Monomeric red fluorescent proteins with a large Stokes shift

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.

[1]  Yan Chen,et al.  Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. , 2006, Biophysical journal.

[2]  Atsushi Miyawaki,et al.  A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy , 2006, Nature Biotechnology.

[3]  D. Bourgeois,et al.  Reverse pH-dependence of chromophore protonation explains the large Stokes shift of the red fluorescent protein mKeima. , 2009, Journal of the American Chemical Society.

[4]  Jeffrey Wyckoff,et al.  Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. , 2006, Cancer research.

[5]  A. Miyawaki,et al.  Two-photon dual-color imaging using fluorescent proteins , 2008, Nature Methods.

[6]  Alexander S. Mishin,et al.  Green fluorescent proteins are light-induced electron donors , 2009, Nature chemical biology.

[7]  Joachim Goedhart,et al.  Bright monomeric red fluorescent protein with an extended fluorescence lifetime , 2007, Nature Methods.

[8]  M. Drobizhev,et al.  Two-photon absorption standards in the 550-1600 nm excitation wavelength range. , 2008, Optics express.

[9]  Jacco van Rheenen,et al.  Intravital imaging of metastatic behavior through a mammary imaging window , 2008, Nature Methods.

[10]  S. Boxer,et al.  Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies. , 2007, Biochemistry.

[11]  A. Gotlieb,et al.  Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation. , 1981, The American journal of pathology.

[12]  Atsushi Miyawaki,et al.  Fluorescence imaging using a fluorescent protein with a large Stokes shift. , 2008, Methods.

[13]  S. Remington,et al.  Excited state proton transfer in the red fluorescent protein mKeima. , 2009, Journal of the American Chemical Society.

[14]  D. Shcherbo,et al.  Bright far-red fluorescent protein for whole-body imaging , 2007, Nature Methods.

[15]  Robert M. Hoffman,et al.  Imaging cancer dynamics in vivo at the tumor and cellular level with fluorescent proteins , 2008, Clinical & Experimental Metastasis.

[16]  Alan Hall,et al.  Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement , 1999, The Journal of cell biology.

[17]  B. O’Rourke,et al.  Two-Photon Microscopy of Cells and Tissue , 2004 .

[18]  A. Bershadsky,et al.  Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[19]  K K Baldridge,et al.  The structure of the chromophore within DsRed, a red fluorescent protein from coral. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Segall,et al.  A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. , 2000, Cancer research.

[21]  Suliana Manley,et al.  Photoactivatable mCherry for high-resolution two-color fluorescence microscopy , 2009, Nature Methods.

[22]  M. Drobizhev,et al.  Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. , 2009, The journal of physical chemistry. B.

[23]  Martin Chalfie,et al.  Green fluorescent protein : properties, applications, and protocols , 2005 .

[24]  Zbigniew Dauter,et al.  A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore* , 2008, Journal of Biological Chemistry.

[25]  R. Pepperkok,et al.  In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum , 2008, Journal of Cell Science.

[26]  J. Zavadil,et al.  Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. , 2002, Cancer research.

[27]  P. Devreotes,et al.  Eukaryotic Chemotaxis: Distinctions between Directional Sensing and Polarization* , 2003, Journal of Biological Chemistry.

[28]  K. König,et al.  Multiphoton microscopy in life sciences , 2000, Journal of microscopy.

[29]  Christian Seebacher,et al.  Mutagenic Stabilization of the Photocycle Intermediate of Green Fluorescent Protein (GFP) , 2003, Chembiochem : a European journal of chemical biology.

[30]  S. Singer,et al.  Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Michael Z. Lin,et al.  Improving the photostability of bright monomeric orange and red fluorescent proteins , 2008, Nature Methods.

[32]  Vladimir N Uversky,et al.  Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes. , 2008, Current protein & peptide science.

[33]  Yarong Wang,et al.  Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. , 2007, Cancer research.