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.
Highlights
Cell migration plays a crucial role in neoangiogenesis and tumor progression
Blocking the autocrine Vascular Endothelial Growth Factor (VEGF) effect on Human Umbilical Vein Endothelial Cells (HUVECs) by using VEGF Trap led to a significant increase in overall MT dynamicity from 4.9 mm/min in control cells to 7.0 mm/min in VEGF Trap-treated cells accompanied by a significant increase in both MT growth rate (11.360.7 mm/min vs 14.361.2 mm/min, p,0.05) and shortening rate (14.161.0 mm/min vs 20.261.3 mm/ min, p,0.001), and in the transition frequencies, i.e catastrophes (2.160.3 m21 vs 1.860.3 m21, p,0.05) and rescues (5.660.8 m21 vs 4.260.8 m21, p,0.01) (Table 1)
In HUVECs co-transfected with GFP-tubulin and DsRed-paxillin, we showed that MT (+) ends localized close to the plasma membrane in control and VEGF treated cells as observed by TIRF microscopy (Figure S2A)
Summary
Cell migration plays a crucial role in neoangiogenesis and tumor progression. Major components of the cytoskeleton, microtubules (MT) are key players involved in cell polarization and migration in many cell types such as fibroblasts and endothelial cells [1,2]. Several works have revealed the crucial role of the (+) end tracking protein EB1 in cell migration [4,5,6]. EB1 is considered as the master regulator at MT (+) ends, since it has been shown to act as a loading factor of other MT-interacting proteins, including those responsible for the MT stabilization at the cell cortex [11,12]. The hallmark of +TIPs is that they form dynamic interaction networks that rely on a limited number of protein modules and linear sequence motifs, such as the CH, EBH and CAP-Gly domains, and EEY/F and SxIP motifs [11]. EB1 C-terminal sequence 2EEY is responsible for interaction with the CAP-Gly domain containing +TIPs such as CLIP-170 and p150Glued, and their localization at MT (+) ends [9]. No post-translational modification of the C-terminal extremity of EB1 has been identified
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
