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RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

Jackson B, Peyrollier K, Pedersen E, Basse A, Karlsson R, Wang Z, Lefever T, Ochsenbein AM, Schmidt G, Aktories K, Stanley A, Quondamatteo F, Ladwein M, Rottner K, van Hengel J, Brakebusch C - Mol. Biol. Cell (2011)

Bottom Line: Rho-inhibiting toxins further increased multinucleation of RhoA- cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA.Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence.These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA- keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark.

ABSTRACT
RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA- keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA- cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA- keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

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Increased number of multinucleated cells in the absence of RhoA. (A) Primary keratinocytes were cultured in vitro and fixed, and nuclei were stained with DAPI. RhoA- keratinocytes (ko; n = 11) showed an increased number of multinucleated cells (arrows) compared to control cells (con; n = 13). Transfection of an EGFP-RhoA fusion protein partially rescued (ko + EGFP-RhoA; n = 2), and a high-cycling mutant form of RhoA (F30L) fully rescued the phenotype (ko + RhoA (F30L); n = 3). RhoA knockout induction in vitro reproduced the multinucleation phenotype (ko in vitro; n = 2). (B) Increased levels of total and GTP-bound active RhoB in cultured RhoA- keratinocytes (n = 3/3). (C) Treatment for 3 h with the Rho inhibitor C2I-NC3 increased multinucleation in both control and ko keratinocytes, whereas the activating toxins CNF1 and CNFY could not rescue the phenotype.
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Figure 6: Increased number of multinucleated cells in the absence of RhoA. (A) Primary keratinocytes were cultured in vitro and fixed, and nuclei were stained with DAPI. RhoA- keratinocytes (ko; n = 11) showed an increased number of multinucleated cells (arrows) compared to control cells (con; n = 13). Transfection of an EGFP-RhoA fusion protein partially rescued (ko + EGFP-RhoA; n = 2), and a high-cycling mutant form of RhoA (F30L) fully rescued the phenotype (ko + RhoA (F30L); n = 3). RhoA knockout induction in vitro reproduced the multinucleation phenotype (ko in vitro; n = 2). (B) Increased levels of total and GTP-bound active RhoB in cultured RhoA- keratinocytes (n = 3/3). (C) Treatment for 3 h with the Rho inhibitor C2I-NC3 increased multinucleation in both control and ko keratinocytes, whereas the activating toxins CNF1 and CNFY could not rescue the phenotype.

Mentions: Rho-mediated activation of ROCK, citron kinase, and mDia were reported to control cytokinesis (Piekny et al., 2005). The specific role of RhoA, however, was not studied. The normal skin maintenance of RhoA ko mice suggested that RhoA is not essential for cytokinesis in keratinocytes. Moreover, analysis of skin sections by staining of the nuclei with 4′,6′-diamidino-2-phenylindole (DAPI) and of the cell borders with E-cadherin did not indicate any obvious multinucleated cells in RhoA- epidermis (Figure 2, e and e′). We then investigated the role of RhoA in cytokinesis in primary keratinocytes in vitro by staining subconfluent cells with DAPI (Figure 6A). In cultures obtained from control mice, the percentage of multinucleated cells was 6.4 ± 2.2%. RhoA ko cells showed a significant increase in multinucleated cells to 13.2 ± 4.2%. To rescue this defect, we transfected RhoA- keratinocytes with an enhanced green fluorescent protein (EGFP)-RhoA fusion protein or an activated form of RhoA (RhoA F30L) coexpressing EGFP. Whereas the EGFP-RhoA fusion partially rescued multinucleation, the activated RhoA fully rescued the phenotype, indicating that the cytokinesis defect in RhoA ko cells is caused by a loss of active RhoA (Figure 6A). Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre reproduced the multinucleation defect (Figure 6A), indicating that it is a cell autonomous defect. These data indicate that RhoA contributes to cytokinesis, without being essential.


RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

Jackson B, Peyrollier K, Pedersen E, Basse A, Karlsson R, Wang Z, Lefever T, Ochsenbein AM, Schmidt G, Aktories K, Stanley A, Quondamatteo F, Ladwein M, Rottner K, van Hengel J, Brakebusch C - Mol. Biol. Cell (2011)

Increased number of multinucleated cells in the absence of RhoA. (A) Primary keratinocytes were cultured in vitro and fixed, and nuclei were stained with DAPI. RhoA- keratinocytes (ko; n = 11) showed an increased number of multinucleated cells (arrows) compared to control cells (con; n = 13). Transfection of an EGFP-RhoA fusion protein partially rescued (ko + EGFP-RhoA; n = 2), and a high-cycling mutant form of RhoA (F30L) fully rescued the phenotype (ko + RhoA (F30L); n = 3). RhoA knockout induction in vitro reproduced the multinucleation phenotype (ko in vitro; n = 2). (B) Increased levels of total and GTP-bound active RhoB in cultured RhoA- keratinocytes (n = 3/3). (C) Treatment for 3 h with the Rho inhibitor C2I-NC3 increased multinucleation in both control and ko keratinocytes, whereas the activating toxins CNF1 and CNFY could not rescue the phenotype.
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Related In: Results  -  Collection

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Figure 6: Increased number of multinucleated cells in the absence of RhoA. (A) Primary keratinocytes were cultured in vitro and fixed, and nuclei were stained with DAPI. RhoA- keratinocytes (ko; n = 11) showed an increased number of multinucleated cells (arrows) compared to control cells (con; n = 13). Transfection of an EGFP-RhoA fusion protein partially rescued (ko + EGFP-RhoA; n = 2), and a high-cycling mutant form of RhoA (F30L) fully rescued the phenotype (ko + RhoA (F30L); n = 3). RhoA knockout induction in vitro reproduced the multinucleation phenotype (ko in vitro; n = 2). (B) Increased levels of total and GTP-bound active RhoB in cultured RhoA- keratinocytes (n = 3/3). (C) Treatment for 3 h with the Rho inhibitor C2I-NC3 increased multinucleation in both control and ko keratinocytes, whereas the activating toxins CNF1 and CNFY could not rescue the phenotype.
Mentions: Rho-mediated activation of ROCK, citron kinase, and mDia were reported to control cytokinesis (Piekny et al., 2005). The specific role of RhoA, however, was not studied. The normal skin maintenance of RhoA ko mice suggested that RhoA is not essential for cytokinesis in keratinocytes. Moreover, analysis of skin sections by staining of the nuclei with 4′,6′-diamidino-2-phenylindole (DAPI) and of the cell borders with E-cadherin did not indicate any obvious multinucleated cells in RhoA- epidermis (Figure 2, e and e′). We then investigated the role of RhoA in cytokinesis in primary keratinocytes in vitro by staining subconfluent cells with DAPI (Figure 6A). In cultures obtained from control mice, the percentage of multinucleated cells was 6.4 ± 2.2%. RhoA ko cells showed a significant increase in multinucleated cells to 13.2 ± 4.2%. To rescue this defect, we transfected RhoA- keratinocytes with an enhanced green fluorescent protein (EGFP)-RhoA fusion protein or an activated form of RhoA (RhoA F30L) coexpressing EGFP. Whereas the EGFP-RhoA fusion partially rescued multinucleation, the activated RhoA fully rescued the phenotype, indicating that the cytokinesis defect in RhoA ko cells is caused by a loss of active RhoA (Figure 6A). Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre reproduced the multinucleation defect (Figure 6A), indicating that it is a cell autonomous defect. These data indicate that RhoA contributes to cytokinesis, without being essential.

Bottom Line: Rho-inhibiting toxins further increased multinucleation of RhoA- cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA.Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence.These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA- keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark.

ABSTRACT
RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA- keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA- cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA- keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

Show MeSH
Related in: MedlinePlus