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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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Actin fiber organization in H4ev and shK8b cells seeded on substratum of increasing rigidity.(A) Maximum projection confocal images of fibrillar actin in 3-day monolayers of cells seeded on FN-gels of increasing rigidity or in FN-coated dishes, showing distinct actin organizations in H4ev versus shK8b monolayers. In both cell monolayers, the number and density of actin fibers increase with substratum rigidity. (B) Average actin fiber length estimates from the corresponding seeding conditions, showing shorter actin fibers in shK8b compared to those in H4ev cells. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
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pone-0038780-g002: Actin fiber organization in H4ev and shK8b cells seeded on substratum of increasing rigidity.(A) Maximum projection confocal images of fibrillar actin in 3-day monolayers of cells seeded on FN-gels of increasing rigidity or in FN-coated dishes, showing distinct actin organizations in H4ev versus shK8b monolayers. In both cell monolayers, the number and density of actin fibers increase with substratum rigidity. (B) Average actin fiber length estimates from the corresponding seeding conditions, showing shorter actin fibers in shK8b compared to those in H4ev cells. Bars denote SE. *, p<0.05 for H4ev versus shK8b.

Mentions: Considering that a change in ECM rigidity modulates fibrillar actin organization in adherent cells [27], [31], we determined here the effect of the K8/K18 IF loss on actin organization in hepatic cells at day-3 post-seeding on FN-gels. H4ev cells seeded on a 1.8 kPa FN-gel showed numerous actin fibers, but did not form a coherent fiber network, which contrasted with the interconnected network formed on 3 kPa FN-gel; it was even more so for H4ev cells on FN-coated glass, where the actin fibers formed a compact network across the monolayer (Fig. 2A). No such fibrillar actin networks formed in shK8b cells seeded on 1.8 and 3 kPa FN-gel but still, seeding on FN-coated glass yielded networks of non-interconnected actin fibers. Quantitative estimates revealed a substrate rigidity-dependent increase in actin fiber length in H4ev cells (Fig. 2B). In contrast, in shK8b cells, the actin fiber length did not increase between the 1.8 kPa and 3 kPa FN-gels, and even though the fiber length increased for cells seeded on FN-coated glass, the actin fibers remained shorter when compared to those in H4ev cells (Fig. 2B). These results demonstrate that K8/K18 IFs constitute a modulator of actin fiber organization over a wide range of ECM rigidity.


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)

Actin fiber organization in H4ev and shK8b cells seeded on substratum of increasing rigidity.(A) Maximum projection confocal images of fibrillar actin in 3-day monolayers of cells seeded on FN-gels of increasing rigidity or in FN-coated dishes, showing distinct actin organizations in H4ev versus shK8b monolayers. In both cell monolayers, the number and density of actin fibers increase with substratum rigidity. (B) Average actin fiber length estimates from the corresponding seeding conditions, showing shorter actin fibers in shK8b compared to those in H4ev cells. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038780-g002: Actin fiber organization in H4ev and shK8b cells seeded on substratum of increasing rigidity.(A) Maximum projection confocal images of fibrillar actin in 3-day monolayers of cells seeded on FN-gels of increasing rigidity or in FN-coated dishes, showing distinct actin organizations in H4ev versus shK8b monolayers. In both cell monolayers, the number and density of actin fibers increase with substratum rigidity. (B) Average actin fiber length estimates from the corresponding seeding conditions, showing shorter actin fibers in shK8b compared to those in H4ev cells. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
Mentions: Considering that a change in ECM rigidity modulates fibrillar actin organization in adherent cells [27], [31], we determined here the effect of the K8/K18 IF loss on actin organization in hepatic cells at day-3 post-seeding on FN-gels. H4ev cells seeded on a 1.8 kPa FN-gel showed numerous actin fibers, but did not form a coherent fiber network, which contrasted with the interconnected network formed on 3 kPa FN-gel; it was even more so for H4ev cells on FN-coated glass, where the actin fibers formed a compact network across the monolayer (Fig. 2A). No such fibrillar actin networks formed in shK8b cells seeded on 1.8 and 3 kPa FN-gel but still, seeding on FN-coated glass yielded networks of non-interconnected actin fibers. Quantitative estimates revealed a substrate rigidity-dependent increase in actin fiber length in H4ev cells (Fig. 2B). In contrast, in shK8b cells, the actin fiber length did not increase between the 1.8 kPa and 3 kPa FN-gels, and even though the fiber length increased for cells seeded on FN-coated glass, the actin fibers remained shorter when compared to those in H4ev cells (Fig. 2B). These results demonstrate that K8/K18 IFs constitute a modulator of actin fiber organization over a wide range of ECM rigidity.

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