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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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RhoA and ROCK-mediated contraction counteract spreading in the presence and absence of Rac1. Area of primary RhoA- (ko) and control (con) keratinocytes was measured (A) in the absence of inhibitors, (B) in the presence of the Rho inhibitor C2I-NC3 (the activating toxins CNFY and CNF1), and (C) in the presence of the ROCK inhibitor Y27632 and the MLCP inhibitor calyculin. Transfection of RhoA- keratinocytes with an EGFP-RhoA fusion (ko + EGFP-RhoA) or a high-cycling mutant form of RhoA (F30L) rescued the increased spreading phenotype (ko + RhoA [F30L]), whereas RhoA knockout induction in vitro reproduced defect (ko in vitro). (D) Reduced levels of GTP-bound active Rac1 in cultured RhoA- keratinocytes (n = 3/3; p < 0.05). (E) Spreading-defective Rac1- keratinocytes treated with Y27632 are able to spread.
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Figure 8: RhoA and ROCK-mediated contraction counteract spreading in the presence and absence of Rac1. Area of primary RhoA- (ko) and control (con) keratinocytes was measured (A) in the absence of inhibitors, (B) in the presence of the Rho inhibitor C2I-NC3 (the activating toxins CNFY and CNF1), and (C) in the presence of the ROCK inhibitor Y27632 and the MLCP inhibitor calyculin. Transfection of RhoA- keratinocytes with an EGFP-RhoA fusion (ko + EGFP-RhoA) or a high-cycling mutant form of RhoA (F30L) rescued the increased spreading phenotype (ko + RhoA [F30L]), whereas RhoA knockout induction in vitro reproduced defect (ko in vitro). (D) Reduced levels of GTP-bound active Rac1 in cultured RhoA- keratinocytes (n = 3/3; p < 0.05). (E) Spreading-defective Rac1- keratinocytes treated with Y27632 are able to spread.

Mentions: During this analysis we noted that keratinocytes lacking RhoA appeared more spread. Quantifying the relative cell area of keratinocytes in culture, we observed an increased number of widely spread cells in RhoA-deficient keratinocytes. On average, the cell area of RhoA ko cells showed a twofold increase compared to control keratinocytes (Figure 8A). To rescue this defect, we transfected RhoA- keratinocytes with an EGFP-RhoA fusion protein or an activated form of RhoA (RhoA F30L) coexpressing EGFP. The increased area phenotype was rescued by the EGFP-RhoA fusion and the activated RhoA, indicating that the altered spreading of RhoA ko cells is caused by a loss of active RhoA (Figure 8C). Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre reproduced the increased spreading (Figure 8C), indicating that it is a cell-autonomous defect.


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)

RhoA and ROCK-mediated contraction counteract spreading in the presence and absence of Rac1. Area of primary RhoA- (ko) and control (con) keratinocytes was measured (A) in the absence of inhibitors, (B) in the presence of the Rho inhibitor C2I-NC3 (the activating toxins CNFY and CNF1), and (C) in the presence of the ROCK inhibitor Y27632 and the MLCP inhibitor calyculin. Transfection of RhoA- keratinocytes with an EGFP-RhoA fusion (ko + EGFP-RhoA) or a high-cycling mutant form of RhoA (F30L) rescued the increased spreading phenotype (ko + RhoA [F30L]), whereas RhoA knockout induction in vitro reproduced defect (ko in vitro). (D) Reduced levels of GTP-bound active Rac1 in cultured RhoA- keratinocytes (n = 3/3; p < 0.05). (E) Spreading-defective Rac1- keratinocytes treated with Y27632 are able to spread.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: RhoA and ROCK-mediated contraction counteract spreading in the presence and absence of Rac1. Area of primary RhoA- (ko) and control (con) keratinocytes was measured (A) in the absence of inhibitors, (B) in the presence of the Rho inhibitor C2I-NC3 (the activating toxins CNFY and CNF1), and (C) in the presence of the ROCK inhibitor Y27632 and the MLCP inhibitor calyculin. Transfection of RhoA- keratinocytes with an EGFP-RhoA fusion (ko + EGFP-RhoA) or a high-cycling mutant form of RhoA (F30L) rescued the increased spreading phenotype (ko + RhoA [F30L]), whereas RhoA knockout induction in vitro reproduced defect (ko in vitro). (D) Reduced levels of GTP-bound active Rac1 in cultured RhoA- keratinocytes (n = 3/3; p < 0.05). (E) Spreading-defective Rac1- keratinocytes treated with Y27632 are able to spread.
Mentions: During this analysis we noted that keratinocytes lacking RhoA appeared more spread. Quantifying the relative cell area of keratinocytes in culture, we observed an increased number of widely spread cells in RhoA-deficient keratinocytes. On average, the cell area of RhoA ko cells showed a twofold increase compared to control keratinocytes (Figure 8A). To rescue this defect, we transfected RhoA- keratinocytes with an EGFP-RhoA fusion protein or an activated form of RhoA (RhoA F30L) coexpressing EGFP. The increased area phenotype was rescued by the EGFP-RhoA fusion and the activated RhoA, indicating that the altered spreading of RhoA ko cells is caused by a loss of active RhoA (Figure 8C). Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre reproduced the increased spreading (Figure 8C), indicating that it is a cell-autonomous defect.

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