Abstract
Intercellular protein–protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques1,2,3 have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Kim, S.A., Tai, C.-Y., Mok, L.-P., Mosser, E.A. & Schuman, E.M. Calcium-dependent dynamics of cadherin interactions at cell-cell junctions. Proc. Natl. Acad. Sci. USA 108, 9857–9862 (2011).
Feinberg, E.H. et al. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).
Liu, D.S., Loh, K.H., Lam, S.S., White, K.A. & Ting, A.Y. Imaging trans-cellular neurexin-neuroligin interactions by enzymatic probe ligation. PLoS One 8, e52823 (2013).
Craig, A.M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43–52 (2007).
Michnick, S.W., Ear, P.H., Manderson, E.N., Remy, I. & Stefan, E. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat. Rev. Drug Discov. 6, 569–582 (2007).
Yamagata, M. & Sanes, J.R. Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP). Front. Mol. Neurosci. 5, 18 (2012).
Kim, J. et al. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat. Methods 9, 96–102 (2012).
Remy, I. & Michnick, S.W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat. Methods 3, 977–979 (2006).
Galarneau, A., Primeau, M., Trudeau, L.-E. & Michnick, S.W. β-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat. Biotechnol. 20, 619–622 (2002).
Rossi, F., Charlton, C.A. & Blau, H.M. Monitoring protein-protein interactions in intact eukaryotic cells by β-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410 (1997).
Luker, K.E. et al. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. USA 101, 12288–12293 (2004).
Li, J., Wang, Y., Chiu, S.-L. & Cline, H.T. Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front. Neural Circuits 4, 6 (2010).
Rhee, H.-W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).
Porstmann, B., Porstmann, T., Nugel, E. & Evers, U. Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or β-galactosidase? J. Immunol. Methods 79, 27–37 (1985).
Li, X.-W. et al. New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. J. Biol. Chem. 289, 14434–14447 (2014).
Azevedo, A.M. et al. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 9, 199–247 (2003).
Wilkinson, B. & Gilbert, H.F. Protein disulfide isomerase. Biochimica et Biophysica Acta (BBA). Proteins and Proteomics 1699, 35–44 (2004).
Choi, J., Chen, J., Schreiber, S.L. & Clardy, J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242 (1996).
Lam, S.S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).
Pinaud, F. & Dahan, M. Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins. Proc. Natl. Acad. Sci. USA 108, E201–E210 (2011).
Tsetsenis, T., Boucard, A.A., Araç, D., Brunger, A.T. & Südhof, T.C. Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation. J. Neurosci. 34, 15083–15096 (2014).
Shekhawat, S.S. & Ghosh, I. Split-protein systems: beyond binary protein-protein interactions. Curr. Opin. Chem. Biol. 15, 789–797 (2011).
Martell, J.D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).
Araç, D. et al. Structures of neuroligin-1 and the neuroligin-1/neurexin-1 β complex reveal specific protein-protein and protein-Ca2+ interactions. Neuron 56, 992–1003 (2007).
Wickersham, I.R. & Feinberg, E.H. New technologies for imaging synaptic partners. Curr. Opin. Neurobiol. 22, 121–127 (2012).
Jagadish, S., Barnea, G., Clandinin, T.R. & Axel, R. Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace. Neuron 83, 630–644 (2014).
Chen, Y. et al. Cell-type-specific labeling of synapses in vivo through synaptic tagging with recombination. Neuron 81, 280–293 (2014).
Hong, Y.K., Kim, I.-J. & Sanes, J.R. Stereotyped axonal arbors of retinal ganglion cell subsets in the mouse superior colliculus. J. Comp. Neurol. 519, 1691–1711 (2011).
McClure, C., Cole, K.L., Wulff, P., Klugmann, M. & Murray, A.J. Production and titering of recombinant adeno-associated viral vectors. J. Vis. Exp. 57, e3348 (2011).
Atasoy, D., Aponte, Y., Su, H.H. & Sternson, S.M. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008).
Kato, S. et al. Selective neural pathway targeting reveals key roles of thalamostriatal projection in the control of visual discrimination. J. Neurosci. 31, 17169–17179 (2011).
Chalupa, L.M. & Williams, R.W. Eye, Retina, and Visual System of the Mouse (Mit Press, 2008).
Lin, Z., Thorsen, T. & Arnold, F.H. Functional expression of horseradish peroxidase in E. coli by directed evolution. Biotechnol. Prog. 15, 467–471 (1999).
Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).
Kügler, S. et al. Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors. Mol. Cell. Neurosci. 17, 78–96 (2001).
Yamagata, M. & Sanes, J.R. Expanding the Ig superfamily code for laminar specificity in retina: expression and role of contactins. J. Neurosci. 32, 14402–14414 (2012).
Lawrence, A., Bouwer, J.C., Perkins, G. & Ellisman, M.H. Transform-based backprojection for volume reconstruction of large format electron microscope tilt series. J. Struct. Biol. 154, 144–167 (2006).
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).
Guo, P. et al. Rapid and simplified purification of recombinant adeno-associated virus. J. Virol. Methods 183, 139–146 (2012).
Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).
Chen, I., Dorr, B.M. & Liu, D.R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl. Acad. Sci. USA 108, 11399–11404 (2011).
Lõoke, M., Kristjuhan, K. & Kristjuhan, A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50, 325–328 (2011).
Colby, D.W. et al. Engineering antibody affinity by yeast surface display. Methods Enzymol. 388, 348–358 (2004).
Acknowledgements
We thank J. Einstein (MIT) for preparing neuron cultures. W. Wang (MIT) provided yeast expressing the LAP peptide and gave helpful advice for yeast display and AAV preparation. P. Stawski, K. Cox, and K. Loh (MIT) provided synaptic fluorescent protein fusion plasmids. F. Touti and H.-W. Rhee (MIT) synthesized biotin-phenol. FACS experiments were performed at the Koch Institute Flow Cytometry Core (MIT). Funding was provided by the US National Institutes of Health (R01-CA186568 to A.Y.T.; R37NS029169 to J.R.S.; P41 GM103412 and R01GM086197 to M.H.E.) and the Howard Hughes Medical Institute Collaborative Initiative Award (A.Y.T. and J.R.S.). J.D.M. was supported by NSFGR and NDSEG fellowships.
Author information
Authors and Affiliations
Contributions
J.D.M. performed all experiments except those explicitly noted below. J.R.S. and M.Y. designed in vivo experiments and analyzed the results. M.Y. performed all in vivo experiments, prepared constructs and viruses for in vivo experiments, and generated stable HEK293T cells. T.J.D. prepared thin sections and performed EM imaging. T.J.D. and S.P. performed electron tomography and processed the data. M.H.E. guided and oversaw EM experiments and analyzed results with J.D.M. and T.J.D. C.G.K. contributed to deglycosylation and HEK293T cell labeling experiments. J.D.M. and A.Y.T. designed the research and analyzed the data. J.D.M., J.R.S., and A.Y.T. wrote the paper. All authors edited the paper.
Corresponding authors
Ethics declarations
Competing interests
Massachusetts Institute of Technology has filed a patent covering part of the information contained in this article.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–11, Supplementary Tables 1–5 and Supplementary Notes 1–6 (PDF 30095 kb)
EM tomographic volume of an sHRP-stained NRX-NLG contact site in HEK293T cells.
In this short AMIRA animation, successive sections of a tomogram are displayed in a back and forth motion, before a segmentation of the region of interest (red color) highlighting some of the system geometry is shown. The segmentation was manually created using local thresholding considerations on the reconstructed images. (MP4 21609 kb)
EM tomographic volume of an sHRP-stained NRX-NLG contact site in HEK293T cells.
The movie shows progression through the tomogram in a back and forth motion. The mitochondrial staining from APEX on the left-hand side indicates that that cell is transfected with sHRPa-NRX. (MP4 12388 kb)
Rights and permissions
About this article
Cite this article
Martell, J., Yamagata, M., Deerinck, T. et al. A split horseradish peroxidase for the detection of intercellular protein–protein interactions and sensitive visualization of synapses. Nat Biotechnol 34, 774–780 (2016). https://doi.org/10.1038/nbt.3563
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nbt.3563
This article is cited by
-
Real-time visualization of structural dynamics of synapses in live cells in vivo
Nature Methods (2024)
-
Green synthesis of stable hybrid biocatalyst using a hydrogen-bonded, π-π-stacking supramolecular assembly for electrochemical immunosensor
Nature Communications (2023)
-
Construction of L-type lectin displaying Saccharomyces cerevisiae for Vibrio parahaemolyticus agglutination
International Microbiology (2023)
-
Enzyme-mediated proximity labeling for mapping molecular interactions
Science China Life Sciences (2023)
-
Strategies for monitoring cell–cell interactions
Nature Chemical Biology (2021)