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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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Defective formation of mature cell–cell contacts in vitro in the absence of RhoA. Formation of mature cell–cell contacts was induced in confluent monolayers by 12-h incubation with high-calcium growth medium. Cells were stained by immunofluorescence for ZO-1, occludin, and F-actin (occludin and F-actin are double stainings). Shown are confocal pictures (A) and for ZO-1sections through z-stacks of confocal pictures (B). Western blot analysis revealed decreased amounts of occludin protein and unchanged levels of ZO-1 in cultured RhoA- keratinocytes (C; n = 6/6; p < 0.05). (C) Immunofluorescence for ZO-1 after RhoA knockout induction in vitro and high calcium treatment.
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Figure 5: Defective formation of mature cell–cell contacts in vitro in the absence of RhoA. Formation of mature cell–cell contacts was induced in confluent monolayers by 12-h incubation with high-calcium growth medium. Cells were stained by immunofluorescence for ZO-1, occludin, and F-actin (occludin and F-actin are double stainings). Shown are confocal pictures (A) and for ZO-1sections through z-stacks of confocal pictures (B). Western blot analysis revealed decreased amounts of occludin protein and unchanged levels of ZO-1 in cultured RhoA- keratinocytes (C; n = 6/6; p < 0.05). (C) Immunofluorescence for ZO-1 after RhoA knockout induction in vitro and high calcium treatment.

Mentions: Although Cdc42 is crucial for the de novo formation of mature tight junctions (Wu et al., 2007; Du et al., 2009), it is not an immediate requirement for their maintenance in the epidermis in vivo (Wu et al., 2006). To investigate whether RhoA is more important for the formation of cell–cell junctions than for their maintenance in epidermis, we isolated primary keratinocytes from control and RhoA mutant mice and induced the formation of adherens and tight junctions in vitro by high calcium treatment. After 12 h, continuous cell–cell contacts had been formed in control keratinocytes, resulting in continuous staining of the cell borders for ZO-1, present in adherens and tight junctions, and occludin, present only in tight junctions (Figure 5, A and B). RhoA-deficient keratinocytes, however, displayed a discontinuous staining for ZO-1 and occludin (Figure 5A). Western blot analysis revealed reduced amounts of occludin protein and normal amounts of ZO-1 in RhoA- keratinocytes (Figure 5B). Microarray gene expression analysis indicated reduced occludin expression in cultured keratinocytes, suggesting that RhoA regulates occludin expression in vitro at the level of mRNA (Supplemental Figure 3). Staining for F-actin revealed focal patches colocalizing with occludin at the cell–cell contacts of differentiated RhoA- keratinocytes (Figure 5C). Parallel bundles of F-actin inserted at these foci and protruded into the cell. In control cells, F-actin was continuous at the cell–cell junctions, and no obvious bundles of F-actin were observed. Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre followed by high calcium treatment reproduced the defective ZO-1 distribution observed with primary RhoA keratinocytes, indicating that this impairment is a cell-autonomous defect (Figure 5D).


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)

Defective formation of mature cell–cell contacts in vitro in the absence of RhoA. Formation of mature cell–cell contacts was induced in confluent monolayers by 12-h incubation with high-calcium growth medium. Cells were stained by immunofluorescence for ZO-1, occludin, and F-actin (occludin and F-actin are double stainings). Shown are confocal pictures (A) and for ZO-1sections through z-stacks of confocal pictures (B). Western blot analysis revealed decreased amounts of occludin protein and unchanged levels of ZO-1 in cultured RhoA- keratinocytes (C; n = 6/6; p < 0.05). (C) Immunofluorescence for ZO-1 after RhoA knockout induction in vitro and high calcium treatment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Defective formation of mature cell–cell contacts in vitro in the absence of RhoA. Formation of mature cell–cell contacts was induced in confluent monolayers by 12-h incubation with high-calcium growth medium. Cells were stained by immunofluorescence for ZO-1, occludin, and F-actin (occludin and F-actin are double stainings). Shown are confocal pictures (A) and for ZO-1sections through z-stacks of confocal pictures (B). Western blot analysis revealed decreased amounts of occludin protein and unchanged levels of ZO-1 in cultured RhoA- keratinocytes (C; n = 6/6; p < 0.05). (C) Immunofluorescence for ZO-1 after RhoA knockout induction in vitro and high calcium treatment.
Mentions: Although Cdc42 is crucial for the de novo formation of mature tight junctions (Wu et al., 2007; Du et al., 2009), it is not an immediate requirement for their maintenance in the epidermis in vivo (Wu et al., 2006). To investigate whether RhoA is more important for the formation of cell–cell junctions than for their maintenance in epidermis, we isolated primary keratinocytes from control and RhoA mutant mice and induced the formation of adherens and tight junctions in vitro by high calcium treatment. After 12 h, continuous cell–cell contacts had been formed in control keratinocytes, resulting in continuous staining of the cell borders for ZO-1, present in adherens and tight junctions, and occludin, present only in tight junctions (Figure 5, A and B). RhoA-deficient keratinocytes, however, displayed a discontinuous staining for ZO-1 and occludin (Figure 5A). Western blot analysis revealed reduced amounts of occludin protein and normal amounts of ZO-1 in RhoA- keratinocytes (Figure 5B). Microarray gene expression analysis indicated reduced occludin expression in cultured keratinocytes, suggesting that RhoA regulates occludin expression in vitro at the level of mRNA (Supplemental Figure 3). Staining for F-actin revealed focal patches colocalizing with occludin at the cell–cell contacts of differentiated RhoA- keratinocytes (Figure 5C). Parallel bundles of F-actin inserted at these foci and protruded into the cell. In control cells, F-actin was continuous at the cell–cell junctions, and no obvious bundles of F-actin were observed. Knockout induction in vitro by transfection of RhoA fl/fl keratinocytes with Cre followed by high calcium treatment reproduced the defective ZO-1 distribution observed with primary RhoA keratinocytes, indicating that this impairment is a cell-autonomous defect (Figure 5D).

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