be stained by YL1/2 antibody, which is specific for ‘‘tyrosinated tubulin’’, indicating that these microtubules are dynamic in nature [74–75]. How TRPV4 stabilizes microtubules in vivo is not clear. But our in vitro work indicates that the C-terminus of TRPV4 is sufficient for microtubule stabilization. The extent of microtubule stabilization by TRPV4-Ct is apparently very strong as TRPV4-Ct stabilizes microtubules are resistant against potent microtubule destabilizers such as Nocodazole and Ca2+. Thus, TRPV4-Ct induces ‘‘Nocodazole-resistant microtubules’’, a class of microtu- bules that has very different physico-chemical properties [76–77]. Notably, a fraction of ‘‘Nocodazole-resistant microtubules’’ are known to be resistant against Ca2+ and cold-induced depolymer- ization [78–79]. The resistance of a subset of microtubules against calcium is of high importance at the base of an ion channel, which among others allows calcium influx if activated. The observed binding of post-translationally modified tubulin such as acetylated tubulin, detyrosinated tubulin and polyglutamylated tubulin to TRPV4 may also explain the stabilization as these tubulins are known to be involved in the formation of stable microtubules [76,80–81]. Regulation of growth cone, neurites and filopodia by TRPV4 Cellular and neuritic morphology is modulated through mutual regulation of the actin and microtubule cytoskeleton. The microtubule cytoskeleton provides an anterograde force while the actin cytoskeleton provides retrograde force. Thus these two opposing forces regulate the morphology and determine the net movement of neurites [32–34,52,82]. As TRPV4 modulates actin as well as microtubule stability, it has the ability to steer growth cone movements. This result matches well with our previous work demonstrating that activation of TRPV1 causes rapid disassembly of microtubules and thus regulates growth cone movement [31–32,51,82]. However, unlike TRPV1, TRPV4 activation-mediated (by using 4aPDD) disassembly of microtu- bules in DRG neurons, in transfected F11 cells as well as in non-neuronal cells seems to be much slower. Therefore, TRPV1 and TRPV4 might be involved in regulation of the neuronal architecture not only in response to different stimuli but also along a different timescale. Regulation of TRPV4 activity by actin and microtubule cytoskeleton TRPV4 not only changes the cellular cytoskeleton but the cellular cytoskeleton is in turn also altering the TRPV4 channel properties. On one hand TRPV4 regulates stability-instability of the microtubules and reorganizes them, on the other hand microtubule dynamics regulate the Ca2+-influx through TRPV4. We now show that stabilization of microtubules by Taxol in vivo results in reduction and delay in TRPV4-mediated Ca2+-influx. Though the values are not statistically significant, these differences show a clear trend of Taxol-mediated inhibition of TRPV4. This result accords well with the fact that Taxol reduces TRPV4- mediated currents in transfected CHO cells [70]. Along the same line, evidence suggests that the actin cytoskeleton can also regulate the TRPV4 channel activity as disruption of the actin cytoskeleton impairs the TRPV4-mediated currents [70] and Ca2+-influx in TRPV4 expressing cells in response to hypotonic shock [68]. However, the molecular mechanism underlying this inhibitory effect of Taxol on TRPV4-mediated Ca2+-influx is not yet clear. Unlike the response to microtubule destabilizers (like Nocodazole), the integrity of microtubule cytoskeleton, microtubule-based transport and membrane distribution of TRPV4 are not compro- TRPV4 Regulates Cytoskeleton mised by this short-term Taxol application. We predict that this modulation is most likely due to the conformational change of the channel complex induced by the dynamic presence or absence of the microtubule cytoskeleton and its associated components, which correlates with either channel closure and/or desensitisation. In agreement with this concept, it is important to note that activation of single TRPV4 ion channel in response to heat is possible in whole cell recordings but not in a cell-free inside-out patch clamp experiments, suggesting that in the latter some cellular factor is missing [83]. In this regard it is tempting to speculate an involvement of microtubules there. While in the course of the excision still some microtubules might be attached to the TRPV4 complex, later on it might disassemble easily as filamentous microtubules are sensitive to dilution [84]. However, further single channel electrophysiological investigations are required. TRPV4 and cytoskeleton in the context of intracellular pain signaling Previously it has been shown that potentiation, spontaneous activity and desensitisation of TRPV4 are Ca2+-dependent processes, suggesting Ca2+-dependent signalling occurs [44,85–86]. Indeed, TRPV4 interacts with calmodulin in a Ca2+-dependent manner [44]. In agreement with that, we also observed the presence of CamKII in the complex formed by TRPV4-Ct, but only in the presence of Ca2+ (Fig. 1c). This is in line with a reported role of CamKII in pain [46]. It also indicates that CamKII may regulate TRPV4 function. However, not all signalling molecules associate with TRPV4 in a Ca2+-dependent manner. PKCe, a Ca2+-independent kinase investigated in various aspects of mechanical sensitization [39–42,87–88] interacts with the C-terminus of TRPV4 in the presence as well as absence of Ca2+. Our results are also in line with a recent report demonstrating that TRPV4 phosphorylation at its N- terminus is important for its activation and PKCs along with PKA are important for this phosphorylation [45]. The same report demonstrates that PKCs form a complex with the TRPV4 in the presence of AKAP proteins which function as scaffolds. Considering the fact that AKAP proteins interact with tubulin [89], our results suggest that the complex we identified has a mechanistic function. Though, direct phosphorylation of the C- terminus of TRPV4 by PKCe in vitro has not been shown so far, but PKC-mediated potentiation of TRPV4 has been observed [1,43] and PKCe-dependent activation of TRPV4 has been described in vivo [90]. In our preliminary in vitro experiments too, we failed to observe any phosphorylation mediated by recombi- nant (and purified) PKCe when purified MBP-TRPV4-Ct was used as a substrate (data not shown). However, a recent study has demonstrated that in vivo condition, S824 position of the TRPV4 is phosphorylated by PKC [91]. However, this phosphorylation might be mediated by other PKC isotypes and may not be by PKCe. Based on our results and data from others, we believe that PKCe most likely sequesters at the C-terminus of the TRPV4 in order to phosphorylate the N-terminus and/or it needs other factors. Nevertheless the complex we isolated in this work appears to be of importance, as cytoskeletal components, CamKII, PKCe and TRPV4; all are known to be involved in pain sensitization [46,58,90]. TRPV4 and microtubule cytoskeleton also share similarities in terms of regulation by Sac tyrosine kinase, a pro-nociceptive regulator at the plasma membrane. On a cellular level, Src tyrosine kinase activity in response to mechanical force is localized mainly at the membrane [53]. In accordance with that, Src tyrosine kinase phosphorylates membrane-associated tubulin of neuronal origin [92]. This kinase activity is important for TRPV4 PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11654