HOP Regulates Cell Migration Figure 4. HOP regulates cell polarization and membrane ruffling in migrating cells. A. HUVECs transfected with HOP or control siRNAs for 96 hours were scratched, fixed 2 hours later and stained with anti-a-tubulin antibody, anti-c-tubulin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue) respectively. Broken white lines indicate the wound directions. B. Experiments were performed as in A, and the percentage of polarized cells was quantified. **, p,0.01 versus control. C. HUVECs were transfected with GFP along with HOP shRNA or the control vector, and the fluorescence of GFP at the leading edge of cells were recorded at 20-second intervals with the use of the TCS SP5 confocal microscope (Leica). Rectangular regions were selected as indicated to analyze membrane ruffle dynamics. D. Experiments were performed as in C, and membrane ruffle dynamics were presented as 3-dimensional surface plots. E. Immunoblotting analysis of the expression of several Hsp90 client proteins involved in cell migration and polarity determination in HUVECs transfected with HOP or control siRNAs. doi:10.1371/journal.pone.0036389.g004 compared with the control vector (Figure 3C and 3D). Thus, HOP plays a critical role in cell polarization and membrane ruffling in migrating cells. It has been previously shown that in pancreatic cancer cells, the knockdown of HOP expression could decrease the expression levels of several Hsp90 client proteins, including HER2, Bcr-Abl, C-MET, and v-Src [39]. We thus studied whether HOP depletion in HUVECs reduces the levels of known Hsp90 client proteins involved in cell migration and polarity determination, such as Cdc42, RhoA, Rac1, Akt, and HDAC6 [40–43]. By immuno- blotting, we found that the downregulation of HOP in HUVECs did not affect the expression levels of these proteins (Figure 4E). HOP Colocalizes with Microtubules and Interacts with Tubulin Microtubules and microtubule-binding proteins play important roles in cell migration and polarization [44]. To test whether HOP functions in cell migration and polarization through interacting with microtubules, we performed immunoprecipitation and GST-pull- down assays. We found that cellular GFP-HOP could coprecipitate with cellular tubulin (Figure 5A). In addition, in the purified system, GST-HOP protein could pulldown tubulin (Figure 5B). We also studied the localization of HOP in HUVECs by immunofluorescent staining. As shown in Figure 5C, HOP could partially colocalize with microtubules in HUVECs. These results thus reveal that HOP interacts with both tubulin and microtubules. To identify the domains of HOP mediating its interaction with tubulin/microtubules, we constructed a series of plasmids that express various truncated forms of HOP. By immunoprecipitation assay, we found that the HOP mutant lacking the TPR2a domain had the strongest binding affinity to tubulin/microtubules, whereas the TPR2a domain did not interact with tubulin/ microtubules (Figure 5D and 5E). Addition of Anti-HOP Antibody or Recombinant HOP to the Culture Medium Does Not Affect Vascular Endothelial Cell Migration or Tube Formation It has been recently reported that Hsp90 and HOP could be released from tumor cells and that extracellular Hsp90 and HOP could promote cell migration and migration [39,45,46]. We therefore examined whether HOP could also act also extracellu- larly in enhancing cell migration. To test this possibility, scratch wound assays were performed by adding anti-HOP antibody, control IgG antibody, purified GST, or purified GST-HOP to the culture medium. As shown in Figure 6A and 6B, the addition of anti-HOP antibody or recombinant HOP to the culture medium does not affect the migration of HUVECs. In addition, we found that the addition of anti-HOP antibody or recombinant HOP to PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36389