4aPDD in some neurites, but did not show immediate retraction or varicosity formation (data not shown). Thus we tested if long- term exposure to a TRPV4 agonist but a low-level activation of TRPV4 can regulate the neurites. For that purpose, DRG neurons were cultured for 36 hours and then treated with a low dose of 4aPDD (0.1 mM) for an additional 36 hours. Neurons were visualized for neuron-specific bIII tubulin. Immunodetection of TRPV4-positive neurons was not conclusive due to the very low endogenous expression of TRPV4 in DRG neurons in this stage. Thus, we categorized the DRG neurons by staining with IB4 lectin, which mark a subsection of nociceptive neurons [49]. We observed that due to long-term exposure of 4aPDD, majority of the IB4-positive neurons developed much shorter neurites if at all (Fig. 4b). In contrast, under the same condition, IB4-negative neurons revealed long neurites similar to non-treated neurons (Fig. 4b). This confirmed that long-term exposure to a TRPV4 agonist resulted in growth cone and neurite retraction, and demonstrates that this effect is restricted to a subpopulation of nociceptors. Activation of TRPV4 results in disassembly of microtubules Previously it has been shown that activation of Ca2+ channels causes rapid reorganization of the cytoskeleton [31,50–51]. Also, rapid disassembly of microtubules causes growth cone retraction [32,34,52]. So, we tested if in contrast to a stabilizing function of TRVP4 at resting stage, activation of this channel leads to destabilization of microtubules. To better differentiate between soluble and polymerized cytoskeleton components, soluble pro- teins were removed by Digitonin-extraction leaving behind only the insoluble intact cytoskeleton. Extraction of unstimulated TRPV4 expressing F11 cells showed fine filamentous microtubules (Fig. S5a). But application of 4aPDD (1 mM) resulted in loss of microtubules from the majority of TRPV4 expressing F11 cells. In contrast, cells, which did not express TRPV4 were unaffected by 4aPDD (Fig. S5a). We observed that the cytoskeleton rearrangement by TRPV4 was not restricted to the neuronal cells only. In TRPV4 expressing CHO-KI cells 4aPDD treatment induced loss of almost all microtubules whereas non-transfected cells retained all their microtubules (Fig. S5b). Similarly, we observed loss of all peripheral microtubules by application of 4aPDD in CHO-KI- TRPV4 cells, which express low level of TRPV4 stably (Fig. 4c). Under the same conditions, CHO-KI mock-transfected cells retained all microtubules. All these results confirm that activation of TRPV4 results in microtubules disassembly and this effect is independent of TRPV4 expression level in whatever cellular context chosen. Activation of TRPV4 results in formation of elongated filopodia We also explored if TRPV4, being present at the filopodia can alter the actin cytoskeletal dynamics upon activation. For that purpose, we expressed TRPV4-GFP and actin-red in F11 cells and monitored the filopodial structures. We observed that activation of TRPV4 by 4aPDD results in rapid change of lamellipodia to filopodia and/or further elongation of filopodial structures (Fig. 5). These changes were observed at filopodial structures emanating from the cell body as well from neurites. We observed that during this change, several lamellipodial actin ribs fused into single filopodial structures and TRPV4-GFP localized at the filopodial tips (Fig. 5b). At the same time, lamellipodial region located between two actin-ribs retract. TRPV4 Regulates Cytoskeleton Stabilization of microtubules by Taxol reduces Ca2+-influx through TRPV4 activation In contrast to a unidirectional effect of TRPV4 activation on microtubule integrity, we explored if microtubule dynamics can also affect the TRPV4 channel properties. We measured Ca2+- influx via TRPV4 as an indicator of TRPV4 channel opening. We compared the Ca2+-influx via TRPV4 in response to 4aPDD in the presence and absence of Taxol. We observed that the normalized average Ca2+-influx due to TRPV4 activation was strongly diminished (but not abolished) if the cells were treated with Taxol (Fig. 6a). This suggests a partial inhibition of TRPV4 channel activity due to stabilization of microtubules. This reduction due to Taxol treatment (30 min) was also observed when cells were activated by 4aPDD for a second time (Fig. 6a). To understand the extent of reduction, we compared the total Ca2+-influx (Fig. 6b). We observed 21.9% reduction in the case of the 1st pulse in presence of Taxol when compared to Taxol-free control condition. However, this difference remain non-signifi- cant at (P.0.05) (Fig. 6b). However, this difference was even higher in the case of the 2nd pulse where a major reduction (61.79%) was observed in the presence of Taxol (compared with Taxol-free 2nd pulse condition). At the level of P,0.05, this difference is significant. To confirm this inhibitory effect of microtubule stabilization on TRPV4 channel opening, we analysed by another parameter, i.e. the time each cell took to reach its maximum response. We observed that Taxol-treatment resulted in a delay of the cells to reach their maximum response level (Fig. 6c). Though this time difference indicates a clear trend of inhibition, the differences remain non-significant (Fig. 6c). By immunofluorescence analysis (Fig. S6) and surface-biotinylation experiments (Fig. S7), we confirmed that the presence of microtubules and TRPV4 at the cell membrane or nearby regions was also not altered by Taxol application. Taxol did not alter the distribution of TRPV4 in the cell surface also. Taken together, these results suggest that microtubule dynamics can regulate TRPV4 channel properties. Discussion A substantial amount of work has been done to identify endogenous and exogenous sensitizing stimuli, their target proteins and elucidate their molecular effects on nociceptive neurons. Yet, the detailed cellular and molecular mechanisms underlying sensitization against mechanical stimuli and the specific molecular components associated with these signaling events are not well understood. A priori, as there is no force without counterforce, scaffold structures like the cytoskeleton and/or the extracellular matrix are intuitively likely to be required for stimulus detection and execution of the responses. In nociception, so far the cytoskeleton has been described as an important regulator of sensitization only on a behavioural level [29,42,53–58]. In contrast, in systems other than nociception, genetic, biochemical, cellular as well as behavioural data are indicative of the importance of the cytoskeleton for mechanosensation in general [29,59–60]. Thus, contrasting the wealth of data in non- nociceptive context, the nearly complete lack of molecular data about an involvement of the microtubule and actin cytoskeleton in pain sensitization is surprising. Though, recent results demon- strated the expression of TRPV4 in Merkel cells and linked TRPV4 function with the mechanotransduction [61,62], in the context of nociceptive and non-nociceptive mechanosensitivity, a direct physical interaction of actin or microtubule cytoskeleton with ion channels has not been reported. Therefore, we now provide the first evidence that both actin and microtubule PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11654