cytoskeleton-dependent/mediated regulatory volume decrease of cells [6,10,24], a process where actin-binding proteins contribute to cell volume regulatory ion channel activation [24–26]. In addition, TRPV4 has a conserved role in mechanotransduction, a complex process that involves both actin and microtubule cytoskeletal components [27–29]. The interplay of TRPV4 with microtubule cytoskeleton also appears on a behavioural level, where alteration of microtubule dynamics by Taxol induces a TRPV4-dependent painful peripheral neuropathy [30]. While all these cellular and behavioural studies strongly suggest that TRPV4 shares a functional relation with the cytoskeleton, so far a direct link of TRPV4 with the cytoskeleton has not been demonstrated. Thus, a molecular mechanism for the role of TRPV4 and the cytoskeleton in pain, mechanosensation as well as other cellular functions remains elusive. Recently, we have established a functional interplay between TRPV1, a close homologue of TRPV4, and the microtubule cytoskeleton [31–35]. We demonstrated the physical interaction of microtubule cytoskeleton with TRPV1 via two novel tubulin- binding motifs [36–37]. Based on our previous experiments done on TRPV1 and the sequence homology between TRPV1 and TRPV4, we predicted that TRPV4 might interact with tubulin via its C-terminal domain. Therefore, in this work we set out to explore if TRPV4 physically and functionally interacts with actin and microtubule cytoskeletal components. TRPV4 Regulates Cytoskeleton Results TRPV4 interacts with endogenous actin and tubulin In order to test if TRPV4 interacts with cytoskeletal proteins like tubulin and actin, we performed co-immunoprecipitation experi- ments with affinity purified TRPV4 antibodies. CHO-KI-TRPV4 stable cell lines were used, which express low levels of TRPV4. In immunoblot analysis, we observed that TRPV4 antibodies precipitated TRPV4 together with actin and tubulin proteins (Fig. 1a). Presence of tubulin and actin was not observed when a similar co-immunoprecipitation was performed from the same cell extract using an antibody, which was not raised against TRPV4. To confirm further that the tubulin interaction is occurring even in endogenous tissues, we isolate DRG neurons from rat and performed similar co-immunoprecipitation experiments with affin- ity purified TRPV4 antibodies. We observed that tubulin co- immunoprecipitated with TRPV4 even from DRG neurons (1b). The C-terminus of TRPV4 is sufficient for interaction with actin and tubulin To identify, which part of the TRPV4 interacts with actin and/ or tubulin proteins, we performed a pull down experiment using maltose-binding-protein (MBP)-fused to the N- and C-termini of TRPV4. According to our prediction [37], at least one tubulin- binding site is located within the C-terminus of TRPV4 (Fig. S1) Figure 1. Interaction of soluble tubulin and actin with TRPV4. a. Co-immunoprecipitation of actin and tubulin with TRPV4. Cell extracts from CHO-KI cells stably expressing TRPV4 (lane 1) was immunoprecipitated by TRPV4 antibody (lane 2) or by a non-specific antibody (lane 3). Blots were probed for TRPV4 (left side), tubulin (middle) and actin (right side). b. Co-immunoprecipitation of tubulin with TRPV4. Extracts from DRG (lane 1) was immunoprecipitated by TRPV4 antibody (lane 2) or by a non-specific antibody (lane 3). Blots were probed for TRPV4 (upper panel) and tubulin (lower side). c. MBP-TRPV4-Ct (lane 2-3) but not MBP-LacZ (lane 4–5) forms specific complexes when incubated with mammalian brain extract (lane 1), both in presence (lane 2 and 4) or absence (lane 3 and 5) of Ca2+ (1 mM). Presence of PKCe, actin and tubulin are observed only in lane 2 and 3. Neurofilament in the pull down samples is visible only after exposing for a prolonged time. Presence of CamKII is noted only in the presence of Ca2+ (lane 2). Note that the amount of MBP-LacZ used, as a negative control for the pull down experiment is much more than MBP-TRPV4-Ct. d. Tubulin interacts with TRPV4-Ct directly. MBP-LacZ (lane 1–2) or MBP-TRPV4-Ct (lane 3–4) was incubated with buffer only (lane 1 and 3) or with purified tubulin (lane 2 and 4). Pulled down samples were probed for different isotype-specific and different post-translationally modified tubulins. e. Actin interacts directly with TRPV4-Ct. MBP-TRPV4-Ct (lane 1–2) or MBP-LacZ (lane 3–4) was incubated with purified actin (lane 1–4) either in the presence (lane 1-and 3) or absence (lane 2 and 4) of Ca2+ (1 mM) and subsequently probed for bound actin. f. Soluble tubulin and actin competes for the C- terminal cytoplasmic fragment of TRPV4. MBP-TRPV4-Ct was incubated with only tubulin (lane 1), with only actin (lane 2), or both tubulin and actin in a sequential manner (lane 3–4). Prior incubation of tubulin inhibits further binding of actin (lane 3). Similarly, prior incubation of actin significantly reduces the further binding of tubulin (lane 4). doi:10.1371/journal.pone.0011654.g001 PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11654