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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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ROCK involvement in K8/K18 IF modulation of cell stiffness.(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant kc obtained in presence of vehicle (Ctrl) or Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant kc obtained in absence (Ctrl) or presence of Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.
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pone-0038780-g005: ROCK involvement in K8/K18 IF modulation of cell stiffness.(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant kc obtained in presence of vehicle (Ctrl) or Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant kc obtained in absence (Ctrl) or presence of Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.

Mentions: The Rho-ROCK pathway is involved in cell adaptation to substratum rigidity by controlling cell stiffness [27]. We thus assessed whether Rho-ROCK was involved in the differential H4ev versus shK8b cell stiffness measured at FAs. As shown in Fig. 5A, a treatment with the ROCK inhibitor Y27632 increased the displacement of 400 FU FN-coated beads attached to either H4ev or shK8b cells cultured on the 3 kPa FN-gel. Intriguingly, following ROCK inhibition in shK8b cells, the beads maintained a discernible creeping motion, which resulted to a slight distortion of the fitting curve. Nevertheless, it remained that the calculated stiffness values were not statistically different between for both Y27632-treated H4ev and shK8b cells (Fig. 5B). Moreover, measurements with 125 FU FN-coated beads attached to either H4ev or shK8b cells seeded on FN-coated glass yielded equivalent bead displacements following Y27632 treatment, when compared to control untreated cells (Fig. 5C). In the same way, the stiffness difference computed for untreated cells was abolished after ROCK inhibition (Fig. 5D). Together, the results indicate that the K8/K18 differential modulation of cell stiffness at FAs is ROCK dependent.


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)

ROCK involvement in K8/K18 IF modulation of cell stiffness.(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant kc obtained in presence of vehicle (Ctrl) or Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant kc obtained in absence (Ctrl) or presence of Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369864&req=5

pone-0038780-g005: ROCK involvement in K8/K18 IF modulation of cell stiffness.(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant kc obtained in presence of vehicle (Ctrl) or Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant kc obtained in absence (Ctrl) or presence of Y27632. The kc difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.
Mentions: The Rho-ROCK pathway is involved in cell adaptation to substratum rigidity by controlling cell stiffness [27]. We thus assessed whether Rho-ROCK was involved in the differential H4ev versus shK8b cell stiffness measured at FAs. As shown in Fig. 5A, a treatment with the ROCK inhibitor Y27632 increased the displacement of 400 FU FN-coated beads attached to either H4ev or shK8b cells cultured on the 3 kPa FN-gel. Intriguingly, following ROCK inhibition in shK8b cells, the beads maintained a discernible creeping motion, which resulted to a slight distortion of the fitting curve. Nevertheless, it remained that the calculated stiffness values were not statistically different between for both Y27632-treated H4ev and shK8b cells (Fig. 5B). Moreover, measurements with 125 FU FN-coated beads attached to either H4ev or shK8b cells seeded on FN-coated glass yielded equivalent bead displacements following Y27632 treatment, when compared to control untreated cells (Fig. 5C). In the same way, the stiffness difference computed for untreated cells was abolished after ROCK inhibition (Fig. 5D). Together, the results indicate that the K8/K18 differential modulation of cell stiffness at FAs is ROCK dependent.

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