and as the expression of the N-terminal cytoplasmic fragment remained poor, we restricted our study to the C-terminus of TRPV4 only. Pull down was performed from adult porcine brain homogenate as well as from F11 cell lysate, a fusion-cell of rat DRG neurons and mouse neuroblastoma cells [38]. In immuno- blot analysis, we observed MBP-TRPV4-C-terminus fusion protein (MBP-TRPV4-Ct) but not the control fusion protein MBP-LacZ to pull down soluble tubulin and actin (Fig. 1c, S2). When probed for the presence of another cytoskeletal element, namely soluble neurofilament proteins, only minimal amounts of NF116 kDa and no NF200 kDa were detected. As activation of TRPV4 results in high Ca2+-influx, we tested if higher concentration of Ca2+ could modulate these interactions. However, we observed that tubulin and actin interaction with TRPV4 do not depend on Ca2+ (Fig. 1c, S2). TRPV4-Ct forms a Ca2+-sensitive supra-molecular signaling complex made of actin, tubulin as well as nociceptive signaling components PKCe and CamKII Previously we have demonstrated that mechanical hyperalgesia induced agonists of novel estrogen receptor GPR30 strongly correlates with the translocation of PKCe in IB4 (+) neurons [39]. Moreover, mechanical hyperalgesia can be induced by activation of the epsilon isoform of PKC (PKCe), a well-described pro- nociceptive signaling molecule [39–43]. In addition, calmodulin, and CamKII function has been linked with the chronic inflamma- tory pain [44–46]. Thus, we tested if the TRPV4-actin/tubulin complex also contains PKCe and/or CamKII. We found CamKII to be present in the eluates of the pull-down material both from soluble brain and F11 cell extract, but only in the presence of Ca2+ (Fig. 1c, Fig. S1). In addition, we also detected PKCe in the MBP- TRPV4-Ct complex, both in presence and absence of Ca2+. Presence of PKCe or CamKII was not observed with the control protein MBP-LacZ. Thus, using two different biological sources, our results indicate that TRPV4-Ct can form supra-molecular complexes consisting of structural and signaling proteins. MBP-TRPV4-Ct interacts directly with soluble tubulin and actin To test if TRPV4-Ct interacts directly with tubulin and actin, we performed pull down experiments with the purified MBP-TRPV4- Ct, tubulin, and actin. As expected, MBP-TRPV4-Ct but not MBP- LacZ pulls down tubulin (Fig. 1d). Tubulin is subjected to different types of post-translational modification, which regulate the process of microtubule stabilization, destabilization and maturation. Thus, we tested for the presence of various post-translationally modified tubulins in the complex formed with MBP-TRPV4-Ct. Indeed we found a large number of these post-translationally modified tubulins and neuron-specific b-III tubulin (Fig. 1d). Next we tested if also soluble actin interacts directly with MBP- TRPV4-Ct. In immunoblot analysis, we observed that the MBP- TRPV4-Ct pulls down purified actin, while under the same conditions MBP-LacZ was unable to pull down any actin (Fig. 1e). Again, we observed no influence of Ca2+ on this interaction. Tubulin and actin compete for binding to MBP-TRPV4-Ct To understand if tubulin binding affects actin binding and vice versa, we performed a competition experiment between soluble tubulin and soluble actin for the binding to MBP-TRPV4-Ct. Soluble tubulin and actin were added to MBP-TRPV4-Ct sequentially before being analysed for their binding to TRPV4 (Fig. 1f). As control either soluble tubulin or soluble actin were used alone and washed in the same manner. We observed that the amount of bound tubulin was TRPV4 Regulates Cytoskeleton strongly reduced if MBP-TRPV4-Ct was initially incubated with actin (Fig. 1f). Inversely, the amount of bound actin is very low if MBP- TRPV4-Ct is initially incubated with tubulin. These results indicate that both actin and tubulin compete for binding to MBP-TRPV4-Ct. MBP-TRPV4-Ct interacts with polymerized actin and tubulin filaments and favours formation of stable microtubules Next we addressed whether TRPV4 can also bind to polymerized filaments. We observed that MBP-TRPV4-Ct co-sedimented with polymerized actin and thus appeared in the pellet fraction (Fig. 2a). Under the same conditions, MBP only showed no interaction with polymerized actin filaments. Here we probed if the MBP-TRPV4-Ct can also interact with polymerized microtubules. We observed that MBP-TRPV4-Ct but not MBP alone co-sedimented with Taxol-stabilized polymerized microtubules (Fig. 2b, left side). In addition, we observed that if MBP-TRPV4-Ct was added to saturated tubulin dimer solution during GTP-induced microtubule formation, co-sedimentation of MBP-TRPV4-Ct with polymerized microtubules was also observed (Fig. 2b, right side). Again, only MBP, the control protein failed to bind polymerized microtubules. These results confirm that MBP- TRPV4-Ct directly interacts with filamentous microtubules. We tested if MBP-TRPV4-Ct can change the physico-chemical properties of the microtubules. For that purpose, we analysed the stability of the microtubules by comparing the amount of microtubules formed under depolymerization conditions, both in the presence or absence of MBP-TRPV4-Ct. We observed that the presence of MBP-TRPV4-Ct favours microtubule formation even in presence of Nocodazole, Ca2+, or both (Fig. 2c). In contrast, in absence of MBP-TRPV4-Ct much lower amount of microtubules was formed. MBP as a control protein does not interact with the polymerised microtubules and thus fails to provide stability to the microtubules. This result suggests a stabilization effect of MBP- TRPV4-Ct on microtubules. TRPV4 localizes to actin- and microtubule-enriched regions in F11 cell Based on a RT-PCR analysis demonstrating the amplification of small mRNA fragment, endogenous expression of TRPV4 in F11 cell has been proposed [47]. However, by immunofluorescence analysis two different polyclonal antibodies which recognise the C- terminal region of TRPV4, we could not observe any specific endogenous expression of TRPV4 in F11 cells. To investigate if the biochemical interaction under in vitro conditions also occurs in vivo, we expressed TRPV4 in F11 cells as well as in other non- neuronal cells and performed co-localization experiments. The purpose of TRPV4 overexpression in F-11 cells is to mimic the physiological situation in DRG neurons. Using fluorescent labelled Phalloidin, we observed co-localiza- tion of TRPV4 with actin at various actin cytoskeleton enriched regions, such as the actin ribs of the cortical membranous regions, and at filopodial and lamellipodial structures (Fig. S3). As some of these structures are very dynamic and sensitive to environmental changes such as temperature drops or media composition, we attempted to confirm that the observed co-localization in these structures was not a fixation artefact. Thus, we performed live cell imaging by expressing TRPV4-GFP and RFP-actin in F11 cells and we observed co-localization at filopodia, at lamellipodia, and also at cortical actin-rich structures (Fig. 3a-c). We also noted co-localization at actin-rich structures, which resemble focal adhesion points (Fig. 3b). This is in agreement with the fact that focal adhesion points are important for cellular mechanosensory functions [48]. PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11654