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


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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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




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.

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)

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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).

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