TRPV4 Regulates Cytoskeleton Figure 2. TRPV4 interacts directly with polymerized actin and microtubule filaments. MBP and MBP-TRPV4-Ct were centrifuged at 70000 g/30 min/4uC and only soluble proteins present in the supernatant were used for all co-sedimentation experiments. a. MBP-TRPV4-Ct co- sediments with polymerized actin filaments. Actin was polymerized either in presence of MBP-TRPV4-Ct (lane 1 and 4), in presence of MBP only (lane 2 and 5) or in buffer only (lane 3 and 6). Polymerized actin filaments and associated proteins were isolated from remaining soluble actin and unbound proteins by centrifugal separation of pellets (P, lane 1–3) from corresponding supernatants (S, lane 4–6). The entire amount of MBP remains in the supernatant (lane 5) while a significant amount of MBP-TRPV4-Ct appears in the pellet (lane 1). Arrows indicate the position of respective proteins. b. MBP-TRPV4-Ct co-sediments with microtubules. Taxol-stabilized microtubules (left panel) were incubated with MBP (lane 1–2), MBP-TRPV4-Ct (lane 3– 4) or with buffer only (lane 5–6) followed by the centrifugal separation of pellet (P) consisting MT and bound proteins from supernatant (S) consisting of soluble tubulin and other unbound proteins (left side panel). In right side panel, soluble tubulin and GTP was incubated with MBP (lane 1–2), MBP- TRPV4-Ct (lane 3–4) or buffer only (lane 5–6) followed by separation of pellet (P) and supernatant (S). Note the specific presence of MBP-TRPV4-Ct in the pellet in both cases (in lane 4). c. MBP-TRPV4-Ct stabilizes microtubules against depolymerizing factors. Microtubules was formed form soluble tubulin in buffer (lane 1), along with MBP (lane 2) or along with MBP-TRPV4-Ct (lane 3) in control condition (left most panel), in presence of Nocodazole (middle left panel), in presence of Ca2+ (middle right side) or in presence of both Nocodazole and Ca2+ (right most). Microtubules and bound proteins present in the pellet fraction (P) were isolated from unpolymerized tubulin and unbound proteins remaining in the supernatant (S) by centrifugal separation. Note the enhancement of polymerized microtubules (represented by tubulin present in lane 3, P fraction in every conditions) due to the presence of MBP-TRPV4-Ct. doi:10.1371/journal.pone.0011654.g002 Next, we addressed if TRPV4 similarly co-localizes with microtubules. We observed co-localization between TRPV4 and microtubules in fixed F11 cells all along neurite-like structures. Co- localization was observed at the plasma membrane and at membrane ruffles that are enriched in TRPV4 (Fig. 3d-e). In addition, we noted the presence of numerous microtubule ends at the submembranous regions. Often these microtubule ends extended to the plasma membrane and seemed to be stabilized at the membranous regions containing TRPV4. Co-localization of tubulin and TRPV4 was also observed in filopodial structures that were developed from growth cones, from neurite-like structures as well as from cell bodies. This kind of submembranous tubulin accumulation was not observed in non-transfected cells (Fig. 3f), implying that this effect is primarily due to the presence of TRPV4. Activation of TRPV4 results in fast retraction of growth cones in F11 cell TRPV4 localizes to growth cones when expressed in F11 cells. So we tested if TRPV4 activation can alter the morphology and movement of growth cones. For that purpose we expressed TRPV4-GFP along with RFP-actin and performed live cell imaging. We observed that in response to 4a-phorbol-didecanoate (4aPDD, 1 mM), a TRPV4-specific agonist [22], neurites from TRPV4-GFP expressing F11 cells show rapid change in its morphology and induce multiple varicosities (Fig. S4). In response to 4aPDD, TRPV4-GFP containing growth cones retract quickly (Fig. 4a). Under the same conditions growth cones developing from non-transfected cells do not show any retraction, thus assuring specificity of the pharmacological treatment. These results strongly suggest that TRPV4 can regulate the growth cone motility. Long-term exposure to a TRPV4 agonist restricts neurite outgrowth in a subset of primary DRG neurons To validate the effect of TRPV4 activation on growth cones in a system with endogenous TRPV4 expression, we used primary neurons and tested the effect of TRPV4 activation. DRG neurons from adult male rat were cultured for 5 days and treated with 4aPDD (1 mM) for short-term (20 minutes). Live cell-imaging revealed indications of slow retraction of neurites in response to PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11654