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Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments.

Velez-delValle C, Marsch-Moreno M, Castro-Muñozledo F, Galván-Mendoza IJ, Kuri-Harcuch W - Sci Rep (2016)

Bottom Line: Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes.Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin.These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Center of Research and Advanced Studies, IPN. Apdo. Postal 14-740, México City, 07000, Mexico.

ABSTRACT
Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Decreased migration of keratinocytes electroporated with plasmid harboring Vimentin mutants in the –YRKLLEGEE- sequence of the Vimentin gene.Keratinocytes were electroporated with a Wt Vim or its mutants and they were seeded in a MRMA as above to measure cell migration. We seeded MRMA epidermal cultures for 24 h and thereafter treated them for 6 h with EGF, with or without the plasmid harboring the Vim genes. Then, after photographing the cultures under phase contrast microscopy, we determined the distance of migration by quantifying the area of the MRMA, and then calculated its radius. From these data, distance of migration was calculated. (A) Representative images of MRMA assay at 6 h. (B) Graph showing the distance of migration under each treatment. Note that cultures treated with the plasmid harboring the mutant Vim genes had a reduced distance of migration in comparison to control cultures. Data is the mean of duplicate experiments with n =16 each, ± SD *p ≤ 0.05.
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f5: Decreased migration of keratinocytes electroporated with plasmid harboring Vimentin mutants in the –YRKLLEGEE- sequence of the Vimentin gene.Keratinocytes were electroporated with a Wt Vim or its mutants and they were seeded in a MRMA as above to measure cell migration. We seeded MRMA epidermal cultures for 24 h and thereafter treated them for 6 h with EGF, with or without the plasmid harboring the Vim genes. Then, after photographing the cultures under phase contrast microscopy, we determined the distance of migration by quantifying the area of the MRMA, and then calculated its radius. From these data, distance of migration was calculated. (A) Representative images of MRMA assay at 6 h. (B) Graph showing the distance of migration under each treatment. Note that cultures treated with the plasmid harboring the mutant Vim genes had a reduced distance of migration in comparison to control cultures. Data is the mean of duplicate experiments with n =16 each, ± SD *p ≤ 0.05.

Mentions: Immunoprecipitation with the mAb against KRT14, V-E405G showed a lower content of immunoprecipitated Vim as compared to wild type, suggesting less association between the two molecules (Fig. 3D), whereas the other two mutants comprising amino acid 407 did not show significant changes in the content of the immunoprecipitated protein (Fig. 3D). Most importantly, we carried out functional experiments by the MRMA assay with the keratinocytes transfected with the plasmids harboring the mutants VE405G and VE407G (Fig. 5A), we found that each mutant reduced migration of the keratinocytes (Fig. 5B). In the MRMA control cultures without EGF cells migrated 34.2 μm and in the EGF treated cultures they migrated 78.1 μm, whereas in the V-E405G and VE407G transfected cultures and treated with EGF keratinocytes migrated 62.1 and 48.8 μm, 20 and 38% lower, respectively than in the control EGF stimulated cultures (Fig. 5B). Together, these results suggested that Vim interaction with keratins is necessary for cell migration, and more specifically, the -YRKLLEGEE- sequence seems to be a point of interaction between the two IFs in diploid keratinocytes.


Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments.

Velez-delValle C, Marsch-Moreno M, Castro-Muñozledo F, Galván-Mendoza IJ, Kuri-Harcuch W - Sci Rep (2016)

Decreased migration of keratinocytes electroporated with plasmid harboring Vimentin mutants in the –YRKLLEGEE- sequence of the Vimentin gene.Keratinocytes were electroporated with a Wt Vim or its mutants and they were seeded in a MRMA as above to measure cell migration. We seeded MRMA epidermal cultures for 24 h and thereafter treated them for 6 h with EGF, with or without the plasmid harboring the Vim genes. Then, after photographing the cultures under phase contrast microscopy, we determined the distance of migration by quantifying the area of the MRMA, and then calculated its radius. From these data, distance of migration was calculated. (A) Representative images of MRMA assay at 6 h. (B) Graph showing the distance of migration under each treatment. Note that cultures treated with the plasmid harboring the mutant Vim genes had a reduced distance of migration in comparison to control cultures. Data is the mean of duplicate experiments with n =16 each, ± SD *p ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829867&req=5

f5: Decreased migration of keratinocytes electroporated with plasmid harboring Vimentin mutants in the –YRKLLEGEE- sequence of the Vimentin gene.Keratinocytes were electroporated with a Wt Vim or its mutants and they were seeded in a MRMA as above to measure cell migration. We seeded MRMA epidermal cultures for 24 h and thereafter treated them for 6 h with EGF, with or without the plasmid harboring the Vim genes. Then, after photographing the cultures under phase contrast microscopy, we determined the distance of migration by quantifying the area of the MRMA, and then calculated its radius. From these data, distance of migration was calculated. (A) Representative images of MRMA assay at 6 h. (B) Graph showing the distance of migration under each treatment. Note that cultures treated with the plasmid harboring the mutant Vim genes had a reduced distance of migration in comparison to control cultures. Data is the mean of duplicate experiments with n =16 each, ± SD *p ≤ 0.05.
Mentions: Immunoprecipitation with the mAb against KRT14, V-E405G showed a lower content of immunoprecipitated Vim as compared to wild type, suggesting less association between the two molecules (Fig. 3D), whereas the other two mutants comprising amino acid 407 did not show significant changes in the content of the immunoprecipitated protein (Fig. 3D). Most importantly, we carried out functional experiments by the MRMA assay with the keratinocytes transfected with the plasmids harboring the mutants VE405G and VE407G (Fig. 5A), we found that each mutant reduced migration of the keratinocytes (Fig. 5B). In the MRMA control cultures without EGF cells migrated 34.2 μm and in the EGF treated cultures they migrated 78.1 μm, whereas in the V-E405G and VE407G transfected cultures and treated with EGF keratinocytes migrated 62.1 and 48.8 μm, 20 and 38% lower, respectively than in the control EGF stimulated cultures (Fig. 5B). Together, these results suggested that Vim interaction with keratins is necessary for cell migration, and more specifically, the -YRKLLEGEE- sequence seems to be a point of interaction between the two IFs in diploid keratinocytes.

Bottom Line: Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes.Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin.These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Center of Research and Advanced Studies, IPN. Apdo. Postal 14-740, México City, 07000, Mexico.

ABSTRACT
Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

No MeSH data available.


Related in: MedlinePlus