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Arginylation-dependent neural crest cell migration is essential for mouse development.

Kurosaka S, Leu NA, Zhang F, Bunte R, Saha S, Wang J, Guo C, He W, Kashina A - PLoS Genet. (2010)

Bottom Line: Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture.Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death.Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

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Ate1 knockout affects cell attachment to the substrate via intracellular and not extracellular mechanisms.(A) Left, extracellular fibronectin staining in a dense monolayer of wild-type (WT) and Ate1 knockout (KO) cultured fibroblasts shows no difference between the two cultures. Right, quantification of the fibronectin level in the two cultures measured as average gray value in the entire field of view confirms that there is no difference between WT and KO cells in the amount of extracellular fibronectin. Bars show the ratio between WT and KO and error bars represent the average of the measurements in 10 different fields of view in each culture. (B) Left, an overlay of the fluorescence staining of the edge of the cell monolayer moving into the wound co-stained with rhodamine-phalloidin (red) to visualize the actin filaments and anti-paxillin (green) to visualize the focal adhesions. Right, quantification of the number of focal adhesions per µm of the wound edge shows that the number of prominent focal adhesion in wild-type exceeds that in the knockout by over 5-fold. Error bars represent the measurements in 21 and 18 different images in WT and KO, respectively. Bar, 20 µm.
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pgen-1000878-g006: Ate1 knockout affects cell attachment to the substrate via intracellular and not extracellular mechanisms.(A) Left, extracellular fibronectin staining in a dense monolayer of wild-type (WT) and Ate1 knockout (KO) cultured fibroblasts shows no difference between the two cultures. Right, quantification of the fibronectin level in the two cultures measured as average gray value in the entire field of view confirms that there is no difference between WT and KO cells in the amount of extracellular fibronectin. Bars show the ratio between WT and KO and error bars represent the average of the measurements in 10 different fields of view in each culture. (B) Left, an overlay of the fluorescence staining of the edge of the cell monolayer moving into the wound co-stained with rhodamine-phalloidin (red) to visualize the actin filaments and anti-paxillin (green) to visualize the focal adhesions. Right, quantification of the number of focal adhesions per µm of the wound edge shows that the number of prominent focal adhesion in wild-type exceeds that in the knockout by over 5-fold. Error bars represent the measurements in 21 and 18 different images in WT and KO, respectively. Bar, 20 µm.

Mentions: Cell migration in culture and in situ is mediated by attachment to the substrate and forming a connection between the intracellular actin cytoskeleton and the extracellular matrix via focal adhesions. To test whether the slow migration speeds in Ate1 knockout cells were due to their impaired adhesion on the intra- or extracellular side, we first tested whether these cells are capable of creating a local extracellular environment that favors migration. To do this, we stained wild-type and Ate1 knockout non-permeabilized cell monolayers for fibronectin, a major extracellular matrix component that directs the migration and adhesion of the mesenchymal cells and is secreted by these cells in culture and in situ (reviewed in [46]). No differences were found in the amount or distribution of fibronectin per area in each culture (Figure 6A), suggesting that Ate1 knockout cells are capable of creating and utilizing the same local extracellular environment as wild-type. Thus, the migration defects in Ate1 knockout cells do not originate at the local extracellular level.


Arginylation-dependent neural crest cell migration is essential for mouse development.

Kurosaka S, Leu NA, Zhang F, Bunte R, Saha S, Wang J, Guo C, He W, Kashina A - PLoS Genet. (2010)

Ate1 knockout affects cell attachment to the substrate via intracellular and not extracellular mechanisms.(A) Left, extracellular fibronectin staining in a dense monolayer of wild-type (WT) and Ate1 knockout (KO) cultured fibroblasts shows no difference between the two cultures. Right, quantification of the fibronectin level in the two cultures measured as average gray value in the entire field of view confirms that there is no difference between WT and KO cells in the amount of extracellular fibronectin. Bars show the ratio between WT and KO and error bars represent the average of the measurements in 10 different fields of view in each culture. (B) Left, an overlay of the fluorescence staining of the edge of the cell monolayer moving into the wound co-stained with rhodamine-phalloidin (red) to visualize the actin filaments and anti-paxillin (green) to visualize the focal adhesions. Right, quantification of the number of focal adhesions per µm of the wound edge shows that the number of prominent focal adhesion in wild-type exceeds that in the knockout by over 5-fold. Error bars represent the measurements in 21 and 18 different images in WT and KO, respectively. Bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2837401&req=5

pgen-1000878-g006: Ate1 knockout affects cell attachment to the substrate via intracellular and not extracellular mechanisms.(A) Left, extracellular fibronectin staining in a dense monolayer of wild-type (WT) and Ate1 knockout (KO) cultured fibroblasts shows no difference between the two cultures. Right, quantification of the fibronectin level in the two cultures measured as average gray value in the entire field of view confirms that there is no difference between WT and KO cells in the amount of extracellular fibronectin. Bars show the ratio between WT and KO and error bars represent the average of the measurements in 10 different fields of view in each culture. (B) Left, an overlay of the fluorescence staining of the edge of the cell monolayer moving into the wound co-stained with rhodamine-phalloidin (red) to visualize the actin filaments and anti-paxillin (green) to visualize the focal adhesions. Right, quantification of the number of focal adhesions per µm of the wound edge shows that the number of prominent focal adhesion in wild-type exceeds that in the knockout by over 5-fold. Error bars represent the measurements in 21 and 18 different images in WT and KO, respectively. Bar, 20 µm.
Mentions: Cell migration in culture and in situ is mediated by attachment to the substrate and forming a connection between the intracellular actin cytoskeleton and the extracellular matrix via focal adhesions. To test whether the slow migration speeds in Ate1 knockout cells were due to their impaired adhesion on the intra- or extracellular side, we first tested whether these cells are capable of creating a local extracellular environment that favors migration. To do this, we stained wild-type and Ate1 knockout non-permeabilized cell monolayers for fibronectin, a major extracellular matrix component that directs the migration and adhesion of the mesenchymal cells and is secreted by these cells in culture and in situ (reviewed in [46]). No differences were found in the amount or distribution of fibronectin per area in each culture (Figure 6A), suggesting that Ate1 knockout cells are capable of creating and utilizing the same local extracellular environment as wild-type. Thus, the migration defects in Ate1 knockout cells do not originate at the local extracellular level.

Bottom Line: Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture.Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death.Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Show MeSH
Related in: MedlinePlus