Genetically encoded tags in the post GFP era for electron microscopy and whole-animal imaging

Genetically encoded tags in the post GFP era for electron microscopy and whole-animal imaging Xiaokun Shu University of California, San Diego

Imaging from single molecule to whole body nm m cm Electron microscopy Fluorescence microscopy Whole-body imaging O2 Genetically encoded tag? Genetically encoded tag? 1O2 miniSOG Visible FPs IR (Singlet Oxygen Generating FP) IFP (Infrared FP) Rational protein design

Part IInfrared fluorescent proteinsfor whole-animal imaging

Long wavelength light (650 - 900 nm) penetrates tissue most efficiently 12 Rice,BW & Contag,CH Nature Biotech (2009)

Hemoglobin absorbs intensely below 600 nm while water absorbs light above 920 nm aaaaaaa NIR window (650-900nm)

Short excitation wavelengths limit in vivo applications of RFPs Needs to be redshifted: >650nm absorbance mPlum fluorescence

Chromophores determine spectra of the FPs GFP RFP Adapted from Zeiss/FSU dehydration oxidation mOrange cyclization Shu et al, Biochemistry (2006)

Protein matrix tunes spectra of the FPs II. H-bond interactions mPlum I. - interactions Em. 610 -> 650 nm Tyr203 YFP Watcher et al, Structure (1998) Shu et al, Protein Science (2009) III. electrostatic interactions mCherry DsRed Em. 510 -> 460 nm Ex. 400 -> 415 nm 2.40Å K70 E215 Thr203 Asp148 Val205 Glu222 Shu et al, Biochemistry (2007) Shu et al, Biochemistry (2006) Shu et al, Protein Science (2007)

Is there a short cut in engineering infrared fluorescent proteins? Protein sequence space IFP Space jump YFP mFruits extension of cro. GFP DsRed 22% seq. idn. 1994 - 1999: no success of engineering GFP to be red fluorescent 1999: DsRed was “borrowed” from corals

Bacterial phytochromes absorb at 700nm DrBphP: bacteriophytochrome from Deinococcus radiodurans GAF PHY PAS GAF PHY HKRD PAS BV BV Cys PDB: 3c2w Heme oxygenase

Structure-based engineering PDB: 1ztu

IFP is in the NIR window IFP1.4 absorbance 92,000M-1cm-1 fluorescence QY = 0.08 PDB: 1ztu

IFP1.4 fusion proteins localize correctly DIC+Fluo IFP1.4/HEK293 Ex: 665/45 nm Em: 725/50 nm 10m DIC+Fluo H2B-IFP1.4 -actinin-IFP1.4 IFP1.1/cortical neurons

Pleckstrin homology (PH)-IFP responds to insulin stimulation IFP1.4 PH (AKT1) time interval = 5 min. Confocal: 635 nm ex. / 650nm LP Insulin stimulation: 5ug/ml

IFP-expressing liver is brightly IR fluorescent in an intact living mouse Imaging channel FluoIFP(t) post IV of BV (250nmol) mKate mKate w/BV IFP 1.1 IFP 1.1 w/ BV GFP IFP IRES GFP Imaging channel mKate IRES GFP IFP mKate GFP IFP (IRES) GFP mKate (IRES) GFP 5x 5x mKate is scaled to visualize

Excitation & emission wavelengths of IFP penetrate tissue efficiently skin on skin off liver exposed rostral IFP caudal caudal rostral IFP mKate mKate Normalized liver brightness skin on skin off exposed IFP liver caudal 0.68 0.91 1 rostral 0.63 0.80 1 caudal 0.42 0.79 1 mKate liver rostral 0.15 0.27 1

IFP in 3D reconstruction of mouse liver Fluorescence molecular tomography (FMT) FMT 2500 (VisEn)

Summary • Infrared FPs are engineered from bacterial phytochromes • IFPs are optimized for imaging deeply buried tissues/organs in whole-animal Hundreds of Laboratories are using IFPs

Genetically encoded tags in the post GFP era for electron microscopy and whole-animal imaging