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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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K8/K18 IF modulation of Rho-mediated actin fiber organization.(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.
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pone-0038780-g006: K8/K18 IF modulation of Rho-mediated actin fiber organization.(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.

Mentions: The RhoA-ROCK signaling pathway is a key regulator of cell stiffness through a control of actin contractility and fiber organization [1], [6]. In this context, we addressed the K8/K18 IF involvement in the Rho-mediated regulation of actin dynamics. H4ev and shK8b cells were seeded on FN-coated glass and the actin fiber organization in absence or presence of ROCK Y27632 inhibitor was assessed. In absence of the inhibitor, the actin fiber distribution was profoundly re-arranged at the cell dorsal surface in shK8b versus H4ev cells, but less affected at their ventral surface (Fig. 6A). While its presence led to a significant overall disruption of the actin fibers in both H4ev and shK8b cells, this ROCK inhibition did not result in a complete disassembly of the actin fibers at the dorsal surface of shK8b cells.


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)

K8/K18 IF modulation of Rho-mediated actin fiber organization.(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038780-g006: K8/K18 IF modulation of Rho-mediated actin fiber organization.(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.
Mentions: The RhoA-ROCK signaling pathway is a key regulator of cell stiffness through a control of actin contractility and fiber organization [1], [6]. In this context, we addressed the K8/K18 IF involvement in the Rho-mediated regulation of actin dynamics. H4ev and shK8b cells were seeded on FN-coated glass and the actin fiber organization in absence or presence of ROCK Y27632 inhibitor was assessed. In absence of the inhibitor, the actin fiber distribution was profoundly re-arranged at the cell dorsal surface in shK8b versus H4ev cells, but less affected at their ventral surface (Fig. 6A). While its presence led to a significant overall disruption of the actin fibers in both H4ev and shK8b cells, this ROCK inhibition did not result in a complete disassembly of the actin fibers at the dorsal surface of shK8b cells.

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