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Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

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Effect of VEGF on EB1 comets.(A) Localization of EB1 in HUVECs by indirect immunofluorescence after 1 h of VEGF treatment at various concentrations. Bar, 10 µm. (B) Dose response effect of VEGF on EB1 comet length. * p≤0.001 (vs control, Student t test). (C) Incubation of HUVECs with VEGF trap (1 h) inhibited VEGF-increase of EB1 comet length. * p≤0.001. (D) Western blotting detection of EB1 and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations.
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pone-0065694-g001: Effect of VEGF on EB1 comets.(A) Localization of EB1 in HUVECs by indirect immunofluorescence after 1 h of VEGF treatment at various concentrations. Bar, 10 µm. (B) Dose response effect of VEGF on EB1 comet length. * p≤0.001 (vs control, Student t test). (C) Incubation of HUVECs with VEGF trap (1 h) inhibited VEGF-increase of EB1 comet length. * p≤0.001. (D) Western blotting detection of EB1 and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations.

Mentions: We then explored the effects of VEGF on EB1 comet length by indirect immunofluorescence (Figure 1). At 10 ng/ml VEGF increased EB1 comet length by 40% (3.03±0.3 µm vs 2.17±0.18 µm, p<0.05 (Figure 1 A, B) and induced, in nearly 20% of cells, a complete MT lattice staining (Figure 1A). HUVECs pre-incubation with VEGF-Trap inhibited VEGF increase of EB1 comets, suggesting that such effect was specific of VEGF signaling (Figure 1C). Interestingly, the modification of EB1 comet length by VEGF was not associated with any change in EB1 expression level (Figure 1D).


Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Effect of VEGF on EB1 comets.(A) Localization of EB1 in HUVECs by indirect immunofluorescence after 1 h of VEGF treatment at various concentrations. Bar, 10 µm. (B) Dose response effect of VEGF on EB1 comet length. * p≤0.001 (vs control, Student t test). (C) Incubation of HUVECs with VEGF trap (1 h) inhibited VEGF-increase of EB1 comet length. * p≤0.001. (D) Western blotting detection of EB1 and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672205&req=5

pone-0065694-g001: Effect of VEGF on EB1 comets.(A) Localization of EB1 in HUVECs by indirect immunofluorescence after 1 h of VEGF treatment at various concentrations. Bar, 10 µm. (B) Dose response effect of VEGF on EB1 comet length. * p≤0.001 (vs control, Student t test). (C) Incubation of HUVECs with VEGF trap (1 h) inhibited VEGF-increase of EB1 comet length. * p≤0.001. (D) Western blotting detection of EB1 and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations.
Mentions: We then explored the effects of VEGF on EB1 comet length by indirect immunofluorescence (Figure 1). At 10 ng/ml VEGF increased EB1 comet length by 40% (3.03±0.3 µm vs 2.17±0.18 µm, p<0.05 (Figure 1 A, B) and induced, in nearly 20% of cells, a complete MT lattice staining (Figure 1A). HUVECs pre-incubation with VEGF-Trap inhibited VEGF increase of EB1 comets, suggesting that such effect was specific of VEGF signaling (Figure 1C). Interestingly, the modification of EB1 comet length by VEGF was not associated with any change in EB1 expression level (Figure 1D).

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

Show MeSH
Related in: MedlinePlus