HOP Regulates Cell Migration Figure 6. Addition of anti-HOP antibody or recombinant HOP to the culture medium does not affect vascular endothelial cell migration or tube formation. A. Scratch wound assays were performed using HUVECs with the addition of anti-HOP antibody, control IgG antibody, purified GST, or purified GST-HOP in the culture medium, and wound margins were imaged 0, 6, or 14 hours later. B. Experiments were performed as in A, and the extent of wound closure was quantified. C. Tube formation assays were performed using HUVECs with the addition of anti-HOP antibody, control IgG antibody, purified GST, or purified GST-HOP in the culture medium, and photographs were taken 1, 3, and 6 hours later. D. Experiments were performed as in C, and the cumulative tube length was measured. doi:10.1371/journal.pone.0036389.g006 microtubule dynamics and mediating endothelial cell migration [49]. In this scenario, it is tempting to speculate that HOP may function in angiogenesis in a microtubule-dependent manner; however, additional studies are warranted to investigate this possibility. In this study, we have found that HOP colocalizes with microtubules in cells and shown that HOP could interact with tubulin both in cells and in vitro. At present, it remains unclear how HOP and microtubules collaborate to promote cell migration and angiogenesis. It was reported previously that the TPR motif of HOP could form scaffolds to help formation of the chaperone complex [50]. Thus, it is possible that microtubules may function together with HOP to form scaffolds in promoting cell migration and angiogenesis. It is also possible that microtubules may mediate the trafficking of HOP and promote its redistribution in the cytoplasm to facilitate cell polarization and migration. Structural studies reveal that the interaction between HOP and its target chaperones, Hsp70 and Hsp90, mainly depends on the EEVD motif of the chaperone peptides and the aspartate residue in TPR domains to form a two-carboxylate clamp [6]. In the present study, we have sought to investigate how HOP binds to microtubules/tubulin. Our data show that the domains mediating HOP interaction with microtubules/tubulin may include both TPR1 and TPR2b; however, there is no EEVD motif in tubulin. Considering that TPR domains are generally flexible in the interaction with partner proteins and may have multiple binding modes, different contact interface and different residues might be involved in the interaction of HOP with microtubules/tubulin. In summary, in the present study we have established a relation- ship between the HOP protein and angiogenesis by a series of experiments. It will be important to further study the role of HOP in angiogenesis using in vivo models. In addition, the anti-angiogenesis strategy has been employed in cancer treatment [19–21]. Given the critical role of HOP in angiogenesis [51], it will be interesting to investigate in the future the potential of HOP as an anti-cancer drug target. Author Contributions Conceived and designed the experiments: JZ ML. Performed the experiments: JL XS ZW LC DL. Analyzed the data: JL XS. Wrote the paper: ZW JZ. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e36389