anti rat antibody and/or alexa 488 labelled anti rabbit antibody were used as secondary antibodies. For labeling IB4-positive neurons, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia was used at 1:15000 dilutions in PBST supplemented with 0.2 mM CaCl2, 0.2 mM MgCl2 and 0.2 mM MnCl2 and incubated for 30 minutes. All images were taken on a confocal laser-scanning microscope (Zeiss Axiovert 100 M) with a 63x-objective and analyzed with the Zeiss LSM image examiner software. TRPV4 activation and live cell imaging For visualizing the effect of TRPV4 activation, F11 cells were seeded on glass cover slips. Two days after transfection with TRPV4, the cells in either complete medium or in HBSS buffer were imaged at RT. Where indicated, TRPV4-GFP was co- expressed with either RFP-Actin or Tubulin-cherry. In experi- ments aiming to activated TRPV4, its agonist 4 a-phorbol 12,13- didecanoate (4aPDD) was used at a final concentration of 1 mm, if not stated otherwise [22]. Calcium imaging For measuring the TRPV4 channel activity; Ca2+-imaging was performed using Cos7 cells at room temperature (25uC). Cos7 cells plated on glass cover slips and transiently transfected with TRPV4 and a yellow fluorescent protein (YFP) reporter plasmid. Cells transfected with only YFP reporter plasmid were used as controls. The transfected cells were used for the experiments at around 48 hour later transfection. Cells were loaded with 3 mM Fura-2 AM and placed in a recording chamber that allowed direct application of buffer at controlled temperature (27 to 30uC). Loading of Fura-2 AM dye and the fluorescence measurements were done in calcium imaging buffer (140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Glucose, 10 mM HEPES, pH 7.4). Cells were activated by 4aPDD (1 mM) for 10 seconds after first 30 seconds of imaging. Images were captured in every 2 seconds for a total of 3 minutes. 10 minutes after the 1st round of activation (1st pulse of 4aPDD) and imaging, cells on the same cover slips were imaged again for the next round (2nd pulse of 4aPDD). In experiments aimed to analyze the effect of microtubule stabilization, Taxol (1 mM) was maintained through- out the experiment in all solutions used, i.e. during the dye loading, during activation by 4aPDD and in between when running calcium-imaging buffer was applied. Ratiometric calcium imaging was performed and analyzed using Tillvision software (Till Photonics, Germany). Fluorescence was measured at excitation wavelengths alternating between 340 nm and 380 nm. Following subtraction of background fluorescence the ratio of fluorescence at 340 nm and 380 nm was calculated. For determination of total Ca2+-influx, we measured the total area of the graph each cell formed after stimulation. Microsoft Excel and Origin software were used to calculate and plot the graphs. All significance tests were performed at the ,0.05 level (one sample t- test). Supporting Information Figure S1 Prediction of tubulin-binding region located within the C-terminus of TRPV4. a. Identification of amino acid stretches located within the TRPV4-Ct with high isoelectric point (pI) values. The C-terminal overhanging region of tubulin contains highly acidic residues and most of the tubulin-binding proteins contain amino acid stretches which have high pI due to the presence of basic residues. Therefore, the C-terminus of TRPV4 (amino acid residues 718 to 871) was analyzed for its isoelectric TRPV4 Regulates Cytoskeleton points. Similarly, different fragments of TRPV4-Ct, each shorter by 10 amino acids from the extreme C-terminal end were also analyzed. This analysis indicates that there are two areas which contain positively charged residues (indicated by shaded area). The corresponding pI of each fragment is mentioned in the right most side. In below, the secondary structure prediction is provided (red indicates helical structure, green arrow indicates beta strand and black line indicates unstructured coil region. Note that the arrows do not indicate the directionality of the strands). All theoretical pI values of the fragments were calculated by using available software (http://www.expasy.org/tools/pi_tool.html). All secondary struc- ture prediction was done by using Advanced Protein Secondary Structure Prediction Server available at http://imtech.res.in/ raghava/apssp/. b. Amino acid residues 746 to 779 contain multiple positively charged amino acids which can potentially form an alpha-helix. The PyMol programme was used to build this peptide structure. Blue and red colours indicate the positive and negative charges at the surface respectively. Notably all the positively charged amino acids (indicated by arrows) are located in one side of the helix and can be important for interaction with the tubulin/microtubule (which contain negative charges at the surface). c. These predicted and important basic charges (indicated by blue) are conserved throughout the evolution. The NCBI accession numbers are indicated. TRPV4, Homo sapiens (NP_ 067638.3); TRPV4, Canis lupus familiaris (XP_543434.2); TRPV4, Bos taurus (XP_001253150.1); TRPV4, Mus musculus (NP_071300.1); TRPV4, Rattus norvegicus (NP_076460.1); TRPV4, Gallus gallus (NP_990023.1); TRPV4, Danio rerio (NP_001036195.1); Putative uncharacterized protein, Ailuropoda melanoleuca (PANDA_020862); Putative uncharacterized protein, Ailuropoda melanoleuca (PAN- DA_018294), Chromosome 12 SCAF14999, Tetraodon nigroviridis (GSTENG00029525001). Found at: doi:10.1371/journal.pone.0011654.s001 (7.95 MB TIF) Figure S2 TRPV4 pulls down cytoskeletal components and nociceptive regulator kinases from F11 cell extract. MBP-TRPV4- Ct (lane 2–3) but not MBP-LacZ (lane 4–5) forms specific complex when incubated with soluble F11 extract (lane 1), both in presence (lane 2 and 4) or absence (lane 3 and 5) of Ca2+ (1 mM). Presence of actin, tubulin and PKCe are observed only in lane 2 and 3 whereas CamKII is present only in lane 2. Found at: doi:10.1371/journal.pone.0011654.s002 (8.31 MB TIF) Figure S3 TRPV4 co-localizes with actin cytoskeleton. Shown are the confocal images of neuronal and non-neuronal cells expressing TRPV4. Cells were stained for TRPV4 (green) and actin (red). Presences of TRPV4 specifically in actin-enriched structures (indicated by arrows) are shown. Enlarged views of filopodial structures developed from HaCat cell (upper panel) and F11 (lower panel) cell are shown. Found at: doi:10.1371/journal.pone.0011654.s003 (0.92 MB TIF) Figure S4 TRPV4 activation can influence the neuritic morphology. Shown are the time-series confocal images of a neurite developed from a F11 cell expressing TRPV4-GFP (green) and Tubulin-cherry (red). This neurite produces varicosities (indicated by arrows) after adding 4aPDD due to disassembly of MT. Note that all the varicosities are formed simultaneously (at 5th minute in this case), indicating a global disassembly of microtubules all over this neurite. Tubulin Cherry construct was a kind gift from Prof. R. Y. Tsien (Shaner et al. 2004). Found at: doi:10.1371/journal.pone.0011654.s004 (0.85 MB TIF) Figure S5 Activation of TRPV4 results in microtubule disas- sembly. a. F11 cells were mock activated with buffer or activated with 4aPDD (1 mM) followed by detergent extraction with isotonic PLoS ONE | www.plosone.org 13 July 2010 | Volume 5 | Issue 7 | e11654