function [66] as it phosphorylates TRPV4 [43,93]. Thus membrane-associated tubulin-TRPV4 complex seems to be important for stimuli that activate Src-tyrosine kinase. Microtubules and TRPV4 in pain sensitization Involvement of cytoskeleton in pain sensitisation has been anticipated for a long time, as microtubule-based chemothera- peutics against cancer are known to induce severe long-lasting neuropathic pain [94]. But, how these chemotherapeutics, like Taxol and Vincristine alter pain sensitivity is not well understood. Microtubules and mitochondrial structures develop abnormalities in nociceptive neurons due to prolonged and systemic application of these chemotherapeutics [95–97]. Even stabilization of microtubules by short-term application of Taxol (30 min) exerts an impact on pain sensitization [58]. It is important to mention that disruption of microtubules by short-term (30 min) application of Nocodazole abolishes/reduces pain sensitization [58]. These suggest that the integrity of microtubules can indeed modulate pain sensitivity [98]. TRPV4 has recently been considered as essential for chemotherapy-induced neuropathic pain as mechan- ical hyperalgesia induced by Taxol and Vincristine was strongly reduced in trpv4 2/2 mice [30,66]. We established a direct physical, cell-biological and functional relationship between cytoskeletal components and TRPV4, which plays an important role in inflammatory, neuropathic and chemotherapeutic-induced pain [30,58,66]. This gives a mecha- nistic base to further study the role of the apparent dynamic signalling complex formed by TRPV4 and its functional implications in various physiological scenarios. Materials and Methods Reagents and antibodies Taxol, Nocodazole, 4aPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia, antibodies against a-tubulin (clone DM1A), b-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), b-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany). Antibodies against neurofilament 200 kDa (clone RT97) and detyrosinated tubulin were purchased from Chemicon (Chandlers Ford, UK). The antibody against actin (clone JLA20) was purchased from Oncogene (Cam- bridge, MA, USA). Antibody against maltose-binding protein (MBP) and the amylose resin were purchased from New England Biolab (Beverly, MD, USA). Purified bovine muscle actin was purchased from Sigma-Aldrich (Taufkirchen, Germany). Protein G-agarose was purchased from Amersham Pharmacia Biotech (Munich, Germany). For some experiments, another affinity purified rabbit polyclonal antibody raised against TRPV4 (kind gift from Jon D. Levine) was used [3]. Anti CamKII antibody was purchased from BD (Heidelberg, Germany). Anti PKCe antibody (KP4) was a kind gift from Dr Robert Messing, University of California San Francisco. All secondary IgG antibodies (alexa-488-labelled anti-mouse, alexa-488- labelled anti-rabbit, alexa-594-labelled anti-rabbit, alexa-594-labelled anti-rat, Fura-2/AM, alexa-488- and alexa-594-labelled Phalloitoxin were purchased from Invitrogen (Karlsruhe, Germany). Constructs Full-length TRPV4 (NCBI accession number AF263521) in pCDNA3.1 vector was a kind gift from Prof. Jon D Levine [3]. For expression of C-terminal cytoplasmic domain (amino acid residue TRPV4 Regulates Cytoskeleton 718 to 871) of TRPV4-fused with MBP, the corresponding cDNA region of TRPV4 (NCBI accession number AF263521) was amplified by using forward primer (59TGACGAATTCATG- GGTGAGACCGTGGGCCA39) and reverse primer (59TGA- CAAGCTTCTACAGTGGTGCGTCCTCCG39). The ampli- fied cDNA was sub cloned into the EcoRI and HindIII restriction site of the pMALc2x vector (NEB, Beverly, MD, USA) and verified by automated nucleotide sequencing. TRPV4-GFP construct (cloned in pEGFPN3 vector) used in this study was kindly provided by Prof. J. Berreiter-Hahn [10]. Tubulin-cherry construct was a kind gift from Prof. R. Y. Tsien [99]. RFP-actin construct was purchased from Clontech (Heidelberg, Germany). Cells and transfection F11 cells [38] were cultured in Ham’s F12 medium (Sigma Aldrich) supplemented with 20% fetal bovine serum (Sigma Aldrich). Cos7 [100] and HaCat [101] cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma Aldrich) with 10% fetal calf serum. CHO-K1 cells [3] stably transfected with TRPV4 (CHO-K1-TRPV4) and the negative control cell line (CHO-K1- MOCK) [3] was cultured in F12 Ham’s (Sigma Aldrich) medium supplemented with 5% fetal bovine serum (FBS) and L-glutamine. These two cell lines were kindly provided by Prof. J. D. Levine [3]. All media were supplemented with streptomycin (100 mg/ml) and penicillin (100 mg/ml) (Invitrogen). Cells were maintained in a humidified atmosphere at 5% CO2 and 37uC. For transient transfection, Lipofectamine (Invitrogen) was used according to the manufacturer’s instructions. Ethics Statement Cell biological experiments were performed in cultured DRG neurons from male Sprague-Dawley rats (200–300 g; Harlan Winkelmann, Borchen, Germany). Care and use of animals were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the LaGeSo, Berlin. All efforts were made to minimize the number of animals used and their suffering. DRG neuronal culture and neurite out growth Dissociated rat DRG neurones were prepared from adult male rats (200–300 g) as recently described [32]. Neurons were grown in 24-well plates at 37uC in 5% CO2 and were grown for a total of 72 h. At that time most untreated neurons grow numerous and long neurites. In some experiments neurons were treated with 4aPDD (0.1 mM for the last 36 hour) before they were fixed with 2% PFA. Subsequently the neurons were visualized for bIII- tubulin (antibody dilution 1:1000) and IB4-lectin (1:15000). Immunoprecipitation For immunoprecipitation, approximately 50 ml of 50% protein G beads-slurry equilibrated with IP buffer (1% sodium dodecyl maltoside, PIPES 50 mM (pH 6.8), 100 mM NaCl, 1 mM EGTA, 0.2 mM MgCl2 and complete protease inhibitors (Roche) was used for each IP. Affinity purified rabbit polyclonal TRPV4 antibody (Sigma Aldrich) (7 mg per IP condition, antibody obtained from Sigma-Aldrich) was used. In control, total serum IgG from rabbit (7 ml which is equivalent to 20 mg of antibody) was used as non- specific antibody. For co-immunoprecipitation, CHO-KI-TRPV4 cells grown for 2 days and then scraped off from the dishes. Cells were harvested by centrifuging (Hettich Rotanta/T, 5 min at 100 g). Cells were washed with PBS once and centrifuged again at the same conditions. Cells were resuspended in 2 ml of IP buffer and homogenized (10 strokes) with a glass-Teflon homogenizer PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11654