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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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Anti-migratory vinflunine favored the tyrosinated form of EB1.(A) Effect of VFL on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 5 h with VFL (10, 100 nM) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin) (B) Detection of EB1-EEY sequence with YL½ antibody increased in a concentration-dependent manner under VFL concentrations. Relative ratios are presented under the blots. (C) Correlation between detection of EB1-EEY sequence and reduction of EB1 comet length by VFL.
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pone-0065694-g005: Anti-migratory vinflunine favored the tyrosinated form of EB1.(A) Effect of VFL on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 5 h with VFL (10, 100 nM) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin) (B) Detection of EB1-EEY sequence with YL½ antibody increased in a concentration-dependent manner under VFL concentrations. Relative ratios are presented under the blots. (C) Correlation between detection of EB1-EEY sequence and reduction of EB1 comet length by VFL.

Mentions: We then assessed whether VFL regulated EB1 at a post-translational level. In endothelial cells, VFL (10 nM) altered EB1 2D profile by increasing focalization at isoelectric point 5.02 (native tyrosinated form) with a concomitant decrease at the isoelectric point 5.03 (detyrosinated form) (Figure 5A). This effect was more pronounced with 100 nM VFL as the two spots were clearly visible.


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)

Anti-migratory vinflunine favored the tyrosinated form of EB1.(A) Effect of VFL on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 5 h with VFL (10, 100 nM) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin) (B) Detection of EB1-EEY sequence with YL½ antibody increased in a concentration-dependent manner under VFL concentrations. Relative ratios are presented under the blots. (C) Correlation between detection of EB1-EEY sequence and reduction of EB1 comet length by VFL.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065694-g005: Anti-migratory vinflunine favored the tyrosinated form of EB1.(A) Effect of VFL on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 5 h with VFL (10, 100 nM) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin) (B) Detection of EB1-EEY sequence with YL½ antibody increased in a concentration-dependent manner under VFL concentrations. Relative ratios are presented under the blots. (C) Correlation between detection of EB1-EEY sequence and reduction of EB1 comet length by VFL.
Mentions: We then assessed whether VFL regulated EB1 at a post-translational level. In endothelial cells, VFL (10 nM) altered EB1 2D profile by increasing focalization at isoelectric point 5.02 (native tyrosinated form) with a concomitant decrease at the isoelectric point 5.03 (detyrosinated form) (Figure 5A). This effect was more pronounced with 100 nM VFL as the two spots were clearly visible.

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