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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 regulates directed keratinocyte migration in vitro. In vitro scratch assays carried out on confluent monolayers in the presence or absence of 25 μM Y27632, as described in Materials and Methods, indicated impaired directed migration of RhoA- keratinocytes. (A) Shown are representative pictures of the gap closure and the kinetics of gap closure over time (n = 3). (B) Movies from gap-closure experiments were analyzed by tracking single migrating cells and calculating migration speed and tortuosity of migration (n > 100). (C) Reduced levels of GTP-bound active Cdc42 in cultured RhoA- keratinocytes (n = 3/3).
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Figure 9: RhoA regulates directed keratinocyte migration in vitro. In vitro scratch assays carried out on confluent monolayers in the presence or absence of 25 μM Y27632, as described in Materials and Methods, indicated impaired directed migration of RhoA- keratinocytes. (A) Shown are representative pictures of the gap closure and the kinetics of gap closure over time (n = 3). (B) Movies from gap-closure experiments were analyzed by tracking single migrating cells and calculating migration speed and tortuosity of migration (n > 100). (C) Reduced levels of GTP-bound active Cdc42 in cultured RhoA- keratinocytes (n = 3/3).

Mentions: To test this hypothesis experimentally, we assessed directed migration of RhoA- keratinocytes by an in vitro wound-closure assay monitored by time-lapse microscopy. As expected, RhoA-deficient keratinocytes showed a reduced wound-closure speed and frequently displayed elongated cells with delayed rear detachment (Figure 9A; Supplemental Movies 1 and 2). To show that this impairment is due to a reduction in cell contractility, we tried to reproduce the wound-closure defect by treating control cells with the ROCK inhibitor Y27632. As expected, ROCK inhibitor–treated control keratinocytes showed defective rear detachment, similar to the RhoA- cells (Figure 9A; Supplemental Movies 3 and 4). Surprisingly, however, ROCK inhibition accelerated wound closure both in control and in RhoA- keratinocytes. Even in the presence of ROCK inhibitor, RhoA- cells showed significantly slower wound closure than did control keratinocytes (Figure 9A, compare “ko+Y27632” with “con+Y27632”). These data suggest that strong cell contraction is not required for efficient keratinocyte migration. Importantly, RhoA regulates directed cell migration via mechanisms independent of ROCK.


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 regulates directed keratinocyte migration in vitro. In vitro scratch assays carried out on confluent monolayers in the presence or absence of 25 μM Y27632, as described in Materials and Methods, indicated impaired directed migration of RhoA- keratinocytes. (A) Shown are representative pictures of the gap closure and the kinetics of gap closure over time (n = 3). (B) Movies from gap-closure experiments were analyzed by tracking single migrating cells and calculating migration speed and tortuosity of migration (n > 100). (C) Reduced levels of GTP-bound active Cdc42 in cultured RhoA- keratinocytes (n = 3/3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046057&req=5

Figure 9: RhoA regulates directed keratinocyte migration in vitro. In vitro scratch assays carried out on confluent monolayers in the presence or absence of 25 μM Y27632, as described in Materials and Methods, indicated impaired directed migration of RhoA- keratinocytes. (A) Shown are representative pictures of the gap closure and the kinetics of gap closure over time (n = 3). (B) Movies from gap-closure experiments were analyzed by tracking single migrating cells and calculating migration speed and tortuosity of migration (n > 100). (C) Reduced levels of GTP-bound active Cdc42 in cultured RhoA- keratinocytes (n = 3/3).
Mentions: To test this hypothesis experimentally, we assessed directed migration of RhoA- keratinocytes by an in vitro wound-closure assay monitored by time-lapse microscopy. As expected, RhoA-deficient keratinocytes showed a reduced wound-closure speed and frequently displayed elongated cells with delayed rear detachment (Figure 9A; Supplemental Movies 1 and 2). To show that this impairment is due to a reduction in cell contractility, we tried to reproduce the wound-closure defect by treating control cells with the ROCK inhibitor Y27632. As expected, ROCK inhibitor–treated control keratinocytes showed defective rear detachment, similar to the RhoA- cells (Figure 9A; Supplemental Movies 3 and 4). Surprisingly, however, ROCK inhibition accelerated wound closure both in control and in RhoA- keratinocytes. Even in the presence of ROCK inhibitor, RhoA- cells showed significantly slower wound closure than did control keratinocytes (Figure 9A, compare “ko+Y27632” with “con+Y27632”). These data suggest that strong cell contraction is not required for efficient keratinocyte migration. Importantly, RhoA regulates directed cell migration via mechanisms independent of ROCK.

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