Automated Analysis of FRET Figure 8. A comparison of the subcellular distribution of RhoA activity between the stationary and migrating cells after EGF stimulation. The normalized number of high-FRET pixels in each wedge is plotted against the angle of the wedge for multiple (A) migrating and (B) stationary cells at 52 minutes after EGF stimulation. The error bars represent standard error values. The migrating cells were rotated and aligned so that they had the same migration direction. The front and back of the migration direction were determined by the relative locations of the wedges in regard to their corresponding centroids. doi:10.1371/journal.pone.0004082.g008 References 1. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, et al. (2003) Cell migration: integrating signals from front to back. Science 302: 1704–1709. 2. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21: 247–269. 3. Raftopoulou M, Hall A (2004) Cell migration: Rho GTPases lead the way. Dev Biol 265: 23–32. 4. Wheeler AP, Ridley AJ (2004) Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 301: 43–49. 5. Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4: 446–456. 6. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3: 906–918. 7. Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440: 1069–1072. 8. Kurokawa K, Matsuda M (2005) Localized RhoA activation as a requirement for the induction of membrane ruffling. Mol Biol Cell 16: 4294–4303. 9. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509–544. 10. Wang Y, Shyy JY-J, Chien S (2008) Fluorescence Proteins, Live Cell Imaging, and Mechanobiology: Seeing is Believing. Annu Rev Biomed Eng 10: In Press. 11. Palmer AE, Jin C, Reed JC, Tsien RY (2004) Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A 101: 17404–17409. 12. Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, et al. (2003) Activity of Rho-family GTPases during cell division as visualized with FRET- based probes. J Cell Biol 162: 223–232. 13. Yoshizaki H, Ohba Y, Parrini MC, Dulyaninova NG, Bresnick AR, et al. (2004) Cell type-specific regulation of RhoA activity during cytokinesis. J Biol Chem 279: 44756–44762. 14. Nakamura T, Aoki K, Matsuda M (2005) FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain. Brain Res Mol Brain Res 139: 277–287. 15. Yamana N, Arakawa Y, Nishino T, Kurokawa K, Tanji M, et al. (2006) The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Mol Cell Biol 26: 6844–6858. 16. El-Sibai M, Pertz O, Pang H, Yip SC, Lorenz M, et al. (2008) RhoA/ROCK- mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells. Exp Cell Res 314: 1540–1552. 17. Wong K, Pertz O, Hahn K, Bourne H (2006) Neutrophil polarization: spatiotemporal dynamics of RhoA activity support a self-organizing mechanism. Proc Natl Acad Sci U S A 103: 3639–3644. 18. Wang Y, Shyy JY-J, Chien S (2008) Fluorescence Proteins, Live-Cell Imaging, and Mechanobiology: Seeing Is Believing. Annual Review of Biomedical Engineering 10: 1–38. 19. Zitova B, Flusser J (2003) Image Registration Methods: A Survey. Image and Vision Computing 21: 977–1000. 20. Jagalur M, Pal C, Learned-Miller E, Zoeller RT, Kulp D (2007) Analyzing in situ gene expression in the mouse brain with image registration, feature extraction and block clustering. BMC Bioinformatics 8 Suppl 10: S5. 21. Chamberlain CE, Kraynov VS, Hahn KM (2000) Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol 325: 389–400. 22. Hodgson L, Nalbant P, Shen F, Hahn K (2006) Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol 406: 140–156. 23. Shen F, Hodgson L, Rabinovich A, Pertz O, Hahn K, et al. (2006) Functional proteometrics for cell migration. Cytometry A 69: 563–572. 24. Rieger B, Molenaar C, Dirks RW, Van Vliet LJ (2004) Alignment of the cell nucleus from labeled proteins only for 4D in vivo imaging. Microsc Res Tech 64: 142–150. 25. Matula P, Matula P, Kozubek M, Dvorak V (2006) Fast point-based 3-D alignment of live cells. IEEE Trans Image Process 15: 2388–2396. 26. Dormann D, Libotte T, Weijer CJ, Bretschneider T (2002) Simultaneous quantification of cell motility and protein-membrane-association using active contours. Cell Motil Cytoskeleton 52: 221–230. PLoS ONE | www.plosone.org 11 December 2008 | Volume 3 | Issue 12 | e4082