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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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LARG and GEF-H1 recruitment to adhesion complex during force decrease during TGF-β–induced EMT. (A, B) Effect of EMT on RhoA GEF recruitment in either NMuMG (A) or PANC-1 (B) cells. Indicated cells were incubated for 30 min with FN-coated beads (Guilluy et al., 2011) and stimulated with a force regimen (50 pN; 5 s force, 10 s recovery) using a rotating permanent magnet for different amounts of time (Materials and Methods). After magnetic separation of the adhesion complex, both the lysate and adhesion complex fractions were analyzed using Western blots. Representative of four independent experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratios), relative to untreated cells without force stimulation, is provided. (C) Effect of EMT on RhoA activation in NMuMG cells. Cells were stimulated with a force regimen using a rotating permanent magnet as in A. RhoA activity in lysates was determined as described (Guilluy et al., 2011). Representative of three experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratio), relative to untreated cells without force stimulation, is provided.
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Figure 5: LARG and GEF-H1 recruitment to adhesion complex during force decrease during TGF-β–induced EMT. (A, B) Effect of EMT on RhoA GEF recruitment in either NMuMG (A) or PANC-1 (B) cells. Indicated cells were incubated for 30 min with FN-coated beads (Guilluy et al., 2011) and stimulated with a force regimen (50 pN; 5 s force, 10 s recovery) using a rotating permanent magnet for different amounts of time (Materials and Methods). After magnetic separation of the adhesion complex, both the lysate and adhesion complex fractions were analyzed using Western blots. Representative of four independent experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratios), relative to untreated cells without force stimulation, is provided. (C) Effect of EMT on RhoA activation in NMuMG cells. Cells were stimulated with a force regimen using a rotating permanent magnet as in A. RhoA activity in lysates was determined as described (Guilluy et al., 2011). Representative of three experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratio), relative to untreated cells without force stimulation, is provided.

Mentions: To test whether EMT affects GEF recruitment to sites of force application, we developed a rotating permanent magnet device (Supplemental Figure S2A) to generate a force regimen consistent in magnitude, duty cycle, and frequency with that produced by the magnetic tweezers (Supplemental Figure S2B) and pulsatile forces to the cells via externally attached FN-coated beads. After force stimulation, we separated the bead fraction containing adhesion complex proteins from the whole lysate (Guilluy et al., 2011) and found that LARG and GEF-H1 were recruited in a time-dependent manner in epithelial-state NMuMG cells. In contrast, TGF-β–induced EMT abrogated this time-dependent recruitment of LARG and GEF-H1 (Figure 5A). Examination of p114, another RhoGEF, showed no recruitment to the adhesion complex and was unchanged after EMT (Figure 5A), suggesting that LARG and GEF-H1 have specific roles in force transduction during EMT. Similar loss of force-dependent recruitment of LARG and GEF-H1 was found in PANC-1 cells treated with BMP-2 (Figure 5B).


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)

LARG and GEF-H1 recruitment to adhesion complex during force decrease during TGF-β–induced EMT. (A, B) Effect of EMT on RhoA GEF recruitment in either NMuMG (A) or PANC-1 (B) cells. Indicated cells were incubated for 30 min with FN-coated beads (Guilluy et al., 2011) and stimulated with a force regimen (50 pN; 5 s force, 10 s recovery) using a rotating permanent magnet for different amounts of time (Materials and Methods). After magnetic separation of the adhesion complex, both the lysate and adhesion complex fractions were analyzed using Western blots. Representative of four independent experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratios), relative to untreated cells without force stimulation, is provided. (C) Effect of EMT on RhoA activation in NMuMG cells. Cells were stimulated with a force regimen using a rotating permanent magnet as in A. RhoA activity in lysates was determined as described (Guilluy et al., 2011). Representative of three experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratio), relative to untreated cells without force stimulation, is provided.
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Related In: Results  -  Collection

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Figure 5: LARG and GEF-H1 recruitment to adhesion complex during force decrease during TGF-β–induced EMT. (A, B) Effect of EMT on RhoA GEF recruitment in either NMuMG (A) or PANC-1 (B) cells. Indicated cells were incubated for 30 min with FN-coated beads (Guilluy et al., 2011) and stimulated with a force regimen (50 pN; 5 s force, 10 s recovery) using a rotating permanent magnet for different amounts of time (Materials and Methods). After magnetic separation of the adhesion complex, both the lysate and adhesion complex fractions were analyzed using Western blots. Representative of four independent experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratios), relative to untreated cells without force stimulation, is provided. (C) Effect of EMT on RhoA activation in NMuMG cells. Cells were stimulated with a force regimen using a rotating permanent magnet as in A. RhoA activity in lysates was determined as described (Guilluy et al., 2011). Representative of three experiments. Associated quantification of amount of protein in adhesion complex (bead-to-lysate ratio), relative to untreated cells without force stimulation, is provided.
Mentions: To test whether EMT affects GEF recruitment to sites of force application, we developed a rotating permanent magnet device (Supplemental Figure S2A) to generate a force regimen consistent in magnitude, duty cycle, and frequency with that produced by the magnetic tweezers (Supplemental Figure S2B) and pulsatile forces to the cells via externally attached FN-coated beads. After force stimulation, we separated the bead fraction containing adhesion complex proteins from the whole lysate (Guilluy et al., 2011) and found that LARG and GEF-H1 were recruited in a time-dependent manner in epithelial-state NMuMG cells. In contrast, TGF-β–induced EMT abrogated this time-dependent recruitment of LARG and GEF-H1 (Figure 5A). Examination of p114, another RhoGEF, showed no recruitment to the adhesion complex and was unchanged after EMT (Figure 5A), suggesting that LARG and GEF-H1 have specific roles in force transduction during EMT. Similar loss of force-dependent recruitment of LARG and GEF-H1 was found in PANC-1 cells treated with BMP-2 (Figure 5B).

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