Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
pubs.acs.org/acschemicalbiology
Gap-Junction-Dependent Labeling of Nascent Proteins in Multicellular Networks Yaya Li,†,‡,# Weibing Liu,†,‡,# Qi Tang,†,‡ Xinqi Fan,†,‡ Yi Hao,†,§ Ling Gao,†,§ Zefan Li,†,‡ Bo Cheng,†,‡ and Xing Chen*,†,‡,§,∥,⊥ †
College of Chemistry and Molecular Engineering, ‡Beijing National Laboratory for Molecular Sciences, §Peking-Tsinghua Center for Life Sciences, ∥Synthetic and Functional Biomolecules Center, and ⊥Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China
ACS Chem. Biol. Downloaded from pubs.acs.org by STOCKHOLM UNIV on 02/05/19. For personal use only.
S Supporting Information *
ABSTRACT: Intercellular communication via gap junctions is crucial for orchestrating behaviors of multicellular systems. Imaging methods and electrophysiological techniques have been widely used to identify gap junctions and map the gap-junctionconnected cell networks. However, analyzing gene expression within a gap-junction network remains challenging. Herein, we report the development of bio-orthogonal recording of translation in adjacent cells connected by gap junctions (BORTAC-GJ), a gap-junction-dependent protein tagging method based on local activation of clickable amino acid analogues that pass through gap junctions and are metabolically incorporated into nascent proteins. We demonstrated that BORTAC-GJ enabled selective labeling of nascent proteomes, thus recording translation, in cell networks connected by gap junctions, leaving unconnected cells not labeled. We further applied BORTAC-GJ to probe bystander STING activation triggered by gap-junction-mediated cGAMP transfer, an important process in innate immune response. BORTAC-GJ provides a means to investigate the gap-junction network at the proteome level and is broadly applicable for various cell types connected by gap junctions.
C
activatable noncanonical amino acid (ncAA) precursors for selective labeling of nascent proteomes within the gapjunction-connected cell network (Figure 1). The ncAA precursors are enzymatically converted to ncAAs in cells ectopically expressing the enzyme. The activated ncAAs can transfer through the gap junctions and be metabolically incorporated into newly synthesized proteins, whereas cells not connected by gap junctions are not labeled. For the enzyme−precursor pair, we exploited the Ncarbobenzyloxy (Cbz or Z) cleaving enzyme (Zcleaver) from Sphingomonas paucimobilis15 and Z-protected ncAAs (ZncAAs). The ncAAs, L-azidohomoalanine (Aha) and Lhomopropargylglycine (Hpg), can be activated by the endogenous methionyl-tRNA synthetase (MetRS) by serving as the surrogates of methionine (Met) and therefore metabolically incorporated into nascent proteins.16,17 We chemically synthesized the corresponding Z-ncAAs, Ncarbobenzoxy-L-azidohomoalanine (Z-Aha) and N-carbobenzoxy-homopropargylglycine (Z-Hpg), with overall yields of 36% and 26%, respectively (Figure 1b and Scheme S1). Using an in vitro enzymatic assay, we determined the kinetics for the Zcleaver-catalyzed conversion of Z-ncAAs (Table S1). With Kcat/Km values of 2.16 and 6.06 mM−1 s−1 for Z-Aha and Z-Hpg, respectively, Zcleaver was capable of efficiently
ell-to-cell communication through gap junctions is important for mediating cellular function and behaviors in various multicellular systems.1 Chemical methods have been developed to visualize intercellular communications.2,3 Gap junctions are connexin-based channels formed between adjacent cells.4,5 Through gap junctions, cells exchange cytoplasmic molecules, including ions, second messengers, metabolites, and microRNAs, which regulate and coordinate signal transduction and gene expression in the connected cell network.6,7 Paired electrophysiological recording, in which the intracellular potential evoked by stimulating an adjacent cell is recorded, has long been used to probe gap-junction communication.8 Alternatively, fluorescent tracers can be microinjected into,9,10 transported into,11 or activated locally inside the cells,12,13 followed by monitoring their diffusion through the gap junctions using fluorescence microscopy. These methods have provided a powerful means of identifying gap-junction-connected cells and mapping the network.14 On the other hand, it is of great interest to study how gapjunction-mediated intercellular communication regulates gene expression within the connected cell network. However, proteomic profiling and transcriptomic profiling are often complicated by cells that are not connected. A method for selective labeling of newly synthesized or nascent proteins of gap-junction-connected cells is highly desired. To address this challenge, we developed bio-orthogonal recording of translation in adjacent cells connected by gap junctions (BORTAC-GJ), a strategy that exploits enzyme© XXXX American Chemical Society
Received: December 9, 2018 Accepted: January 30, 2019
A
DOI: 10.1021/acschembio.8b01065 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 2. BORTAC-GJ labeling by Z-Aha in the gap-junctionconnected cell network. Cocultures of HEK293T Zcleaver-T2A-GFP and WT HEK293T cells were incubated with 0.5 mM Z-Aha or 0.5 mM Z-Aha in the presence of CBX for 2 h, followed by reaction with alkyne-Cy5 and staining of the nuclei with Hoechst 33342. Arrows highlight the network of WT HEK293T cells (white arrows) connected with HEK293T Zcleaver-T2A-GFP cells (green arrows), which were labeled by Z-Aha (Cy5). Scale bars, 10 μm.
Figure 1. BORTAC-GJ for selective labeling of nascent proteomes in the gap-junction-connected cell network. (a) In multicellular systems, cells (blue) send signals (e.g., ions and small molecules) to the neighboring cells (green) that are connected to the sender cells by gap junctions. Other cells (gray) do not form gap junctions with the sender cells and, thus, do not receive signals. To record protein synthesis selectively in the gap-junction network, the sender cells are expressed with Zcleaver, which converts Z-ncAA to ncAA. The locally produced ncAA then passes through gap junctions to the connected cells, where ncAA is metabolically incorporated into newly synthesized proteins. (b) Chemical structures of Aha, Hpg, Z-Aha, and Z-Hpg.
both Zcleaver-expressing HeLa CX43 and HeLa CX43 cells in the coculture of those two cells (Figure S4, right panel). Furthermore, Z-Hpg could also be used for BORTAC-GJ labeling of the gap-junction-connected cell network (Figure S5). Next, we constructed a heterogeneous multicellular system by mixing HEK293T Zcleaver, WT HEK293T, and B16-F10 cells (Figure 3a). While HEK293T Zcleaver and WT HEK293T cells formed a gap-junction-connected network, B16-F10 cells, with minimal expression of connexins, including CX43 and CX45, should not form gap junctions with
converting Z-ncAAs to the corresponding ncAAs. To evaluate whether Zcleaver functions in live cells, HEK293T cells stably expressing Zcleaver (HEK293T Zcleaver) were treated with 0.5 mM Z-Aha or Z-Hpg for 4 h, followed by reaction with alkyne-Cy5 or azide-Cy5 via Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC or click chemistry). Confocal fluorescence microscopy of labeled cells and in-gel fluorescence scanning of labeled cell lysates showed that proteins in HEK293T Zcleaver cells were metabolically incorporated with Aha/Hpg in a Z-Aha/Z-Hpg concentration- and time-dependent manner (Figure S 1). Furthermore, Z-ncAAs did not cause significant cytotoxicity (Figure S2). To demonstrate BORTAC-GJ, we constructed a gapjunction-connected cell network by coculturing HEK293T Zcleaver cells with wild-type (WT) HEK293T cells. HEK293T cells endogenously express connexin 43 (CX43) and 45 (CX45) and form gap junctions between adjacent cells18,19 (Figure S3). The cell network was treated with 0.5 mM Z-Aha for 2 h, followed by reaction with alkyne-Cy5 and confocal fluorescence microcopy to visualize nascent proteins metabolically incorporated with Aha. Not only the HEK293T Zcleaver cells but also the adjacent WT HEK293T cells with no Zcleaver expression were fluorescently labeled, indicating that Z-Aha was converted to Aha in the Zcleaver-expressing cells and diffused through gap junctions to the connected cells (Figure 2, left panel). Upon treatment with carbenoxolone (CBX), a gap-junction inhibitor, Z-Aha labeling of nascent proteins was confined to the HEK293T Zcleaver cells (Figure 2, right panel). In the coculture of Zcleaver-expressing HeLa cells and WT HeLa cells, which express a minimal level of CX43 and CX45 (Figure S3), only the Zcleaver-expressing HeLa cells were labeled and no gap-junction-mediated diffusion of Aha was observed (Figure S4, left panel). When CX43 was ectopically expressed in HeLa cells (Figure S3), we observed gap-junction-mediated labeling of nascent proteins of
Figure 3. Selective recording of translation within the gap-junctionconnected cell network in a heterogeneous multicellular system. (a) HEK293T Zcleaver-T2A-mCherry cells (magenta) were cocultured with WT HEK293T cells (gray) and cell tracker CMFDA-labeled B16-F10 cells (green) in a 1:5:5 ratio. A cell network consisting of HEK293T Zcleaver-T2A-mCherry and WT HEK293T cells was formed via gap-junction connection. B16-F10 cells were not connected. (b) The coculture was treated with 2 mM Z-Aha or 10 μM Aha, followed by reaction with alkyne-Cy5 and staining of the nuclei with Hoechst 33342. Magenta and white arrows highlight HEK293T Zcleaver-T2A-mCherry cells and WT HEK293T cells connected with the network, respectively. Green and yellow arrows indicate the positions of B16-F10 cells and WT HEK293T cells not connected with the network, respectively. Scale bar, 20 μm. B
DOI: 10.1021/acschembio.8b01065 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology HEK293T cells20 (Figure S3a). Moreover, B16-F10 cells have an Aha labeling efficiency similar to that of HEK293T cells (Figure S6). The coculture was treated with Z-Aha and reacted with alkyne-Cy5. Confocal fluorescence imaging showed that HEK293T Zcleaver cells and WT HEK293T cells connected with HEK293T Zcleaver cells were labeled with Aha, while the B16-F10 cells and WT HEK293T cells not connected with HEK293T Zcleaver cells were not significantly labeled (Figure 3b, bottom panel). As a control, the coculture was treated with Aha, which resulted in a global labeling of all cell populations (Figure 3b, top panel). Because HEK293T cells and B16-F10 cells have similar Aha incorporation efficiencies, the observed selective labeling was due to gap-junction-dependent incorporation. These results demonstrate that BORTAC-GJ can selectively record translation within the gap-junction cellular network in complex multicellular systems. Finally, we applied BORTAC-GJ to study the bystander effect in the cGAS−STING-mediated innate immune response. Upon microbial infection, the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) responds to cytosolic DNA and catalyzes the synthesis of the second messenger cGAMP.21 cGAMP activates STING (stimulator of interferon genes), leading to the expression of type I interferons (IFNs) and interferon-stimulated genes (ISGs). Interestingly, cGAMP produced in the infected cells was found to spread into neighboring cells through gap junctions, triggering STING activation in bystander cells.22 The bystander STING activation was also observed between brain metastatic cells and astrocytes, which supported tumor growth and chemoresistance.23 HeLa cells have endogenous expression of both cGAS and STING, and the cGAS−STING pathway can be activated by transfection with plasmid DNA.20,22,23 The STING deficient HeLa cells (HeLa STINGKO) can produce cGAMP in response to cytosolic DNA but with no STING activation or downstream gene expression. Conversely, HeLa cGASKO cells can be activated by cGAMP but not by cytosolic DNA. We expressed CX43 in these two cells and constructed a gapjunction-connected network consisting of HeLa CX43 STINGKO and HeLa CX43 cGASKO cells, in which bystander STING activation was observed (Figure S7). To probe protein expression during bystander STING activation, HeLa CX43 STINGKO cells were activated by transfection with the Zcleaver-T2A-mCherry plasmid DNA and cocultured with HeLa reporter cells [HeLa CX43 cGASKO cells containing a stable IFN-stimulated response element-GFP (ISRE-GFP) reporter plasmid], in the presence of Z-Aha (Figure 4a). In the cGAS-activated cells, as indicated by the mCherry fluorescence (Figure 4b), cGAMP was synthesized by cGAS. At the same time, the activated cells expressed Zcleaver, which locally produced Aha. cGAMP, alone with Aha, passed across gap junctions into the bystander HeLa CX43 cGASKO cells. Bystander STING activation, as indicated by ISRE-GFP, was observed in the gap-junction-connected HeLa CX43 cGASKO cells (Figure 4b, GFP fluorescence). Inside the gap-junctionconnected cell networks, Aha was metabolically incorporated into the nascent proteins (Figure 4b, Cy5 fluorescence). To evaluate whether Z-Aha was able to label gene expression in the network, the Z-Aha-treated cells were lysed, reacted with alkyne-biotin, and captured by streptavidin beads, followed by immunoblot analysis. Both GFP and mCherry were enriched with Z-Aha, confirming metabolic labeling of nascent proteins in the cGAS-activated cells and the connected reporter cells
Figure 4. BORTAC-GJ recording of protein expression during bystander STING activation. (a) HeLa CX43 STINGKO cells transfected with the Zclevear-T2A-mCherry plasmid DNA were cocultured with HeLa reporter cells (1:40 ratio) in the presence of ZAha. The locally produced Aha passes through gap junctions along with cGAMP and labels nascent proteins in bystander cells. (b) Confocal fluorescence microscopy of the coculture treated with 1.5 mM Z-Aha for 2 h, followed by reaction with alkyne-Cy5 and staining of the nuclei with Hoechst 33342. Magenta and green arrows highlight the cGAMP-producing and bystander cells with STING activation, respectively. Scale bar, 10 μm. (c) After bystander STING activation and Z-Aha labeling, the cell lysates were reacted with alkyne-biotin, enriched with streptavidin beads, and analyzed by immunoblotting. WCL, whole cell lysate.
(Figure 4c). Furthermore, the expression of ISGs, such as ISG54 and Viperin, was strongly induced and enriched by ZAha, indicating that nascent proteins induced by bystander STING activation could be metabolically labeled by localized activation of Z-Aha within the network (Figure 4c). To further illustrate the specificity and enrichment capability of BORTAC-GJ in heterogeneous cell networks, we constructed two separated networks in a cell culture dish: the central network consisting of interconnected HeLa CX43 STINGKO cells transfected with the Zcleaver plasmid DNA and HeLa CX43 cGASKO cells and the peripheral network containing connected HeLa CX43 cGASKO cells (Figure S8a). The STING pathway was activated in HeLa CX43 cGASKO cells within the central network due to the bystander effect, while no activation occurred in the peripheral network. The enrichment ratio of ISG54 and Viperin was much higher with the use of Z-Aha to selectively label the central network than upon treating the cell networks directly with Aha, which labeled both networks (Figure S8b). BORTAC-GJ is a conditional protein labeling method for monitoring gene expression in gap-junction-connected cell networks. Complementary to current methods for mapping gap-junction connectivity, our strategy provides the capability to investigate the cell network at the proteome level, namely to visualize, enrich, and identify nascent proteins, during gapjunction communication. Although gene expression in both the sender and the connected recipient cells is labeled, gapjunction inhibitors can be used in conjunction with quantitative proteomic analysis to probe gene expression selectively in the recipient cells if necessary. BORTAC-GJ is broadly applicable to various cell types communicating through gap junctions, thus providing a versatile tool for understanding gap-junction-regulated biological and pathological processes. C
DOI: 10.1021/acschembio.8b01065 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
■ ■
(12) Dakin, K., Zhao, Y., and Li, W.-H. (2005) LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling. Nat. Methods 2, 55−62. (13) Tian, L., Yang, Y., Wysocki, L. M., Arnold, A. C., Hu, A., Ravichandran, B., Sternson, S. M., Looger, L. L., and Lavis, L. D. (2012) Selective esterase-ester pair for targeting small molecules with cellular specificity. Proc. Natl. Acad. Sci. U. S. A. 109, 4756−4761. (14) Abbaci, M., Barberi-Heyob, M., Blondel, W., Guillemin, F., and Didelon, J. (2008) Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication. BioTechniques 45, 33−62. (15) Nanduri, V. B., Goldberg, S., Johnston, R., and Patel, R. N. (2004) Cloning and expression of a novel enantioselective Ncarbobenzyloxy-cleaving enzyme. Enzyme Microb. Technol. 34, 304− 312. (16) Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A., and Schuman, E. M. (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. U. S. A. 103, 9482− 9487. (17) Beatty, K. E., Liu, J. C., Xie, F., Dieterich, D. C., Schuman, E. M., Wang, Q., and Tirrell, D. A. (2006) Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew. Chem. Int. Ed. 45, 7364−7367. (18) Butterweck, A., Gergs, U., Elfgang, C., Willecke, K., and Traub, O. (1994) Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells. J. Membr. Biol. 141, 247−256. (19) Langlois, S., Cowan, K. N., Shao, Q., Cowan, B. J., and Laird, D. W. (2008) Caveolin-1 and −2 interact with connexin43 and regulate gap junctional intercellular communication in keratinocytes. Mol. Biol. Cell 19, 912−928. (20) Ito, A., Katoh, F., Kataoka, T. R., Okada, M., Tsubota, N., Asada, H., Yoshikawa, K., Maeda, S., Kitamura, Y., Yamasaki, H., and Nojima, H. (2000) A role for heterologous gap junctions between melanoma and endothelial cells in metastasis. J. Clin. Invest. 105, 1189−1197. (21) Chen, Q., Sun, L., and Chen, Z. J. (2016) Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142−1149. (22) Ablasser, A., Schmid-Burgk, J. L., Hemmerling, I., Horvath, G. L., Schmidt, T., Latz, E., and Hornung, V. (2013) Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530−534. (23) Chen, Q., Boire, A., Jin, X., Valiente, M., Er, E. E., Lopez-Soto, A., S. Jacob, L., Patwa, R., Shah, H., Xu, K., Cross, J. R., and Massagué, J. (2016) Carcinoma−astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533, 493−498.
METHODS
Details of experimental materials and methods are provided in the Supporting Information.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b01065.
■
Experimental details, compound synthesis, and supporting figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Xing Chen: 0000-0002-3058-7370 Author Contributions #
Y.L. and W.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank Z. Jiang at Peking University for providing HeLa cGASKO and HeLa STINGKO cells. This work is supported by the National Key R&D Program of China (2018YFA0507600) and the National Natural Science Foundation of China (21425204, 91753206, and 21521003).
■
REFERENCES
(1) Mittelbrunn, M., and Sánchez-Madrid, F. (2012) Intercellular communication: diverse structures for exchange of genetic information. Nat. Rev. Mol. Cell Biol. 13, 328−335. (2) Porterfield, W. B., Jones, K. A., McCutcheon, D. C., and Prescher, J. A. (2015) A “Caged” Luciferin for Imaging Cell-Cell Contacts. J. Am. Chem. Soc. 137, 8656−8659. (3) Porterfield, W. B., and Prescher, J. A. (2015) Tools for visualizing cell-cell ’interactomes’. Curr. Opin. Chem. Biol. 24, 121− 130. (4) Kumar, N. M., and Gilula, N. B. (1996) The gap junction communication channel. Cell 84, 381−388. (5) Sáez, J. C., Berthoud, V. M., Branes, M. C., Martinez, A. D., and Beyer, E. C. (2003) Plasma membrane channels formed by connexins: their regulation and functions. Physiol. Rev. 83, 1359−1400. (6) Lecanda, F., Towler, D. A., Ziambaras, K., Cheng, S. L., Koval, M., Steinberg, T. H., and Civitelli, R. (1998) Gap junctional communication modulates gene expression in osteoblastic cells. Mol. Biol. Cell 9, 2249−2258. (7) Lin, L., Rodrigues, F. S. L. M., Kary, C., Contet, A., Logan, M., Baxter, R. H. G., Wood, W., and Baehrecke, E. H. (2017) Complement-Related Regulates Autophagy in Neighboring Cells. Cell 170, 158−171. (8) Neyton, J., and Trautmann, A. (1985) Single-channel currents of an intercellular junction. Nature 317, 331−335. (9) Kanno, Y., and Loewenstein, W. R. (1964) Intercellular Diffusion. Science 143, 959−960. (10) Stewart, W. W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14, 741−759. (11) Qiao, M., and Sanes, J. R. (2016) Genetic Method for Labeling Electrically Coupled Cells: Application to Retina. Front. Mol. Neurosci. 8, 81. D
DOI: 10.1021/acschembio.8b01065 ACS Chem. Biol. XXXX, XXX, XXX−XXX
