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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.


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Mutations in the-YRKLLEGEE-region of Vimentin disrupts IFs arrangement.Representative images of the colonies measured in Fig. 3, showing disruption of Vim filaments. Scale bar = 25 μm; inset scale bar 7.5 μm.
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f4: Mutations in the-YRKLLEGEE-region of Vimentin disrupts IFs arrangement.Representative images of the colonies measured in Fig. 3, showing disruption of Vim filaments. Scale bar = 25 μm; inset scale bar 7.5 μm.

Mentions: The-YRKLLEGEE- sequence of Type-II keratins, such as KRT5, is required for interaction and co-polymerization with Type-I keratins, such as KRT14, that have a similar but not identical sequence (-YRRLLEGE-)2425. The -YRKLLEGEE- sequence is essential to maintain the functional integrity of the keratin network, as a single mutation in KRT5 (-YRKLLGGEE-) can cause epidermolysis bullosa simplex2627. Vim, at the end of the helical 2B-domain, has the same amino acid sequence -YRKLLEGEE- found in the type-II keratins17, and this sequence contributes to the IF dimer stability as it was reported in in vitro studies25. This raises the possibility that Vim could interact in vivo with KRT14 or other acidic keratins through such amino acid sequence. We tested this possibility by introducing point mutations by site-directed mutagenesis in the Vim sequence. We tested the mutation that occurred in KRT5 of epidermolysis bullosa by changing Glu 405 to Gly (V-E405G)26; a mutation in which the E407 was replaced by G (V-E407G); and a third mutation containing both modifications (V-E405G-E407G) (Fig. 3A). To our knowledge, these specific mutations have not been tested before in the Vim molecule. However, the deletion of the full coil 2B region of Vim in fibroblasts disrupts the cytoskeleton and delays apoptosis28. We carried out two types of experiments; one testing the colony expansion of growing keratinocytes, and two, the cell migration assay (MRMA) with 3T3 feeder cells (See Materials and Methods). By real-time PCR we showed a 2–6-fold increase in Vim mRNA in the transfected cells, in comparison with controls (Fig. 3B). We also found that all these three Vim mutants exerted a significant reduction in colony size (Fig. 3C) and, as it was expected, disrupted the Vim IFs (Fig. 4) since the -YRKLLEGEE- sequence contributes to the IF dimer stability25, and since we also showed the interaction between Vim with KRT14 (Fig. 1), it was also expected that the keratin filaments would be partially disrupted in the Vim+ keratinocytes (Fig. 4)), demonstrating that mutations in this Vim sequence inhibit colony expansion of the cells. The colony size of keratinocytes transfected with wild type Vim cDNA was similar to the controls, demonstrating that forced expression of Vim per se did not affect colony size (Fig. 3C). However, keratinocytes transfected with the mutated Vim genes showed about 50% smaller colony size (Fig. 3C).


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)

Mutations in the-YRKLLEGEE-region of Vimentin disrupts IFs arrangement.Representative images of the colonies measured in Fig. 3, showing disruption of Vim filaments. Scale bar = 25 μm; inset scale bar 7.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Mutations in the-YRKLLEGEE-region of Vimentin disrupts IFs arrangement.Representative images of the colonies measured in Fig. 3, showing disruption of Vim filaments. Scale bar = 25 μm; inset scale bar 7.5 μm.
Mentions: The-YRKLLEGEE- sequence of Type-II keratins, such as KRT5, is required for interaction and co-polymerization with Type-I keratins, such as KRT14, that have a similar but not identical sequence (-YRRLLEGE-)2425. The -YRKLLEGEE- sequence is essential to maintain the functional integrity of the keratin network, as a single mutation in KRT5 (-YRKLLGGEE-) can cause epidermolysis bullosa simplex2627. Vim, at the end of the helical 2B-domain, has the same amino acid sequence -YRKLLEGEE- found in the type-II keratins17, and this sequence contributes to the IF dimer stability as it was reported in in vitro studies25. This raises the possibility that Vim could interact in vivo with KRT14 or other acidic keratins through such amino acid sequence. We tested this possibility by introducing point mutations by site-directed mutagenesis in the Vim sequence. We tested the mutation that occurred in KRT5 of epidermolysis bullosa by changing Glu 405 to Gly (V-E405G)26; a mutation in which the E407 was replaced by G (V-E407G); and a third mutation containing both modifications (V-E405G-E407G) (Fig. 3A). To our knowledge, these specific mutations have not been tested before in the Vim molecule. However, the deletion of the full coil 2B region of Vim in fibroblasts disrupts the cytoskeleton and delays apoptosis28. We carried out two types of experiments; one testing the colony expansion of growing keratinocytes, and two, the cell migration assay (MRMA) with 3T3 feeder cells (See Materials and Methods). By real-time PCR we showed a 2–6-fold increase in Vim mRNA in the transfected cells, in comparison with controls (Fig. 3B). We also found that all these three Vim mutants exerted a significant reduction in colony size (Fig. 3C) and, as it was expected, disrupted the Vim IFs (Fig. 4) since the -YRKLLEGEE- sequence contributes to the IF dimer stability25, and since we also showed the interaction between Vim with KRT14 (Fig. 1), it was also expected that the keratin filaments would be partially disrupted in the Vim+ keratinocytes (Fig. 4)), demonstrating that mutations in this Vim sequence inhibit colony expansion of the cells. The colony size of keratinocytes transfected with wild type Vim cDNA was similar to the controls, demonstrating that forced expression of Vim per se did not affect colony size (Fig. 3C). However, keratinocytes transfected with the mutated Vim genes showed about 50% smaller colony size (Fig. 3C).

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