phenotype in terms of stiffness and stiffening response, we calculated the time-dependent compliance of the cell and fitted it to a Jeffrey model for viscoelastic liquids (Figure 1B; Larson 1999). The spring constant obtained during the first pulse of force provided a measure of stiffness, and by normalizing the spring constants of subsequent force pulses to the first, we obtained the stiffness-response to force, or stiffening response. Two classifications of mechanical response were observed: a stiffening response (Figure 1C) and a softening response (Figure 1D). TGF-β–induced EMT was verified by monitoring reduced E-cadherin levels (see later discussion of Figure 4A) and actin reorganization (insets, Figure 2, 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