Found at: doi:10.1371/journal.pone.0005716.s005 (0.01 MB PDF) Table S3 FWHM changes in the expansion of the cell wall autofluorescence in BRI1-GFP expressing hypocotyl and root cells before (0 min) and 30 min after application of 10 nM BL (30 min). Found at: doi:10.1371/journal.pone.0005716.s006 (0.01 MB PDF) Table S4 FWHM changes in the expansion of Calcofluor-stained cell walls in BRI1-GFP expressing hypocotyl and root cells before (0 min) and 30 min after application of 10 nM BL (30 min). Found at: doi:10.1371/journal.pone.0005716.s007 (0.01 MB PDF) Table S5 Statistics of 72 periclinal cell wall width measurements, each, derived from ultrathin TEM sections of BRI-GFP expressing, high-pressure-frozen root cells, which were either mock-treated or treated for 30 min with 10 nM BL. Found at: doi:10.1371/journal.pone.0005716.s008 (0.01 MB PDF) Table S6 FWHM changes in the widening of the plasmalemma- bound GFP fluorescence signal in aquaporin-GFP expressing hypocotyl and root cells before (0 min) and 30 min after application of 10 nM BL (30 min.). References 1. Hood L, Heath JR, Phelps ME, Lin B (2004) Systems biology and new technologies enable predictive and preventative medicine. Science 306: 640–643. 2. Liu ET (2005) Systems biology, integrative biology, predictive biology. Cell 121: 505–506. 3. Miyawaki A, Sawano A, Kogure T (2003) Ligthing up cells: labelling proteins with fluorophores. Nature Cell Biol 5: S1–7. 4. Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440: 935–939. 5. Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, et al. (2008) Video-rate far-field optical nanoscopy dissects synaptic versicle movement. Science 320: 246–249. 6. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642–1645. 7. Heinlein T, Biebricher A, Schl u¨ter P, Herten DP, Wolfram J, et al. (2005) High- resolution colocalization of single molecules within the resolution gap of far-field microscopy. Chem Phys Chem 6: 949–955. 8. Ries J, Schwille P (2008) New concepts for fluorescence correlation spectroscopy on membranes. Phys Chem Chem Phys 10: 3487–3497. 9. Vale RD (2008) Microscopes for Fluorimeters: The Era of Single Molecule Measurements. Cell 135: 779–785. 10. Fernandez-Suarez, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9 (12): 929–943. 11. Slaughter BD, Schwartz JW, Li R (2007) Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging. Proc Natl Acad Sci USA 104: 20320–20325. 12. Slaughter BD, Huff JM, Wiegraebe W, Schwartz JW, Li R (2008) SAM domain- based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy. PLoS One 3 (4): e1931. 13. Maeder CI, Hink MA, Kinkhabwala A, Mayr R, Bastiaens PIH, et al. (2007) Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling. Nature Cell Biol 9: 1319–1326. 14. Muto H, Kinjo M, Yamamoto KT (2009) Fluorescence cross-correlation spectroscopy of plant proteins. Methods Mol Biol 479: 203–215. 15. Hink MA, Shah K, Russinova E, de Vries SC, Visser AJWG (2008) Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid sensitive 1 receptor oligomerization. Biophysical J 94: 1052–1062. 16. Martini J, Schmied K, Palmisano R, Toensing K, Anselmetti D, et al. (2007) Multifocal two-photon laser scanning microscopy combined with photo- activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. J Struct Biol 158: 401–409. 17. Li J, Wen JQ, Lease KA, Doke JT, Tax FE, et al. (2002) BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signalling. Cell 110: 213–222. 18. Bo¨se G, Schwille P, Lamparter T (2004) The mobility of phytochrome within protonemal tip cells of the moss Ceratodon purpureus, monitored by fluorescence correlation spectroscopy. Biophys J 87 (3): 2013–2021. Subcellular Dynamics in Plants Found PDF) at: doi:10.1371/journal.pone.0005716.s009 (0.01 MB Table S7 FWHM changes in the widening of the cell wall autofluorescence in wildtype hypocotyl and root cells before (0 min) and 30 min after application of 10 nM BL (30 min). Found at: doi:10.1371/journal.pone.0005716.s010 (0.01 MB PDF) Text S1 Calculation of the width of plasmalemma-cell wall sections from apparent GFP fluorescence data (FWHM values). Found at: doi:10.1371/journal.pone.0005716.s011 (0.23 MB PDF) Acknowledgments We thank Felicity de-Courcy for proofreading the manuscript, N Geldner for seeds and K Schumacher for helpful suggestions. Author Contributions Conceived and designed the experiments: KE FS YDS AM KH. Performed the experiments: KE KC YDS. Analyzed the data: KE KC FS YDS AM KH. Wrote the paper: KE AM KH. 19. Blum C, Stracke F, Becker S, Mu¨llen K, Meixner AJ (2001) Discrimination and Interpretation of Spectral Phenomena by Room-Temperature Single-Molecule Spectroscopy. J Phys Chem A 105: 6983–6990. 20. Schleifenbaum F, Blum C, Elgass K, Subramanian V, Meixner AJ (2008) New insights into the photophysics of DsRed by multiparameter spectroscopy on single proteins. J Phys Chem B 112: 7669–7674. 21. Wang Z-Y, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasmamembrane receptor for plant steroids. Nature 410: 380–383. 22. Geldner N, Hyman DL, Wang X, Schumacher K, Chory J (2007) Endosomal signaling of plant steroid receptor BRI1. Genes Develop 21: 1598–1602. 23. Robert S, Chary SN, Drakakaki G, Li SD, Yang ZB, et al. (2008) Endosidin 1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and Aux1. Proc Natl Acad Sci U S A 105: 8464–8469. 24. Clouse SD (1998) Brassinosteroids: Essential Regulators of Plant Growth and Development. Annu Rev Plant Physiol Plant Mol Biol 49: 427–451. 25. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509–544. 26. Lakowicz JR (1999) Principles of fluorescence spectroscopy. New York: Kluwer Academic. 27. Striker G, Subramaniam V, Seidel CAM, Volkmer A (1999) Photochromicity and fluorescence lifetimes of green fluorescent protein. J Phys Chem B 103: 8612–8617. 28. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP: cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci U S A 97: 3718–3723. 29. Nakabayashi T, Wang HP, Kinjo M, Ohta N (2008) Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements. Photochem Photobiol Sci 7: 668–670. 30. van Manen HJ, Verkuijlen P, Wittendorp P, Subramaniam V, van den Berg TK, et al. (2008) Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy. Biophysical J 94: L67–L69. 31. Zander C, Sauer M, Drexhage KH, Ko DS, Schulz A, et al. (1996) Detection and characterization of single molecules in aqueous solution. Appl Phys B 63: 517–523. 32. Anders N, Ju¨rgens G (2008) Large ARF guanine nucleotide exchange factors in membrane trafficking. Cell Mol Life Sci; doi: 10.1007/s00018-008-8227-7. 33. Robinson DG, Jiang LW, Schumacher K (2008) The endosomal system of plants: Charting new and familiar territories. Plant Phys 147: 1482–1492. 34. Haubrick LL, Assmann SM (2006) Brassinosteroids and plant function: some clues, more puzzles. Plant Cell Environment 29 (3): 446–457. 35. Haubrick LL, Torsethaugen G, Assmann SM (2006) Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts. Physiol Plantarum 128: 134–143. 36. Horcas I, Fernandez R, Gomez-Rodriguez JM, Colchero J, Gomez-Herrero J, et al. (2007) WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev Sci Instrum 78: e013705. 37. Dettmer J, Hong-Hermesdorf A, Stierhof Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18: 715–730. PLoS ONE | www.plosone.org 13 May 2009 | Volume 4 | Issue 5 | e5716