buffer and fixed. Cells were immunostained for TRPV4 (green) and tubulin (red). In absence of activation, TRPV4 expressing cells retains all the microtubules (upper panel, i-iii), but lost all the distal microtubules if activated with 4aPDD (middle panel, iv-vi). The stable MTOC region is marked with an arrow. Note that non- transfected cells retain all the microtubules even after addition of 4aPDD (lower panel). b. CHO-KI cells expressing TRPV4 (green) were activated with 4aPDD (1 mM) and were further extracted with detergent in isotonic buffer and fixed. Under this condition, nontransfected cells show microtubules all over the cells, while transfected cells contain very little microtubules. Found at: doi:10.1371/journal.pone.0011654.s005 (1.67 MB TIF) Figure S6 TRPV4 localization at membrane is not affected by Taxol. Shown are the confocal images depicting the distribution and co-localization of TRPV4 (green) and tubulin (red) in Cos7 cells. Cells expressing TRPV4 were fixed under control condition (i), fixed after incubation with Taxol for 30 minute (ii) or incubation with Nocodazole for 30 minutes (iii). Cells were immunostained for TRPV4 (green) and tubulin (red). The white line indicates the periphery of the cell. All these images were acquired at an identical laser power and gain value. Found at: doi:10.1371/journal.pone.0011654.s006 (1.62 MB TIF) Figure S7 Microtubule stabilization by Taxol does not alter the surface distribution of TRPV4. To confirm that Taxol-treated cells still contain TRPV4 at the cell surface, we performed surface biotinylation of TRPV4-GFP expressing Cos7 cells that were References 1. Gao X, Wu L, O’Neil RG (2003) Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways. J Biol Chem 278: 27129–37. 2. Mizuno A, Matsumoto N, Imai M, Suzuki M (2003) Impaired osmotic sensation in mice lacking TRPV4. Am J Physiol Cell Physiol 285: C96–101. 3. Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, et al. (2003) Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 39: 497–511. 4. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD (2000) OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2: 695–702. 5. Liedtke W, Tobin DM, Bargmann CI, Friedman JM (2003) Mammalian TRPV4 (VR-OAC) directs behavioural responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proc Natl Acad Sci U S A 100: 14531–14536. 6. Pan Z, Yang H, Mergler S, Liu H, Tachado SD, et al. (2008) Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells. Cell Calcium 44: 374–385. 7. Ma YY, Huo HR, Li CH, Zhao BS, Li LF, et al. (2008) Effects of cinnamaldehyde on PGE2 release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin-1beta. Biol Pharm Bull 31: 426–430. 8. Benfenati V, Amiry-Moghaddam M, Caprini M, Mylonakou MN, Rapisarda C, et al. (2007) Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes. Neuroscience 148: 876–892. 9. Lorenzo IM, Liedtke W, Sanderson MJ, Valverde MA (2008) TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells. Proc Natl Acad Sci U S A 105: 12611–12616. 10. Becker D, Blase C, Bereiter-Hahn J, Jendrach M (2005) TRPV4 exhibits a functional role in cell-volume regulation. J Cell Sci 118: 2435–2440. 11. Kohler R, Heyken WT, Heinau P, Schubert R, Si H, et al. (2006) Evidence for a functional role of endothelial transient receptor potential V4 in shear stress- induced vasodilatation. Arterioscler Thromb Vasc Biol 26: 1495–1502. 12. Cuajungco MP, Grimm C, Heller S (2007) TRP channels as candidates for hearing and balance abnormalities in vertebrates. Biochim Biophys Acta 1772: 1022–1027. 13. Tabuchi K, Suzuki M, Mizuno A, Hara A (2005) Hearing impairment in TRPV4 knockout mice. Neurosci Lett 382: 304–308. 14. Alessandri-Haber N, Joseph E, Dina OA, Liedtke W, Levine JD (2005) TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator. Pain 118: 70–79. TRPV4 Regulates Cytoskeleton either treated with Taxol (1 mM, 30 minutes) or left untreated. We isolated TRPV4-GFP by immunoprecipitation and found that TRPV4-GFP is similarly labelled with biotin in both conditions. These results confirm that short-term Taxol application does not alter the distribution of TRPV4 in the membrane. Total cell extract in control condition (lane 1) or under Taxol-stabilized condition (lane 2), TRPV4 immunoprecipitates from control condition (lane 3) or from Taxol-stabilized condition (lane 4) were blotted with TRPV4 antibody (upper panel) and with HRP-Avidin (lower panel). Arrow indicates the position of biotinylated TRPV4. Found at: doi:10.1371/journal.pone.0011654.s007 (1.19 MB TIF) Acknowledgments We thank Prof. J.D Levine, Prof. J. Berreiter-Hahn, Prof. F. Hucho, Prof. R.Y. Tsien and Dr. Franck Perez for their generous help with reagents. We thank Prof. H.H. Ropers for supporting this work. We thank Dr. Luna Goswami and Dr. K.V.S. Badireenath for their help with numerical data analysis and bioinformatics. Author Contributions Conceived and designed the experiments: CG PH TH. Performed the experiments: CG JK PH. Analyzed the data: CG PH TH. Contributed reagents/materials/analysis tools: CG PH TH. Wrote the paper: CG TH. Performed all experiments except figure 1b: CG. Performed experiments required for Figure 6 jointly: CG PH. Contributed in preparation of figure 1b: JK. Majority of the work was done in TH’s lab. 15. Levine JD, Alessandri-Haber N (2007) TRP channels: targets for the relief of pain. Biochem Biophys Acta 1772: 989–1003. 16. Kwan HY, Huang Y, Yao X (2007) TRP channels in endothelial function and dysfunction. Biochim Biophys Acta 1772: 907–914. 17. Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, et al. (2008) Gain-of- function mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet 40: 999–1003. 18. Arniges M, Va´zquez E, Ferna´ndez-Ferna´ndez JM, Valverde MA (2004) Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Biol Chem 279: 54062–54068. 19. Gradilone SA, Masyuk AI, Splinter PL, Banales JM, Huang BQ, et al. (2007) Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion. Proc Natl Acad Sci U S A 104: 19138– 19143. 20. Teilmann SC, Byskov AG, Pedersen PA, Wheatley DN, Pazour GJ, et al. (2005) Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs. Mol Reprod Dev 71: 444–452. 21. Andrade YN, Fernandes J, Va´zquez E, Ferna´ndez-Ferna´ndez JM, Arniges M, et al. (2005) TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity. J Cell Biol 168: 869–874. 22. Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, et al. (2002) Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J Biol Chem 277: 13569–13577. 23. Waning J, Vriens J, Owsianik G, Stuwe L, Mally S, et al. (2007) A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration. Cell Calcium 42: 17–25. 24. Liu X, Bandyopadhyay B, Nakamoto T, Singh B, Liedtke W, et al. (2006) A role for AQP5 in activation of TRPV4 by hypotonicity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery. J Biol Chem 281: 15485–15495. 25. Cantiello HF, Prat AG, Bonventre JV, Cunningham CC, Hartwig JH, et al. (1993) Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells. J Biol Chem 268: 4596–4599. 26. Blase C, Becker D, Kappel S, Bereiter-Hahn J (2009) Microfilament dynamics during HaCaT cell volume regulation. Eur J Cell Biol 88: 131–139. 27. Alenghat FJ, Nauli SM, Kolb R, Zhou J, Ingber DE (2004) Global cytoskeletal control of mechanotransduction in kidney epithelial cells. Exp Cell Res 301: 23–30. 28. Kaazempur Mofrad MR, Abdul-Rahim NA, Karcher H, Mack PJ, Yap B, et al. (2005) Exploring the molecular basis for mechanosensation, signal transduction, and cytoskeletal remodeling. Acta Biomater 1: 281–293. 29. Chen CS (2008) Mechanotransduction - a field pulling together? J Cell Sci 121: 3285–3292. PLoS ONE | www.plosone.org 14 July 2010 | Volume 5 | Issue 7 | e11654