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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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Substratum rigidity differentially affects H4ev and shK8b cell shapes.(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
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pone-0038780-g001: Substratum rigidity differentially affects H4ev and shK8b cell shapes.(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. Bars denote SE. *, p<0.05 for H4ev versus shK8b.

Mentions: Since the shape of adherent cells largely depends on an interplay between cytoskeleton and ECM [27], we first assessed to which extend a loss of the K8/K18 IF network in hepatic cells affects their shape in response to FN-gel rigidities that are representative of normal and fibrotic hepatic tissue [28], [29], [30]. H4ev and shK8b cells were seeded at an intermediate density on FN-gel of 0.8, 1.8 and 3 kPa or on FN-coated glass (>3 GPa). As shown in Fig. 1A, the shape of H4ev cells at day-1 post-seeding appeared similar over the whole range of FN-gel rigidity. In contrast, shK8b cells seeded on a 0.8 kPa gel maintained a round shape despite their firm adhesion on the gel, even for those cells that maintained cell-cell contacts, whereas their shape was similar to that of H4ev cells on a 1.8 kPa or 3 kPa FN-gel (Fig. 1A). In fact, cell area measurements revealed that the spreading ability of shK8b cells remained below that of H4ev cells over the whole range of FN-gel rigidity (Fig. 1B). Of note, shK8b cells plated on 0.8 kPa FN-gels exhibit a size equivalent to that we observed previously for the same cell type held in suspension [24]. It is also worth noticing that the present finding using the FN-coated glass substratum confirmed our previous data using the same seeding condition [22], [24]. At day-3, H4ev cells formed confluent monolayers on all gels. This was not the case for shK8b cells seeded on a 0.8 kPa FN-gel, in which case the cells maintained a rounded shape, even when plated at higher density (data not shown). Together, these observations readily pointed to an impaired cell mechanosensing as a result of the K8/K18 IF loss.


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)

Substratum rigidity differentially affects H4ev and shK8b cell shapes.(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. 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-g001: Substratum rigidity differentially affects H4ev and shK8b cell shapes.(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. Bars denote SE. *, p<0.05 for H4ev versus shK8b.
Mentions: Since the shape of adherent cells largely depends on an interplay between cytoskeleton and ECM [27], we first assessed to which extend a loss of the K8/K18 IF network in hepatic cells affects their shape in response to FN-gel rigidities that are representative of normal and fibrotic hepatic tissue [28], [29], [30]. H4ev and shK8b cells were seeded at an intermediate density on FN-gel of 0.8, 1.8 and 3 kPa or on FN-coated glass (>3 GPa). As shown in Fig. 1A, the shape of H4ev cells at day-1 post-seeding appeared similar over the whole range of FN-gel rigidity. In contrast, shK8b cells seeded on a 0.8 kPa gel maintained a round shape despite their firm adhesion on the gel, even for those cells that maintained cell-cell contacts, whereas their shape was similar to that of H4ev cells on a 1.8 kPa or 3 kPa FN-gel (Fig. 1A). In fact, cell area measurements revealed that the spreading ability of shK8b cells remained below that of H4ev cells over the whole range of FN-gel rigidity (Fig. 1B). Of note, shK8b cells plated on 0.8 kPa FN-gels exhibit a size equivalent to that we observed previously for the same cell type held in suspension [24]. It is also worth noticing that the present finding using the FN-coated glass substratum confirmed our previous data using the same seeding condition [22], [24]. At day-3, H4ev cells formed confluent monolayers on all gels. This was not the case for shK8b cells seeded on a 0.8 kPa FN-gel, in which case the cells maintained a rounded shape, even when plated at higher density (data not shown). Together, these observations readily pointed to an impaired cell mechanosensing as a result of the K8/K18 IF loss.

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