Enzyme-mediated intercellular proximity labeling for detecting cell-cell

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Enzyme-mediated intercellular proximity labeling for detecting cell-cell interactions Yun Ge, Long Chen, Shibo Liu, Jingyi Zhao, Heng Zhang, and Peng R. Chen J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Enzyme-mediated intercellular proximity labeling for detecting cell-cell interactions Yun Ge†,#, Long Chen†,#,Shibo Liu†, Jingyi Zhao†, Heng Zhang†, Peng R. Chen*,†,‡. †

Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. ‡

Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.

Supporting Information Placeholder cell-cell interactions in a proximity-dependent fashion (Scheme 1a). Prompted by the fact that the mono-glycine residue is a much more abundant N-terminal residue on cell surface proteins than tri-glycine17 (Figure S1), we evolved a “promiscuous” SrtA variant (mgSrtA) capable of labeling the exposed N-terminal mono-glycine residue on membrane proteins (Scheme 1b). By displaying mgSrtA on the surface of cells of interest, our EXCELL strategy allows tagging and tracking the interacting cells without prior knowledge of cell identity involved.

ABSTRACT: Cell-cell interactions and communications play fundamental roles in life processes but remain largely uncharacterized. We developed an enzyme-mediated proximity cell labeling (EXCELL) strategy as a general method to detect and record cell-cell interactions under living conditions. EXCELL relies on an evolved Staphylococcus aureus transpeptidase sortase A variant (mgSrtA) capable of promiscuous labeling of various cell surface proteins containing a mono-glycine residue at the Nterminus. Displaying mgSrtA on the surface of a cell of interest allows the labeling and detection of interacting cells in a proximity-dependent fashion. A variety of fundamental biological processes are known to rely on dynamic and complex cell-cell interactions that still remain poorly defined, largely due to the difficulty in identifying and monitoring such interactions in vivo1-3. A panel of proximitydependent labeling strategies has emerged in recent years as a powerful tool to profile the spatially organized proteome within a cell4,5. However, these approaches are not suitable for studying cell-cell interactions as they rely on toxic reagents (eg. H2O2 in the APEX (engineered ascorbate peroxidase) method)6,7, endogenous metabolites (eg. Biotin in the BioID8,9 (proximitydependent biotin identification) /TurboID10 methods) or lowdiffusive molecules (eg. an intact protein in the PUP-IT (pupylation-based interaction tagging) method)11 that may perturb cell physiology, interactions and/or result in low labeling efficiency. Moreover, the reactive radicals or high energy molecules generated by the APEX/BioID/Horseradish peroxidase (HRP)12,13 approaches result in a relatively large labeling radius, causing inevitable false positive results (see detailed comparison of the current methods in Table S1). Alternatively, Staphylococcus aureus transpeptidase Sortase A14,15 (SrtA) represents a promising enzyme for proximal-dependent intercellular labeling as it uses a small peptide (a LPXTG pentapeptide) as the substrate and has recently been applied to monitor cell-cell interactions in living mice16. However, since wild-type SrtA can only label the N-terminal tri-glycine moiety that needs to be pre-installed onto a cell surface protein, this method cannot label intercellular interactions without pre-engineering the prey cells. We herein developed an enzyme-mediated proximity cell labeling strategy (EXCELL) as a general approach for detecting

Scheme 1. Design and development of the enzyme-mediated proximity cell labeling (EXCELL) strategy. a. Schematic representation of the EXCELL strategy. An evolved, promiscuous SrtA variant-mgSrtA is displayed on the surface of a cell of interest, which can transfer the 'Biotin-LPETG'-containing peptide tag onto the N-terminal mono-glycine residues on diverse membrane proteins on interacting cells in a proximitydependent fashion. EXCELL offers a general approach to label and track intercellular interactions without prior knowledge of cell types involved. b. “Promiscuous” protein labeling enabled by mgSrtA-mediated N-terminal mono-glycine conjugation. Whereas wild type SrtA can only recognize and label the N-terminal oligoglycine motif (generally tri-/penta-glycines) that is much less abundant than the mono-glycine residue on cell surface proteins, the promiscuous mgSrtA variant possesses an expanded substrate tolerance and improved catalytic activity, which can catalyze efficient Nterminal mono-glycine conjugation. X equals any amino acids.

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We first performed directed evolution on SrtA by utilizing our recently developed FRET (Fluorescence Resonance Energy Transfer)-based selection assay18 (Figure 1a) that is constituted of an EGFP module bearing the C-terminal SrtA sorting motif LPETG (EGFP-LPETG) and a circular permutated Venus module bearing an N-terminal mono-glycine residue (G1-cpVenus). Ligation between these two modules by SrtA leads to an increased FRET signal that is proportional to the mono-glycine conjugation efficiency. We used error-prone PCR to mutagenize the starting WT-SrtA template and generate a random library. E. coli cells transformed with this library were subject to the FRET-based high throughput screening assay. After several rounds of screening, a panel of SrtA variants with increased FRET signal was obtained, with two mutations D124G, F200L highly enriched (red in Figure 1b). The FRET results (Figure S2) and kinetics characterization (Figure S3, Table S2 and S3) showed that D124G was the most beneficial mutation and F200L exhibited a synergistic effect with D124G that further increased SrtA's activity by over 2 folds than the previous variant without this mutation. Interestingly, as D124G was also identified from our previous evolution work on the oligoglycine substrate18, we included two additional mutations Y187L and E189R identified from the same study, and indeed, the resulting SrtA variant with all four mutations (named 4Mmg) showed a further enhanced efficiency for N-terminal monoglycine conjugation with an over 6-fold increase of kcat/Km LPETG value than that of WT-SrtA (Figure S2 and S3). Finally, since two previously reported SrtA variants (4M: P94S/D160N/D165A/K196T) (5M: P94R/D160N/D165A/K190E/K196T)19 possess significantly increased kcat/Km LPETG values towards oligoglycine, we tested whether adding these mutations would further enhance 4Mmg's ability for mono-glycine conjugation. The efficiency of these new variants in catalyzing mono-glycine conjugation was examined by labeling G1-cpVenus with a LPETG-containing fluorescent peptide (Figure 1c). Both '4Mmg+4M' and '4Mmg+5M' gave higher efficiency than their parent 4M and 5M variants, with the latter one exhibiting a higher efficiency. We renamed this variant as mgSrtA which showed a kcat/Km LPETG activity 5-fold and 78fold higher than that of 5M and WT-SrtA, respectively (Table S2). To further demonstrate the specificity and efficiency of mgSrtA, we conducted mgSrtA-mediated ligation on peptides and proteins. A total of 20 peptides with the N-terminal residue being any of the 20 natural amino acids were incubated with the TAMRAALPETGG probe in the presence of mgSrtA followed by LC-MS analysis. Only the peptide containing the N-terminal Gly residue but not other amino acids can form efficient ligation product (Figure S4a). Next, since the sequences of cell surface proteins bearing N-terminal glycine residues are quite random, we made a series of protein substrates with the N-terminal sequence as MGX to examine the labeling efficiency (X represents a randomly chosen amino acid at the P2 position). These proteins were then incubated with the TAMRA-ALPETGG probe in the presence of mgSrtA and the labeling products were monitored by LC-MS. The results showed that mgSrtA-mediated labelling was effective regardless of the identity of the X residue (Figure S4). Taken together, we demonstrated that mgSrtA can specifically and efficiently label peptides and proteins containing an N-terminal mono-glycine residue. We next applied mgSrtA to label intact cells through membrane proteins containing surface exposed N-terminal mono-glycine residues (Figure 1d). GFP containing a C-terminal LPETG motif was first incubated with Jurkat cells in the presence of various SrtA variants. Successful attachment of GFP on Jurkat cell

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surfaces was monitored by flow cytometry and further validated by western blotting analysis (Figure 1e, Figure S5a and S5b). As expected, mgSrtA exhibited the highest labeling efficiency compared to other SrtA variants. We further detected the attachment of biotin-AALPETG*G20 (referred as biotin-LPETG hereafter) on diverse cell types via mgSrtA, which all showed intense labeling bands on cell surface proteins and confirmed that many cell types have abundant surface exposed N-terminal glycine residues that can be labeled by SrtA (Figure S5c). Jurkat cells equipped with a T-cell receptor (TCR) responsive NFAT transcriptional reporter21 were next labeled with biotin-LPETG by mgSrtA and subject to T cell activation assay. The successful activation of this TCR responsive reporter illustrated that the physiology of T cells was not affected after our mgSrtA-mediated cell-surface labeling. Additionally, we genetically fused the gene encoding a Her2-specific affibody22,23 (ZHER) with the mgSrtA gene and the expressed fusion protein (ZHER-mgSrtA) showed the specificity on Her2 positive (Her2+) cells, which exhibited much higher biotin modification than Her2 negative (Her2-) as examined by flow cytometry (Figure S5e).

Figure 1. Directed evolution of SrtA for promiscuous protein and cell labeling. a. Schematic representation of the FRET-based screening system for directed evolution of SrtA. Green: EGFP-LPETG, Yellow: G1cpVenus. SrtA mediated ligation (SML) will lead to enhanced FRET efficiency on the EGFP-cpVenus pair. b. Crystal structure of SrtA in complex with its substrate peptide (PDB: 2KID). Cyan: Peptide substrate LPAT. Blue: Residues involved in substrate-enzyme intermediate formation H120, C184, R197. Magenta: residues from previously reported penta-mutation (5M). Red: residues evolved in this paper, D124G and F200L. Orange: residues evolved from the site-saturation mutagenesis library, Y187L and E189R. Yellow sphere: calcium ion. c. Fluorescence labeling of template protein G1-cpVenus with SrtA variants. FL: Fluorescence. CBB: Coomassie brilliant blue. d. Schematic of SrtA mediated cell surface labeling. Target molecules can be proteins, peptides or small molecules. e. Histograms of labeling EGFP bearing C-terminal LPETG sequence on Jurkat cell surfaces in the presence (blue line) and absence (red line) of mgSrtA.

Next, we employed mgSrtA to label and detect cell-cell interactions (Figure 2a). ZHER-mgSrtA was first displayed on the surface of HEK 293T cells, with the acquired affinity to Her2+

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cells due to direct ZHER-Her2 interaction (Figure S7a, S11). Flow cytometric analysis showed that Her2+ cells were significantly biotinylated only when incubated with the ZHERmgSrtA displaying cells, which is consistent with the direct connection between ZHER and Her2 (Figure 2b and S7c). In contrast, HEK 293T cells transfected with the empty vector or mgSrtA gene alone led to a much lower labelling of Her2+ cells. We also incubated the ZHER-mgSrtA displaying HEK 293T cells with Her2- cells and observed negligible biotin labelling (Figure 2c and S7e). Certain background labelling on Her2+/- MDA-MB231 cells were observed when displaying mgSrtA alone, largely due to non-specific cell-cell interactions. Furthermore, we cotransfected mgSrtA and ZHER as two separate genes into HEK293T cells, and as expected, Her2+ cells can still be efficiently labeled (Figure S12). Taken together, we demonstrated that our EXCELL strategy relying on surface-displayed mgSrtA can transfer biotin to cells in close proximity and thus record the cell-cell interaction history.

the aforementioned receptor-ligand interactions (Figure S8c). Flow cytometric analysis further confirmed biotin labeling of Raji cells after incubating with mgSrtA-displaying cells expressing CD40L or CTLA4 (Figure 3c, Figure S8a and S8b). Moreover, the activity of mgSrtA was validated by an enhanced biotin modification on HEK 293T surfaces. Finally, the biotin modification sites on Raji cell surface were fluorescent labeled via Streptavidin Alexa Fluor488 and clearly observed by fluorescent microscopy, which further confirmed the cell-cell contact and EXCELL-enabled labeling (Figure S9). As a control, when incubating Raji cells with mgSrtA-displaying cells that express an irrelevant ligand (eg. ZHER) or a lose-of-function CD40L mutant that is incapable of CD40 (CD40L*)16 binding, no obvious biotin labeling was observed (Figure S8a).

Figure 2. Validation and EXCELL-enabled detection of cell-cell interactions induced by ZHER and Her2. a. Schematic representation of EXCELL-enabled labeling of 'ZHER-Her2'-induced cell-cell interactions. Green: Her2 positive cells with EGFP expression. Gray: Her2 negative cells. Blue: Cells expressing ZHER-mgSrtA fusion protein on the surface. b. Flow cytometric analysis of EXCELL-labeled cell-cell interactions through the ZHER/Her2 affibody-antigen pair. HEK 293T cells expressing the empty vector, mgSrtA alone or the ZHER-mgSrtA fusion protein were incubated with the Her2+ MDA-MB-231 cells respectively before the addition of the biotin-AALPETG*G probe. The biotin-tagged cells were stained with Streptavidin PE and subject to flow cytometry. The Her2+ MDA-MB-231 cells stained with eFluor670 were gated and the percentage of biotin-labeled cells were quantified. c. Histograms of biotin staining in MDA-MB-231 Her2+ and Her2- cells shown respectively. PE, phycoerythrin.

Figure 3. EXCELL-enabled labeling and recording of cell-cell interactions. a. EXCELL-enabled labeling and recording of interactions between B cells and cells expressing CTLA4 or CD40L. b. Constructs used in c. PDGFR TM refers to the transmembrane domain of PDGFR. CTLA4-mgSrtA and CD40L-mgSrtA express a bicistronic gene that encodes a fluorescent reporter protein tdTomato. c. Flow cytometric analysis of enzymatic labeling on interacting Raji cells. HEK 293T cells expressing the empty vector, mgSrtA alone, CTLA4-mgSrtA or CD40LmgSrtA were incubated with Raji cells, respectively. Cells were stained with Streptavidin PE to reveal biotin attachment on cell surface. Raji cells stained with eFluor670 were gated and the percentage of biotin-labeled cells (in the rectangle) were quantified. PE, phycoerythrin.

Finally, we applied EXCELL to record cell-cell interactions induced by receptor-ligand recognitions that are essential for immune activations or suppression24. CD40 is a costimulatory receptor found primarily on B cells that is required for B cell activation through binding to CD40L on T cells25. CTLA4 expressed on T cells functions as an immune checkpoint protein and downregulates immune responses by binding CD80/86 (B7) on the surface of antigen presenting cells26. Therefore, cells expressing CD40L or CTLA4 were expected to directly interact with Raji B lymphocytes, which could be labeled and recorded via EXCELL (Figure 3a and 3b). Indeed, HEK 293T cells expressing CD40L or CTLA4 were found to interact with Raji cells through

In summary, we developed a proximal labeling strategyEXCELL that relies on cell-surface displaying of an evolved Nterminal mono-glycine recognition SrtA variant (mgSrtA) to label

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Wang, H.; Zhuang, M. Nat. Meth. 2018, 55, 332. (12) Kotani, N.; Gu, J.; Isaji, T.; Udaka, K.; Taniguchi, N.; Honke, K. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (21), 7405. (13) Bar, D. Z.; Atkatsh, K.; Tavarez, U.; Erdos, M. R.; Gruenbaum, Y.; Collins, F. S. Nat. Meth. 2018, 15 (2), 127. (14) Pishesha, N.; Ingram, J. R.; Ploegh, H. L. Annu. Rev. Cell Dev. Biol. 2018, 34 (17), 1. (15) Zong, Y.; Bice, T. W.; Ton-That, H.; Schneewind, O.; Narayana, S. V. L. J. Biol. Chem. 2004, 279 (30), 31383. (16) Pasqual, G.; Chudnovskiy, A.; Tas, J. M. J.; Agudelo, M.; Schweitzer, L. D.; Cui, A.; Hacohen, N.; Victora, G. D. Nature 2018, 553 (7689), 496. (17) Swee, L. K.; Lourido, S.; Bell, G. W.; Ingram, J. R.; Ploegh, H. L. ACS Chem. Biol. 2015, 10 (2), 460. (18) Chen, L.; Cohen, J.; Song, X.; Zhao, A.; Ye, Z.; Feulner, C. J.; Doonan, P.; Somers, W.; Lin, L.; Chen, P. R. Sci. Rep. 2016, 6 (1), 31899. (19) Chen, I.; Dorr, B. M.; Liu, D. R. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (28), 11399. (20) Williamson, D. J.; Fascione, M. A.; Webb, M. E.; Turnbull, W. B. Angew. Chem. Int. Ed. Engl. 2012, 51 (37), 9377. (21) Wei, P.; Wong, W. W.; Park, J. S.; Corcoran, E. E.; Peisajovich, S. G.; Onuffer, J. J.; Weiss, A.; Lim, W. A. Nature 2012, 488 (7411), 384. (22) Orlova, A.; Magnusson, M.; Eriksson, T. L. J.; Nilsson, M.; Larsson, B.; Höidén-Guthenberg, I.; Widström, C.; Carlsson, J.; Tolmachev, V.; Ståhl, S.; Nilsson, F. Y. Cancer Res. 2006, 66 (8), 4339. (23) Wikman, M.; Steffen, A. C.; Gunneriusson, E.; Tolmachev, V.; Adams, G. P.; Carlsson, J.; Stahl, S. Protein Eng. Des. Sel. 2004, 17 (5), 455. (24) Friedl, P.; Boer, den, A. T.; Gunzer, M. Nat. Rev. Immunol. 2005, 5 (7), 532. (25) Elgueta, R.; Benson, M. J.; de Vries, V. C.; Wasiuk, A.; Guo, Y.; Noelle, R. J. Immunol. Rev. 2009, 229 (1), 152. (26) Alegre, M. L.; Frauwirth, K. A.; Thompson, C. B. Nat. Rev. Immunol. 2001, 1 (3), 220. (27) Minamihata, K.; Goto, M.; Kamiya, N. Bioconjugate Chem. 2011, 22 (11), 2332.

and record cell-cell interactions. By targeting exposed N-terminal mono-glycine on cell surface, EXCELL may cause less perturbation to modified cells than other proximity-labeling methods relying on enzymes that modify all lysine residues or electron-rich residues (eg. tyrosine27 in APEX) on proteins. Mechanistic investigation on the mono-glycine recognition feature of our evolved mgSrtA may help to further enhance the cell labelling efficiency and/or signal-to-noise ratio. Nevertheless, by avoiding the pre-installation of oligoglycine on the surface of prey cells, EXCELL holds the potential to detect unknown cellcell interactions. In particular, since the small penta-peptide 'LPETG' can be readily conjugated with other molecules and has been administrated to live animals previously16, EXCELL may become a powerful tool to detect and discover cell-cell interactions within more complicated in vivo settings.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected]

Author Contributions #Y.G.

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and L.C. contributed equally to this work.

Notes The authors declare the following competing financial interest(s): The SrtA variants and applications described in the manuscript are under patent pending in People’s Republic of China.

ACKNOWLEDGMENT We thank Dr. Jian Lin and Dr. Ping Wei in Peking University for providing cell lines; and Dr. Meng Xu in Tsinghua University for useful discussion. This work was supported by the National Key Research and Development Program of China (2016YFA0501500) and National Natural Science Foundation of China (21521003, 21432002 and 21740001).

REFERENCES (1) Mittelbrunn, M.; Sánchez-Madrid, F. Nat. Rev. Mol. Cell Biol. 2012, 13 (5), 328. (2) Porterfield, W. B.; Prescher, J. A. Curr. Opin. Chem. Biol. 2015, 24, 121. (3) Germain, R. N.; Robey, E. A.; Cahalan, M. D. Science 2012, 336 (6089), 1676. (4) Kim, D. I.; Roux, K. J. Trends Cell Biol. 2016, 26 (11), 804. (5) Weeks, A. M.; Wells, J. A. Nat. Chem. Biol. 2018, 14 (1), 50. (6) Martell, J. D.; Deerinck, T. J.; Sancak, Y.; Poulos, T. L.; Mootha, V. K.; Sosinsky, G. E.; Ellisman, M. H.; Ting, A. Y. Nat. Biotechnol. 2012, 30 (11), 1143. (7) Lam, S. S.; Martell, J. D.; Kamer, K. J.; Deerinck, T. J.; Ellisman, M. H.; Mootha, V. K.; Ting, A. Y. Nat. Meth. 2015, 12 (1), 51. (8) Roux, K. J.; Kim, D. I.; Raida, M.; Burke, B. J. Cell Biol. 2012, 196 (6), 801. (9) Kim, D. I.; Jensen, S. C.; Noble, K. A.; Kc, B.; Roux, K. H.; Motamedchaboki, K.; Roux, K. J. Mol. Biol. Cell 2016, 27 (8), 1188. (10) Branon, T. C.; Bosch, J. A.; Sanchez, A. D.; Udeshi, N. D.; Svinkina, T.; Carr, S. A.; Feldman, J. L.; Perrimon, N.; Ting, A. Y. Nat. Biotechnol. 2018, 6, e02357. (11) Liu, Q.; Zheng, J.; Sun, W.; Huo, Y.; Zhang, L.; Hao, P.;

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Table of content

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Scheme 1. Design and development of the enzyme-mediated proximity cell labeling (EXCELL) strategy. a. Schematic representation of the EXCELL strategy. An evolved, promiscuous SrtA variant-mgSrtA is displayed on the surface of a cell of interest, which can transfer the 'Biotin-LPETG'-containing peptide tag onto the Nterminal mono-glycine residues on diverse membrane proteins on interacting cells in a proximity-dependent fashion. EXCELL offers a general approach to label and track intercellular interactions without prior knowledge of cell types involved. b. “Promiscuous” protein labeling enabled by mgSrtA-mediated N-terminal mono-glycine conjugation. Whereas wild type SrtA can only recognize and label the N-terminal oligoglycine motif (generally tri-/penta-glycines) that is much less abundant than the mono-glycine residue on cell surface proteins, the promiscuous mgSrtA variant possesses an expanded substrate tolerance and improved catalytic activity, which can catalyze efficient N-terminal mono-glycine conjugation. X equals any amino acids.

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Figure 1. Directed evolution of SrtA for promiscuous protein and cell labeling. a. Schematic representation of the FRET-based screening system for directed evolution of SrtA. Green: EGFP-LPETG, Yellow: G1-cpVenus. SrtA mediated ligation (SML) will lead to enhanced FRET efficiency on the EGFP-cpVenus pair. b. Crystal structure of SrtA in complex with its substrate peptide (PDB: 2KID). Cyan: Peptide substrate LPAT. Blue: Residues involved in substrate-enzyme intermediate formation H120, C184, R197. Magenta: residues from previously reported penta-mutation (5M). Red: residues evolved in this paper, D124G and F200L. Orange: residues evolved from the site-saturation mutagenesis library, Y187L and E189R. Yellow sphere: calcium ion. c. Fluorescence labeling of template protein G1-cpVenus with SrtA variants. FL: Fluorescence. CBB: Coomassie brilliant blue. d. Schematic of SrtA mediated cell surface labeling. Target molecules can be proteins, peptides or small molecules. e. Histograms of labeling EGFP bearing C-terminal LPETG sequence on Jurkat cell surfaces in the presence (blue line) and absence (red line) of mgSrtA. 81x90mm (300 x 300 DPI)

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Figure 2. Validation and EXCELL-enabled detection of cell-cell interactions induced by ZHER and Her2. a. Schematic representation of EXCELL-enabled labeling of 'ZHER-Her2'-induced cell-cell interactions. Green: Her2 positive cells with EGFP expression. Gray: Her2 negative cells. Blue: Cells expressing ZHER-mgSrtA fusion protein on the surface. b. Flow cytometric analysis of EXCELL-labeled cell-cell interactions through the ZHER/Her2 affibody-antigen pair. HEK 293T cells expressing the empty vector, mgSrtA alone or the ZHERmgSrtA fusion protein were incubated with the Her2+ MDA-MB-231 cells respectively before the addition of the biotin-AALPETG*G probe. The biotin-tagged cells were stained with Streptavidin PE and subject to flow cytometry. The Her2+ MDA-MB-231 cells stained with eFluor670 were gated and the percentage of biotinlabeled cells were quantified. c. Histograms of biotin staining in MDA-MB-231 Her2+ and Her2- cells shown respectively. PE, phycoerythrin.

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Figure 3. EXCELL-enabled labeling and recording of cell-cell interactions. a. EXCELL-enabled labeling and recording of interactions between B cells and cells expressing CTLA4 or CD40L. b. Constructs used in c. PDGFRβ TM refers to the transmembrane domain of PDGFRβ. CTLA4-mgSrtA and CD40L-mgSrtA express a bicistronic gene that encodes a fluorescent reporter protein tdTomato. c. Flow cytometric analysis of enzymatic labeling on interacting Raji cells. HEK 293T cells expressing the empty vector, mgSrtA alone, CTLA4-mgSrtA or CD40L-mgSrtA were incubated with Raji cells, respectively. Cells were stained with Streptavidin PE to reveal biotin attachment on cell surface. Raji cells stained with eFluor670 were gated and the percentage of biotin-labeled cells (in the rectangle) were quantified. PE, phycoerythrin. 83x126mm (300 x 300 DPI)

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