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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 FN-gel rigidity.Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant kc, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.
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pone-0038780-g004: H4ev and shK8b cell stiffness as function of FN-gel rigidity.Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant kc, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.

Mentions: In light of the actin fiber organization modulation we observed over the different FN-gel rigidities (Fig. 1), we determined whether a correlation was taking place between actin fiber organization and cell stiffness at the bead adhesion site. To eliminate a potential difference related to bead mechanical coupling, we selected here the 400 FU FN-coated beads, based on the finding showing equivalent local stiffness for both H4ev and shK8b cell seeded on FN-coated glass (see Fig. 3F). As seen in Fig. 4A, H4ev and shK8b cells seeded on 1.8 kPa FN-gels yielded comparable induced FN-coated bead displacements. However, when the FN-gel rigidity was increased to 3 kPa, the average FN-coated bead displacement was significantly reduced on H4ev cells, and to a lower extent on shK8b cells (Fig. 4B). As a result, the computed cell elastic constant on a 3 kPa FN-gel was significantly lower for shK8b cells than for H4ev cells (Fig. 4C). Thus, hepatic cells lacking K8/K18 IFs are unable to adequately match their stiffness with the rigidity of the underlying FN-gel substratum.


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 FN-gel rigidity.Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant kc, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038780-g004: H4ev and shK8b cell stiffness as function of FN-gel rigidity.Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant kc, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.
Mentions: In light of the actin fiber organization modulation we observed over the different FN-gel rigidities (Fig. 1), we determined whether a correlation was taking place between actin fiber organization and cell stiffness at the bead adhesion site. To eliminate a potential difference related to bead mechanical coupling, we selected here the 400 FU FN-coated beads, based on the finding showing equivalent local stiffness for both H4ev and shK8b cell seeded on FN-coated glass (see Fig. 3F). As seen in Fig. 4A, H4ev and shK8b cells seeded on 1.8 kPa FN-gels yielded comparable induced FN-coated bead displacements. However, when the FN-gel rigidity was increased to 3 kPa, the average FN-coated bead displacement was significantly reduced on H4ev cells, and to a lower extent on shK8b cells (Fig. 4B). As a result, the computed cell elastic constant on a 3 kPa FN-gel was significantly lower for shK8b cells than for H4ev cells (Fig. 4C). Thus, hepatic cells lacking K8/K18 IFs are unable to adequately match their stiffness with the rigidity of the underlying FN-gel substratum.

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