TRPV4 Regulates Cytoskeleton Figure 6. Microtubule cytoskeleton regulates Ca2+-influx via TRPV4. a. Taxol (1 mM, 30 minutes) reduces TRPV4-mediated Ca2+-influx in Cos7 cells. Shown are the normalized average ratiometric Ca2+-influx in arbitrary units (AU). TRPV4 was activated by a pulse (indicated by a black line) of 4aPDD for 10 sec. The dark blue line (C1) represents average response due to first pulse in control conditions (n = 32), the light blue line (C2) represents average response due to second pulse in control conditions (n = 32), the blackish green line (T1) represents average response due to first pulse under the influence of Taxol-stabilized microtubules (n = 42) and the light green line (T2) represents the average response due to second pulse under the Taxol-stabilized microtubules (n = 30). b. Cells with Taxol-stabilized microtubules reveal reductions in total Ca2+-influx due to TRPV4 activation. Total Ca2+-influx was calculated from the total area appeared by the ratiometric calcium-influx graph (as shown in figure a) for each cell and was calculated by Origin 7G software. At the ,0.05 level, the difference of the population means between 2nd pulse (untreated and taxol-treated, one sample t-test) is significant (*). The difference in case of 1st pulse is non-significant. c. Taxol stabilized cells reveal a trend for time delay to reach in maximum response. The time (in seconds) each cell took to reach its maximum response (indicated by filled triangles) was plotted. Times needed during first pulse in control conditions (dark blue), second pulse in control conditions (light blue), first pulse with Taxol (blackish green) and second pulse with Taxol (light green) are indicated. The average values (indicated by filled squares) and the standard deviations are also shown for each condition. The average time differences remain non-significant. doi:10.1371/journal.pone.0011654.g006 C-terminal cytoplasmic domain. Thus, our biochemical study confirms and extends a recent fluorescence resonance energy transfer (FRET) analysis performed on live cells demonstrating close proximity of actin and TRPV4 [67]. Interestingly, a recent study shows that disruption of the actin cytoskeleton increases the intracellular mobility of TRPV4-GFP and results in loss of co- localization of TRPV4 with actin [68]. Our findings suggest that the direct interaction of TRPV4 and actin is a general feature observed in various cellular systems. This indicates the importance of the interaction far beyond the nociceptive system. Recent EM study also reveals that TRPV4 forms a ‘‘hanging gondola’’-like structure which undergoes different structural conformations representing open and closed state of the channel [69]. According to this structure, the C-terminus of TRPV4 is exposed outside and accessible for interaction with other proteins. In that context, it is tempting to speculate that interaction of TRPV4 with the actin and tubulin may have an importance for such conformational changes. It is also important to note that the presence of GFP tag (240 amino acid) at the C-terminus of TRPV4 retains perfect co-localization of TRPV4-GFP with actin and microtubule cytoskeleton, indicating that the tubulin-binding region located with the TRPV4-Ct is most likely not present at the extreme C-terminal free end of the TRPV4. Previously, we identified two novel tubulin-binding motifs, which we mapped to the C-terminal cytoplasmic domain of TRPV1 [36–37]. They consist of two short basic amino acid-rich sequence stretches, which are thought to interact directly with the acidic overhangs of tubulin. Indeed, positively charged amino acids in one of these two novel motifs are partially conserved in the C-termini of TRPV4 and TRPV2 [37]. Though we did not perform deletion mapping, based on other experimental evidences and best of our understanding, we applied a similar conceptual basis and predicted that the basic amino acid residues present within the C-terminus of TRPV4, especially within the 746 to 779 amino acids may interact with tubulin (Fig. S1). Though the ability of this small region to interact with tubulin needs to be experimentally verified, we found that indeed the total C-terminus of TRPV4 interacts with tubulin and/or microtubules. Thus, beyond the reported indirect binding through MAP7 [70], we find direct interaction of TRPV4 to microtubule cytoskeleton. In this study we have not tested if the N-terminal region of TRPV4 can interacts with actin and tubulin. However, considering the fact that the N-terminus of TRPV4 contains multiple ankyrin repeats which are presumably important for protein interaction possible interaction of tubulin and actin at the N-terminus cannot be ruled out completely. In this context, it is worth mentioning that recent studies demonstrate that point mutations located at these ankyrin repeats at the N-terminus either increase or decrease the TRPV4 activity and results in genetic disorders [71–73]. In this work we investigated if TRPV4 can affect microtubule properties. Binding of different post-translationally modified tubulins suggests that TRPV4 can modulate the stability of the microtubules. Indeed, we show that TRPV4 stabilizes microtu- bules at the membrane. These microtubules at the membrane can PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11654