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

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

2. Part IIA genetically encoded tag for electron microscopy ~20nm EM image of synapse, courtesy of Thomas Deerinck

3. Previous labeling methods for EM • Immuno-EM labeling • Post-embedding => Limited preservation of antigenicity and ultrastructure, 2-D stochiometric labeling only • Pre-embedding => Limited preservation of ultrastructure and potential reaction product diffusion, limited 3-D labeling • Ultracryosections => Limited preservation of ultrastructure, 2-D stochiometric labeling only, staining restricted to sectioned tissue surface • Genetically targetable ReAsH-tetracysteine • Excellent ultrastructure and 3-D amplified labeling • Highly correlated live-cell LM and subsequent EM imaging • High resolution EM labeling ReAsH tetracysteine Immunogold-labeled Cx43 ReAsH-labeled Cx43-TC 100nm 100nm Gaietta et al, Science, 2002

4. Limitations of biarsenical-tetracysteine • Modest 1O2 generation efficiency (2%) limits sensitivity • Requires exogenous application of the biarsenical dye • Difficult to apply to multicellular organisms a Genetically encoded, efficient singlet oxygen generator (SOG) requiring no exogenous cofactors is desired a

5. GFP is intrinsically inefficient in 1O2 generation Abandoned GFP and TC • The chromophore is inefficient in photogenerating1O2 • The -barrel may prevent release of 1O2 QY ~0.004 vs 0.02 of ReAsH-TC Jimenez-Banzo et al, Biophysical J. (2008) KillerRed: GFP-like, engineered to generate reactive oxygen species (ROS) Bulina et al, Nat. Biotech. (2006) 1O2: undetected

6. A blue light photoreceptor binds to flavin mononucleotide (FMN) Cys426 Phototropin2 (Arabidopsis thaliana) LOV2 PKD FMN Cys426 PDB: 2v1b

7. Further engineering also increases fluorescence miniSOG Kd = 170.3 ± 8.4 pM very tight binding 106-residue QY (fluo) = 0.30 KillRed: 0.000 QY (1O2) = 0.47 20 times more efficient than ReAsH ReAsH: 0.024

8. miniSOG fluorescence is well correlated with DAB photoconversion ISC Fluorescence DIC before after HEK293 DAB photoconversion Ex. 488nm; Em. 530/30nm Varda Lev-Ram

9. Large # of miniSOG fusions are correctly localized both at LM/EM monomeric; smaller than half of GFP Better performance of the fusion proteins Michael Davidson Ericka Ramko

10. miniSOG in mitochondrial matrix generates large signal/background Fused to mitochondrial targeting seq. of cytochrome c Transmitted light 200 nm 2 microns

11. Cx43-miniSOG assembles into gap junctions miniSOG Cx43 GJ Connexin43_miniSOG 50 nm

12. a-actinin_miniSOG cross-links F-actin 2 micron

13. Pre or post-synaptic localization of synaptic cell-adhesion molecules (SynCAMs)? • SynCAMs  Ig superfamily; Ca2+-independent; • Synaptogenesis, synaptic plasticity: e.g. overexpression of SynCAM1 in hippocampus increases synapse number/spontaneous activity (Poster by Biederer lab, SfN 2009) • SynCAM1, 2, 3 & 4 in vertebrate; highly conserved Presynaptic SynCAM1 initially reported at both pre & post-synapse. (Ab -> SynCAM1, 2) 1? 2? Pre 1? 2? post Postsynaptic Biederer et al (Thomas C. Sudhof), Science (2002) 297, 1525-1531 Nils Bros, Neuron, 2009

14. SynCAM1: only found in pre-synaptic membrane (cultured cortical neurons)

15. SynCAM2: found in post-synaptic membrane (cultured cortical neurons)

16. Current data suggests pre and post-synaptic localization of SynCAM1 & 2 respectively Strong heterophilic interaction found between SynCAM1 & 2 (Fogel et al, J. Neuronscience, 2007) Presynaptic SynCAM1 SynCAM2 Postsynaptic Adapted from Nils Bros, Neuron, 2009

17. miniSOG expressed in intact mouse brain photoconverts DAB efficiently Expression of miniSOG-labeled SynCAM1 &2 by in utero electroporation at E15 SynCAM1-miniSOG photoconverted at P7 SynCAM2-miniSOG photoconverted at P21

18. SynCAM2: post-synaptic localization (intact mice) Excellent preservation of ultrastructure and labeling of target protein Serial block-face Scanning EM 500 nm

19. miniSOG can also be expressed in C. elegans Mito-miniSOG expressed in muscle (myo-3)

20. Genetically targeted light-induced cell ablation by mito-miniSOG miniSOG expressed in the mitochondrial matrix (fused to mitochondrial targeting seq. from cytochrome c) IFP (cotransfected) Hoechst Caspase activity

21. Mito-miniSOG photoablates GABAergic motor neurons of C. elegans Control (no light) Mito-miniSOG under Punc-25 expressed in 19 GABAergic motor neurons # of the surviving neurons Blue light

22. Summary • miniSOG is engineered from a blue-light photoreceptor • Genetically encoded requiring no exogenous cofactors • Super-efficient in 1O2 generation • Green fluorescent • miniSOG is demonstrated to be as useful for EM as GFP for FM • 3D reconstruction of neural circuits using serial block-face SEM • Correlative super-resolution imaging/EM • Other applications include: • Light-induced cell ablation • Acute photoinactivation of proteins

23. Acknowledgement Infrared fluorescent proteins miniSOG • Tsien lab • Varda Levram-Ellisman • Collaborators • UCSD (EM) • Mark Ellisman; Thomas Deerinck • Florida State Univ. (constructs) • Michael Davidson; Ericka Ramko • UCSD (C. elegans) • Billy Qi; YiShi Jin • Tsien lab • Antoine Royant • Michael Lin • Todd Anguila • Varda Levram-Ellisman Roger Tsien Financial support: HHMI & NIH