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A split horseradish peroxidase for the detection of intercellular protein–protein interactions and sensitive visualization of synapses

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.

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Figure 1: Protein engineering of sHRP.
Figure 2: Intercellular reconstitution of sHRP for fluorescent and EM labeling.
Figure 3: Synapse detection in cultured neurons using sHRP.
Figure 4: Detection of reconstituted sHRP in vivo.

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References

  1. 1

    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).

    CAS  Article  Google Scholar 

  2. 2

    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).

    CAS  Article  Google Scholar 

  3. 3

    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).

    CAS  Article  Google Scholar 

  4. 4

    Craig, A.M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43–52 (2007).

    CAS  Article  Google Scholar 

  5. 5

    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).

    CAS  Article  Google Scholar 

  6. 6

    Yamagata, M. & Sanes, J.R. Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP). Front. Mol. Neurosci. 5, 18 (2012).

    CAS  Article  Google Scholar 

  7. 7

    Kim, J. et al. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat. Methods 9, 96–102 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Remy, I. & Michnick, S.W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat. Methods 3, 977–979 (2006).

    CAS  Article  Google Scholar 

  9. 9

    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).

    CAS  Article  Google Scholar 

  10. 10

    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).

    CAS  Article  Google Scholar 

  11. 11

    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).

    CAS  Article  Google Scholar 

  12. 12

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Rhee, H.-W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).

    CAS  Article  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    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).

    CAS  Article  Google Scholar 

  16. 16

    Azevedo, A.M. et al. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 9, 199–247 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Wilkinson, B. & Gilbert, H.F. Protein disulfide isomerase. Biochimica et Biophysica Acta (BBA). Proteins and Proteomics 1699, 35–44 (2004).

    CAS  Article  Google Scholar 

  18. 18

    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).

    CAS  Article  Google Scholar 

  19. 19

    Lam, S.S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    CAS  Article  Google Scholar 

  20. 20

    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).

    CAS  Article  Google Scholar 

  21. 21

    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).

    Article  Google Scholar 

  22. 22

    Shekhawat, S.S. & Ghosh, I. Split-protein systems: beyond binary protein-protein interactions. Curr. Opin. Chem. Biol. 15, 789–797 (2011).

    CAS  Article  Google Scholar 

  23. 23

    Martell, J.D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    CAS  Article  Google Scholar 

  24. 24

    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).

    Article  Google Scholar 

  25. 25

    Wickersham, I.R. & Feinberg, E.H. New technologies for imaging synaptic partners. Curr. Opin. Neurobiol. 22, 121–127 (2012).

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    Chen, Y. et al. Cell-type-specific labeling of synapses in vivo through synaptic tagging with recombination. Neuron 81, 280–293 (2014).

    CAS  Article  Google Scholar 

  28. 28

    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).

    CAS  Article  Google Scholar 

  29. 29

    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).

    Google Scholar 

  30. 30

    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).

    CAS  Article  Google Scholar 

  31. 31

    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).

    CAS  Article  Google Scholar 

  32. 32

    Chalupa, L.M. & Williams, R.W. Eye, Retina, and Visual System of the Mouse (Mit Press, 2008).

  33. 33

    Lin, Z., Thorsen, T. & Arnold, F.H. Functional expression of horseradish peroxidase in E. coli by directed evolution. Biotechnol. Prog. 15, 467–471 (1999).

    CAS  Article  Google Scholar 

  34. 34

    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).

    CAS  Article  Google Scholar 

  35. 35

    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).

    Article  Google Scholar 

  36. 36

    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).

    CAS  Article  Google Scholar 

  37. 37

    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).

    CAS  Article  Google Scholar 

  38. 38

    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).

    CAS  Article  Google Scholar 

  39. 39

    Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Guo, P. et al. Rapid and simplified purification of recombinant adeno-associated virus. J. Virol. Methods 183, 139–146 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).

    CAS  Article  Google Scholar 

  42. 42

    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).

    CAS  Article  Google Scholar 

  43. 43

    Lõoke, M., Kristjuhan, K. & Kristjuhan, A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50, 325–328 (2011).

    Article  Google Scholar 

  44. 44

    Colby, D.W. et al. Engineering antibody affinity by yeast surface display. Methods Enzymol. 388, 348–358 (2004).

    CAS  Article  Google Scholar 

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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.

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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

Correspondence to Joshua R Sanes or Alice Y Ting.

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Massachusetts Institute of Technology has filed a patent covering part of the information contained in this article.

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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)

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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

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