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Keratin 8/18 regulation of cell stiffness-extracellular matrix interplay through modulation of Rho-mediated actin cytoskeleton dynamics.

Bordeleau F, Myrand Lapierre ME, Sheng Y, Marceau N - PLoS ONE (2012)

Bottom Line: Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum.We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface.In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche en cancérologie and Centre de Recherche du Centre hospitalier de Québec, Quebec City, Quebec, Canada.

ABSTRACT
Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.

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H4ev and shK8b cell stiffness as function of bead FN-coating density.(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant kc and (G) viscosity constant γc are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
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pone-0038780-g003: H4ev and shK8b cell stiffness as function of bead FN-coating density.(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant kc and (G) viscosity constant γc are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.

Mentions: The cell elasticity at FAs, created by the binding of 3–5 µm FN-coated beads, has been assessed before [7], [8], based on the cell mechanics model described by Fung [32]. Here, we added a spring, representing the optical tweezers, to the Voigt body comprised of a spring in parallel with a dashpot as originally proposed in Fung's model (Fig. 3A). Since the optical tweezers-generated force F(t) is a function of the bead displacement ε(t), the force can be expressed as:(1)where the initial force applied to the bead depends on kt, the trap elastic constant, and ξ, the distance of the bead from the trap center (at t = 0). We can then write the differential equation for a Voigt body by substituting the force with Eq.(1):(2)which introduces the cell local elastic parameter kc and viscous parameter γc (Fig. 3A). The corresponding solution for the bead displacement ε(t) is given by:(3)The local cell parameters kc and γc, are numerically extracted from Eq. (3), using a least square fit on the experimental data measured for the bead displacement ε(t).


Keratin 8/18 regulation of cell stiffness-extracellular matrix interplay through modulation of Rho-mediated actin cytoskeleton dynamics.

Bordeleau F, Myrand Lapierre ME, Sheng Y, Marceau N - PLoS ONE (2012)

H4ev and shK8b cell stiffness as function of bead FN-coating density.(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant kc and (G) viscosity constant γc are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369864&req=5

pone-0038780-g003: H4ev and shK8b cell stiffness as function of bead FN-coating density.(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant kc and (G) viscosity constant γc are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
Mentions: The cell elasticity at FAs, created by the binding of 3–5 µm FN-coated beads, has been assessed before [7], [8], based on the cell mechanics model described by Fung [32]. Here, we added a spring, representing the optical tweezers, to the Voigt body comprised of a spring in parallel with a dashpot as originally proposed in Fung's model (Fig. 3A). Since the optical tweezers-generated force F(t) is a function of the bead displacement ε(t), the force can be expressed as:(1)where the initial force applied to the bead depends on kt, the trap elastic constant, and ξ, the distance of the bead from the trap center (at t = 0). We can then write the differential equation for a Voigt body by substituting the force with Eq.(1):(2)which introduces the cell local elastic parameter kc and viscous parameter γc (Fig. 3A). The corresponding solution for the bead displacement ε(t) is given by:(3)The local cell parameters kc and γc, are numerically extracted from Eq. (3), using a least square fit on the experimental data measured for the bead displacement ε(t).

Bottom Line: Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum.We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface.In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche en cancérologie and Centre de Recherche du Centre hospitalier de Québec, Quebec City, Quebec, Canada.

ABSTRACT
Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.

Show MeSH
Related in: MedlinePlus