An improved monomeric infrared fluorescent protein for neuronal and tumor brain imaging

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.

[1]  Smaroula Dilioglou,et al.  Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector , 2004, Nature Biotechnology.

[2]  Kami Kim,et al.  Bright and stable near infra-red fluorescent protein for in vivo imaging , 2011, Nature Biotechnology.

[3]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[4]  Lily Yeh Jan,et al.  Branching out: mechanisms of dendritic arborization , 2010, Nature Reviews Neuroscience.

[5]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[6]  A. Merlo,et al.  Frequent Co‐Alterations of TP53, p16/CDKN2A, p14ARF, PTEN Tumor Suppressor Genes in Human Glioma Cell Lines. , 1999, Brain pathology.

[7]  Philip R. Evans,et al.  An introduction to data reduction: space-group determination, scaling and intensity statistics , 2011, Acta crystallographica. Section D, Biological crystallography.

[8]  B. Glasheen,et al.  Distinct functions for the catalytic and hemopexin domains of a Drosophila matrix metalloproteinase , 2009, Proceedings of the National Academy of Sciences.

[9]  Roger Y Tsien,et al.  Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). , 2009, Angewandte Chemie.

[10]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[11]  Michael Z. Lin,et al.  Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome , 2009, Science.

[12]  R. Weissleder,et al.  Imaging in the era of molecular oncology , 2008, Nature.

[13]  D. A. Thayer,et al.  Increased neuronal activity fragments the Golgi complex , 2013, Proceedings of the National Academy of Sciences.

[14]  Y. Jan,et al.  Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron–glia interactions in Drosophila , 2011, Proceedings of the National Academy of Sciences.

[15]  Katrina T Forest,et al.  Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes* , 2008, Journal of Biological Chemistry.

[16]  S. Lukyanov,et al.  Fluorescent proteins and their applications in imaging living cells and tissues. , 2010, Physiological reviews.

[17]  R. W. Draft,et al.  Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system , 2007, Nature.

[18]  R. Vierstra,et al.  A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome , 2005, Nature.

[19]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[20]  Vasilis Ntziachristos,et al.  Shedding light onto live molecular targets , 2003, Nature Medicine.

[21]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Timm Schroeder,et al.  Imaging stem-cell-driven regeneration in mammals , 2008, Nature.

[23]  Mark J Schnitzer,et al.  An infrared fluorescent protein for deeper imaging , 2011, Nature Biotechnology.

[24]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[25]  R. Vierstra,et al.  Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors. , 2005, The Biochemical journal.

[26]  A. Verméglio,et al.  Bacteriophytochromes in anoxygenic photosynthetic bacteria , 2008, Photosynthesis Research.

[27]  David W Piston,et al.  Fluorescent proteins at a glance , 2011, Journal of Cell Science.

[28]  M. Affolter,et al.  Cytonemes Show Their Colors , 2011, Science.

[29]  Sougata Roy,et al.  Specificity of Drosophila Cytonemes for Distinct Signaling Pathways , 2011, Science.

[30]  F. Jöbsis.  Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatory parameters. , 1977, Science.

[31]  C. Contag,et al.  The importance of being red , 2009, Nature Biotechnology.

[32]  S. Jacques,et al.  Optical reflectance and transmittance of tissues: principles and applications , 1990 .

[33]  Frederick E. Petry,et al.  Principles and Applications , 1997 .