Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Programming cell-surface signaling by phase-separation-controlled compartmentalization

Nature Chemical Biology volume 18, pages 1351–1360 (2022)Cite this article

Subjects

This article has been updated

Abstract

The landscape of cell-surface signaling is formidably complex. Robust tools capable of manipulating the spatiotemporal distribution of cell-surface proteins (CSPs) for dissecting signaling are in high demand. Some CSPs are regulated via multivalency-driven liquid–liquid phase separation (LLPS). Employing the robustness and versatility of LLPS, we decided to engineer LLPS-based tools for precisely manipulating CSPs. We generated membrane-tethering LLPS systems by fusing multivalent modular phase-separation scaffold pairs with CSP binders. Phase separation of the scaffold pairs, concomitant compartmentalization of CSPs on membranes, and cluster-dependent signaling outputs of CSPs require membrane recruitment of one or both scaffolds. We also engineered orthogonal phase-separation systems to segregate CSPs into mutually exclusive compartments. The engineered phase-separation systems can robustly cluster individual CSPs, co-cluster two or more CSPs, or segregate different CSPs into distinct compartments on cell surfaces. These tools will enable the dissection of complicated cell-signaling landscapes with high precision.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Clustering of CSPs via multivalent interactions.
Fig. 2: Co-clustering of two types of CSPs via multivalent interactions.
Fig. 3: Co-clustering two types of CSPs via an OR-gate phase-separation system.
Fig. 4: Co-clustering two types of CSPs via an AND-gate phase-separation system.
Fig. 5: Orthogonal condensates mediate the segregation of underlying CSPs.

Similar content being viewed by others

Data availability

All data are available in the main text or the supplementary materials. Expression plasmids and cell lines are available upon request. Source data are provided with this paper.

Change history

  • 01 December 2022

    In the version of this article initially published online, the word “Biology” mistakenly appeared at the start of the first affiliation, which has been amended to read “Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China” in the HTML and PDF versions of the article.

References

  1. Hubbard, S. R. & Miller, W. T. Receptor tyrosine kinases: mechanisms of activation and signaling. Curr. Opin. Cell Biol. 19, 117–123 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lin, C. W. et al. A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane. Proc. Natl Acad. Sci. USA 119, e2122531119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Xu, G. W. & Shi, Y. F. Apoptosis signaling pathways and lymphocyte homeostasis. Cell Res. 17, 759–771 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Komiya, Y. & Habas, R. Wnt signal transduction pathways. Organogenesis 4, 68–75 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Cadigan, K. M. & Liu, Y. I. Wnt signaling: complexity at the surface. J. Cell Sci. 119, 395–402 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Chen, X., Wu, X., Wu, H. & Zhang, M. Phase separation at the synapse. Nat. Neurosci. 23, 301–310 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Feng, Z., Chen, X., Zeng, M. & Zhang, M. Phase separation as a mechanism for assembling dynamic postsynaptic density signalling complexes. Curr. Opin. Neurobiol. 57, 1–8 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ditlev, J. A. et al. A composition-dependent molecular clutch between T cell signaling condensates and actin. eLife 8, e42695 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Case, L. B., Zhang, X., Ditlev, J. A. & Rosen, M. K. Stoichiometry controls activity of phase-separated clusters of actin signaling proteins. Science 363, 1093–1097 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zeng, M., Bai, G. & Zhang, M. Anchoring high concentrations of SynGAP at postsynaptic densities via liquid-liquid phase separation. Small GTPases 10, 296–304 (2019).

    CAS  PubMed  Google Scholar 

  12. Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tian, F. F., Lv, Y. G., Zhou, P. & Yang, L. Characterization of PDZ domain–peptide interactions using an integrated protocol of QM/MM, PB/SA, and CFEA analyses. J. Comput. Aid. Mol. Des. 25, 947–958 (2011).

    Article  CAS  Google Scholar 

  15. Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. Identification of an 11-Kda Fkbp12-rapamycin-binding domain within the 289-Kda Fkbp12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl Acad. Sci. USA 92, 4947–4951 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jahnichen, S. et al. CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc. Natl Acad. Sci. USA 107, 20565–20570 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huet, H. A. et al. Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction. Mabs 6, 1560–1570 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Mongkolsapaya, J. et al. Structure of the TRAIL–DR5 complex reveals mechanisms conferring specificity in apoptotic initiation. Nat. Struct. Biol. 6, 1048–1053 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Liu, T. et al. Rational design of CXCR4 specific antibodies with elongated CDRs. J. Am. Chem. Soc. 136, 10557–10560 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jura, N. et al. Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell 137, 1293–1307 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Needham, S. R. et al. EGFR oligomerization organizes kinase-active dimers into competent signalling platforms. Nat. Commun. 7, 13307 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Pan, L. et al. Higher-order clustering of the transmembrane anchor of DR5 drives signaling. Cell 176, 1477–1489 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chong, B. F. & Mohan, C. Targeting the CXCR4/CXCL12 axis in systemic lupus erythematosus. Expert Opin. Ther. Targets 13, 1147–1153 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I. & Littman, D. R. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Chatterjee, S., Behnam Azad, B. & Nimmagadda, S. The intricate role of CXCR4 in cancer. Adv. Cancer Res 124, 31–82 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Muller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Su, L. P. et al. Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells. Clin. Cancer Res. 11, 8273–8280 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Rempel, S. A., Dudas, S., Ge, S. & Gutierrez, J. A. Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma. Clin. Cancer Res. 6, 102–111 (2000).

    CAS  PubMed  Google Scholar 

  29. Barbolina, M. V. et al. Microenvironmental regulation of chemokine (C-X-C-motif) receptor 4 in ovarian carcinoma. Mol. Cancer Res 8, 653–664 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Darash-Yahana, M. et al. Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J. 18, 1240–1242 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sako, Y., Minoguchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168–172 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Williams, J. Z. et al. Precise T cell recognition programs designed by transcriptionally linking multiple receptors. Science 370, 1099–1104 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. De Miguel, D. et al. High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer. Cancer Lett. 383, 250–260 (2016).

    Article  PubMed  Google Scholar 

  35. Huang, K. H. et al. Caspase-3, a key apoptotic protein, as a prognostic marker in gastric cancer after curative surgery. Int J. Surg. 52, 266–271 (2018).

    Article  Google Scholar 

  36. Casao, A. et al. Cleaved PARP-1, an apoptotic marker, can be detected in ram spermatozoa. Reprod. Domest. Anim. 50, 688–691 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Tiacci, E. et al. The NPM1 wild-type OCI-AML2 and the NPM1-mutated OCI-AML3 cell lines carry DNMT3A mutations. Leukemia 26, 554–557 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Hosokawa, T. et al. CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation. Nat. Neurosci. 24, 777–785 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Dine, E., Reed, E. H. & Toettcher, J. E. Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch. Cell Rep. 35, 109280 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schwayer, C. et al. Mechanosensation of Tight Junctions Depends on ZO-1 phase separation and flow. Cell 179, 937–952 (2019).

    Article  CAS  PubMed  Google Scholar 

  41. Beutel, O., Maraspini, R., Pombo-Garcia, K., Martin-Lemaitre, C. & Honigmann, A. Phase separation of zonula occludens proteins drives formation of tight junctions. Cell 179, 923–936 (2019).

    Article  CAS  PubMed  Google Scholar 

  42. Zeng, M. et al. Phase separation-mediated TARP/MAGUK complex condensation and AMPA receptor synaptic transmission. Neuron 104, 529–543 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wu, X. et al. RIM and RIM-BP form presynaptic active-zone-like condensates via phase separation. Mol. Cell 73, 971–984 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Huang, W. Y. C. et al. A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS. Science 363, 1098–1103 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wong, L. E. et al. Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness. Nat. Commun. 11, 848 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhu, L. et al. Controlling the material properties and rRNA processing function of the nucleolus using light. Proc. Natl Acad. Sci. USA 116, 17330–17335 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lin, C. C. et al. Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state. Mol. Cell 82, 1089–1106 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Niehrs, C. The complex world of WNT receptor signalling. Nat. Rev. Mol. Cell Bio 13, 767–779 (2012).

    Article  CAS  Google Scholar 

  49. Decaillot, F. M. et al. CXCR7/CXCR4 heterodimer constitutively recruits β-arrestin to enhance cell migration. J. Biol. Chem. 286, 32188–32197 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to members of the Du and Li groups for helpful discussions. We thank D. Sun (Tsinghua) for her discussions and suggestions about the paper. We thank W. Xu for his help with protein purification. We are grateful to the SLSTU-Nikon Biological Imaging Center and the Core Facility of the Center of Biomedical Analysis, Tsinghua University, especially J. Ji, for providing technical support. This work was supported by grants from the Natural Science Foundation of China (32150023 to P.L.) National Key R&D Program (2019YFA0508403 to P.L. and 2018YFA0209700 to J.D.) and Natural Science Foundation of China (32125010, 31871443 to P.L.).

Author information

Authors and Affiliations

  1. Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Ru Li, Genzhe Lu, Zhi Cao, Bowen Chen & Pilong Li

  2. School of Pharmaceutical Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing, China

    Tiantian Li & Juanjuan Du

  3. The State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Yalong Wang

Authors
  1. Ru Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tiantian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Genzhe Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhi Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bowen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yalong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Juanjuan Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pilong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.L. performed all cell biology experiments and performed biochemical and genetic experiments with support from T.L., G.L., Z.C., and Y.W. B.C. conducted the simulation experiment. P.L. conceived the ideas. J.D. and P.L. designed and supervised the experiments, and wrote the paper with input from all other authors.

Corresponding authors

Correspondence to Juanjuan Du or Pilong Li.

Ethics declarations

Competing interests

The Tsinghua University has filed a patent application on the basis of this work. The authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Hong Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 General information about the system (Related to Figs. 1, 2).

(A) The domain structures of the two engineered cell-surface proteins for expression in mammalian cells. SP (signal peptide) and TM (transmembrane domain) of the fusion proteins were from EGFR. The domain structures of SUM5, SIM6, SIM6-R4L, SUM5-TRAIL, SUM5-FRB, SIM6-PV, SIM6-mEGF, SIM6-GFP, SIM6-GFP-mEGF, SUM5-mCherry, SUM5-R4L-mCherry and GFP-mEGF. (C) Phase separation diagrams for SIM6 (Alexa594-red) alone, SUM5 (Alexa647-green) alone, and SIM6 together with SUM5. Scale bar, 10 µm. (D) Time-lapse imaging of droplet fusion as in D (SIM6 and SUM5 at 10 µM each). Scale bar, 1 µm. (E) HEK cells expressing PDZ-TM-GFP were treated with SIM6-PV(Alexa405-blue). (F) HEK cells expressing FKBP-TM-mCherry were treated with SUM5-FRB(Alexa647-pink) and rapamycin (100 µM). (G) Time-lapse imaging of puncta formation on cell membranes after sequentially adding SIM6-PV and SUM5. The green channel (488 nm) was for PDZ-TM-GFP; the red channel shows (405 nm) SIM6-PV (Alexa405); the pink channel (647 nm) shows SUM5 (Alexa647). (H) Fluorescence intensity recovery after photobleaching (FRAP) of a droplet formed in HEK cells expressing PDZ-TM-GFP treated with SIM6-PV and SUM5. The green channel (488 nm) shows PDZ-TM-GFP. The red channel (405 nm) shows SIM6-PV (Alexa405). The pink channel (647 nm) shows SUM5 (Alexa647). Right upper: time series of GFP signal in the boxed region on the left images; right lower: quantification of fluorescence intensity recovery of PDZ-TM-GFP in bleached puncta (n = 3). Data was presented as mean ± SD. (I, J) Time-lapse imaging of puncta formation on plasma membranes of cells expressing FKBP-TM-mCherry after adding SUM5-FRB, SIM6 and rapamycin (B) or DMSO (C). The green channel (405 nm) shows SIM6 (Alexa405). The red channel (594 nm) shows FKBP-TM-mCherry. The pink channel (647 nm) shows SUM5-FRB (Alexa647). (K) Western blotting assay of expression levels of DR5, EGFR, and CXCR4 in different cell lines (OCI-AML3, HL-60, SY5Y, Colo-205, SJSA-1, and HEK293). A membrane protein, ATP1A1, serves as a loading control. (E–J) Final concentrations of added proteins were 200 nM. Rapamycin was used at 100 µM. Scale bar, 10 µm.

Source data

Extended Data Fig. 2 Multivalency-fused mEGF clusters and activates EGFR via phase separation (Related to Fig. 1).

(A) Left: SJSA-1 cells were treated with SIM6-mEGF (Alexa594, red) and SUM5 (Alexa647, green). Final protein concentrations were 200 nM. Scale bar, 10 µm. Right: green, red, and merged images of the boxed region in the image on the left. Scale bar, 1 µm. (B) Immunofluorescence staining of pEGFR of SJSA-1 cells after 40-minute treatment with SIM6-mEGF+SUM5. Right panel shows fluorescence intensity line-scanning plots from the merged image on the left, showing that endogenous pEGFR is concentrated in the puncta. Final protein concentrations were 200 nM. Scale bar, 1 µm. (C) Western blotting of pEGFR in SJSA-1 cells treated with different proteins combinations at different concentrations. (D) Statistical analysis of intensities of pEGFR in SJSA-1 cells treated with different proteins combinations. The intensities of pEGFR were normalized with Tubulin signal (n = 3). P values were evaluated by Student’s t test (two-tailed). Data was presented as mean ± SEM. (E) Immunofluorescence staining of total EGFR of A431 cells after 20-minute treatment with SIM6-mEGF+SUM5 (up), SIM6-mEGF (middle), and negative control (bottom). Final protein concentrations were 200 nM. Scale bar, 10 µm. (F) A merged image of A431 cells treated with SIM6-mEGF(Alexa594, red) + SUM5 (Alexa647, green). Bottom: green, red, and merged images of the boxed region in the image above. Final protein concentrations were 200 nM. Scale bar, 10 µm.

Source data

Extended Data Fig. 3 Multivalency-fused ligands co-cluster two CSPs (Related to Fig. 2).

(A) A scheme depicting clustering of two types of CSPs via recruitment of a multivalent protein pair. (B) Left: A scheme depicting the treatment of cells expressing PDZ-TM-GFP and FKBP-TM-mCherry with multivalent scaffold proteins. Right: Time-lapse imaging of cells co-expressing PDZ-TM-GFP and FKBP-TM-mCherry after adding SIM6-PV, SUM5-FRB + rapamycin. The red channel (594 nm) shows FKBP-TM-mCherry. The green channel (488 nm) shows PDZ-TM-GFP. The blue channel shows (405 nm) SIM6-PV (Alexa405). The pink channel (647 nm) shows SUM5-FRB (Alexa647). The protein concentrations were 200 nM. Scale bar, 5 µm. (C) Left, fluorescence intensity recovery after photobleaching (FRAP) of a droplet on a HEK cell expressing PDZ-TM-GFP and FKBP-TM-mCherry treated with SIM6-PV (blue) and SUM5-FRB + rapamycin (pink). Scale bar, 10 µm. Right upper: snapshots of mCherry and GFP channels of the box in the left image. Scale bar, 1 µm. Right lower: quantification of fluorescence intensity recovery of PDZ-TM-GFP and FKBP-TM-mCherry of bleached droplets (n = 3). Data was presented as mean ± SD (D) Left, Image of SJSA-1 cells treated with SIM6-GFP-mEGF+SUM5-R4L-mCherry. The green channel shows SIM6-GFP-mEGF and the red channel shows SUM5-R4L-mCherry. Final concentrations of proteins were 200 nM. Scale bar, 10 µm. Right, the green, red, and merged images of the boxed region in the image on the left. (E) Immunofluorescence staining of EGFR (upper) and CXCR4 (middle) of SJSA-1 cells after 40-minute treatment with SIM6-R4L + SUM5-TRAIL. Bottom: fluorescence intensity line-scanning plots from panels above showing that endogenous EGFR and CXCR4 are concentrated in the puncta. (F) Immunofluorescence staining of DR5 (upper) and pEGFR (middle) of SJSA-1 cells after 40-minute treatment with SIM6-mEGF (red, 594 nm)+SUM5-TRAIL (green, 647 nm). Bottom: fluorescence intensity line-scanning plots from panels above showing that endogenous DR5 and pEGFR are concentrated in the puncta. (G) Immunofluorescence staining of DR5 (upper) and CXCR4 (middle) of SJSA-1 cells after 40-minute treatment with SIM6-R4L + SUM5-TRAIL. Bottom: fluorescence intensity line-scanning plots from panels above showing that endogenous DR5 and CXCR4 are concentrated in the puncta. (E–G) Scar bar, 1 µm.

Source data

Extended Data Fig. 4 Extended information about the OR-gate system (Related to Fig. 3).

(A) In vitro phase separation assay of SIM6-CNB and SUM5-DNB. The green channel (488 nm) shows extracellular domain of DR5 (Alexa488-green) is recruited into puncta formed by SIM6-CNB and SUM5-DNB. For each protein, 10% was labeled, that is the blue channel (405 nm) shows SIM6-CNB containing 10% SIM6-CNB labeled with Alexa405 and the pink channel (647 nm) shows SUM5-DNB containing 10% SUM5-DNB labeled with Alexa647. Scale bar, 10 µm. (B) TIRF images of SJSA-1 cells subjected to different treatments: SUM5-DNB(Alexa647, green), or SIM6-CNB(Alexa594, red), or CNB(Alexa594, red) + DNB(Alexa647, green). Images were taken using the SIM/STORM-TIRF 20 minutes after treatment. Scale bar, 1 µm. (C) Statistical analysis of the condensate area formed in Fig. 3F. Only the condensate contains both signals of Alexa647 and Alexa594 were counted. Statistical significance was evaluated by Student’s t test (two-tailed). (SIM6-CNB + SUM5-DNB and SIM6 + SUM5-DNB,n = 20; SIM6-CNB + SUM5,n = 2; SIM6 + SUM5,n = 11; CNB + DNB, n = 7). P values were evaluated by Student’s t test (two-tailed). Data was presented as mean ± SEM. (D) Fluorescence intensity recovery after photobleaching (FRAP) of a droplet formed in SJSA-1 cells treated with SUM5-DNB + SIM6-CNB. Scale bar, 10 µm. (E) Flow cytometry detection of apoptotic SJSA-1 cells after treatment with the indicated proteins. Cells were treated for 4 hours. (F) Representative images of cells at 3 hours after treatment. The protein concentrations used were 100 nM. (B–D, E, F) The concentrations of the proteins were 100 nM.

Source data

Extended Data Fig. 5 Extended information about the OR-gate system (Related to Fig. 3).

(A) TIRF images of SJSA-1 cells 20 minutes after treatments with SIM6-CNB + SUM5-DNB under different concentrations. The green channel shows (the signals in 647 nm) SUM5-DNB (Alexa Fluor 647) and the red channel (the signals in 594 nm) shows SIM6-CNB (Alexa Fluor 594). (B, C) Statistical analysis of the condensate number and condensate area formed under different treatment. Only condensate contains both Alexa647 and Alexa594 would be counted. The total condensate number and condensate area from cell number as (2 nM, n = 77, 5 nM, n = 66, 10 nM, n = 154, 20 nM, n = 106, 50 nM, n = 48, 100 nM, n = 145). The dotted line in panel C means the zero in Y-axis. (D) Apoptosis of SJSA-1 cells 4 hours after different treatments. n = 4. P values were evaluated by Student’s t test (two-tailed). (E) Cell survival analysis of SJSA-1 cells under different treatments. Time-lapse imaging with an interval of 30 minutes was performed with IncuCyte ZOOM. n = 4. (F) Representative images of cells in (E) at 3 hours after treatment. (G) An engineered two-receptor OR-gate phase separation system involving stimulation of DR5 and CXCR4. (B-D) P values were evaluated by Student’s t test (two-tailed). (B–E) Data was presented as mean ± SEM.

Source data

Extended Data Fig. 6 Extended information about the AND-gate system (Related to Fig. 4).

(A) An engineered two-receptor AND-gate phase separation system involving stimulation of DR5 and CXCR4. (B) In vitro phase diagrams of SIM3-CNB and SUM3-DNB. The ECD of DR5 (Alexa488, green) is recruited into the puncta formed by SIM3-CNB and SUM3-DNB. For SIM3-CNB and SUM3-DNB, 10% of the protein was labeled, that is the red channel (594 nm) shows SIM3-CNB containing 10% SIM3-CNB labeled with Alexa594 and the pink channel (647 nm) shows SUM3-DNB containing 10% SUM3-DNB labeled with Alexa647. Scale bar, 10 µm. (C) TIRF images of SJSA-1 cells 20 minutes after treatments with different protein pairs. (D)Statistical analysis of the condensate area formed in C. (SIM3-CNB + SUM3-DNB and SIM3 + SUM3-DNB,n = 21; SIM3-CNB + SUM3, n = 1; SIM3 + SUM3, n = 1). (E) FRAP analysis of a droplet formed by treatment of SJSA-1 cells with SUM3-DNB + SIM3-CNB. Scale bar, 2 µm. (F)TIRF images of SJSA-1 cells 20 minutes after treatments with SIM3-CNB + SUM3-DNB under different concentrations. The green channel shows (647 nm) SUM3-DNB (Alexa Fluor 647) and the red channel (594 nm) shows SIM3-CNB (Alexa Fluor 594). (G-H) Statistical analysis of the condensate number and condensate area formed under different treatment. The total condensate number and condensate area from cell number as (2 nM, n = 107, 5 nM, n = 226, 10 nM, n = 127, 20 nM, n = 137, 50 nM, n = 222, 100 nM, n = 258). (I) Apoptosis of SJSA-1 cells 4 hours after different treatments. n = 4. (J) Representative images of cells at 3 hours after treatment, Protein concentration 100 nM. The dotted line in panel G and H means the zero in Y-axis. (D, G, H) Only the condensate contains both signals of Alexa647 and Alexa594 were counted. (D, G–I) P values were evaluated by Student’s t test (two-tailed). Data was presented as mean ± SEM. The protein concentrations in (B, C, E, G) were 100 nM. (C, F) Scale bar, 1 µm.

Source data

Extended Data Fig. 7 Different outputs of the AND-gate system with different cell lines (Related to Fig. 4).

(A) Western blot assay of activation of the DR5 pathway. DR5 signaling is only activated upon treatment with both proteins, SIM3-CNB and SUM3-DNB. Cells were treated with different combinations of proteins for 4 hours. The protein concentrations were 50 nM. (B) Western blotting assay of the total expression levels of DR5 and CXCR4 in HEK293 cells and U2OS cells. (C) Cell survival analysis of SY5Y cells under different treatments. The protein concentrations were 50 nM. Time-lapse imaging with an interval of 0.5 hours was performed with IncuCyte ZOOM. n = 4. U2OS (D) and HEK293cells (E) were treated with different combinations of proteins for 4 hours. n = 4. (F) Cell survival analysis of Colo-205 cells under different treatments. The protein concentrations were 50 nM. Time-lapse imaging was performed with IncuCyte ZOOM. Representative images at 1.5 hours after treatment are shown. (G) Colo-205 cells were treated with different combinations of proteins for 4 hours. The apoptosis ratios are shown. n = 4. (H) Apoptosis ratios of HL-60 and OCI-AML3 cells 4 hours after different treatments. n = 4. (D, E, G, H) P values were evaluated by Student’s t test (two-tailed). (C–E, G, H) Data was presented as mean ± SEM.

Source data

Extended Data Fig. 8 PRM5-CNB and SH35-DNB undergo phase separation and induce apoptosis (Related to Fig. 5).

(A) In vitro analysis of the protein properties. Puncta did not form with only one protein. (B) The blue channel shows (the signals in 405 nm) ECD of DR5(Alexa405) is recruited into condensates formed by PRM5-CNB and SH35-DNB. The red channel (594 nm) shows PRM5-CNB contained 10% PRM5-CNB labeled with Alexa594 and the green channel (647 nm) shows SH35-DNB contained 10% SH35-DNB labeled with Alexa647. Scale bar, 10 µm. (C) Time-lapse imaging of droplet fusion as in C (20 µM), Scale bar, 1 µm. (D) SJSA-1 cells were treated with PRM5-CNB (Alexa594, red) and SH35-DNB (Alexa647, green). Images were taken after incubation for 5 minutes. The final protein concentrations were 100 nM. Scale bar, 10 µm. (E) Flow cytometry analysis of apoptosis of SJSA-1 cells. SJSA-1 cells were treated with PRM5-CNB, SH35-DNB or PRM5-CNB + SH35-DNB. The final protein concentrations were 100 nM. After incubation for 4 hours, the apoptosis ratios were analyzed with PI/Annexin V-FITC through flow cytometry. (F) Summary of the apoptosis ratios of SJSA-1 cells after treatment with the specified protein combinations and concentrations. n = 4. P values were evaluated by Student’s t test (two-tailed). Data was presented as mean ± SEM.

Source data

Extended Data Fig. 9 Extended information about the Orthogonal system (Related to Fig. 5).

Time-lapse imaging of puncta formation on cell membrane after separately adding SIM6-mEGF + SUM5 and SH35-DNB + PRM5. The green channel shows (the signals in 647 nm) SH35-DNB (Alexa Fluor 647) + PRM5 and the red channel (the signals in 594 nm) shows SIM6-mEGF (Alexa Fluor 594) + SUM5.The protein concentration was 150 nM, scar bar 10 µm.

Supplementary information

Supplementary Information

Supplementary Figure 1, Supplementary Tables 1 to 2, Supplementary Videos 1 to 5.

Reporting Summary

Unmodified gels for Figs. 1–5 and Extended Data Figs. 1–9.

Supplementary Video 1

SJSA-1 cells were treated with SIM6-CNB(Alexa594-red) and SUM5-DNB(Alexa647-green). After adding protein, time-lapse imaging was performed immediately with SIM/STOM-TRIF microscope. A representative movie after treatment is shown. The protein concentrations were 100 nM. Scale bar, 1 µm.

Supplementary Video 2

Cell survival analysis of SJSA-1 cells under different treatments. The protein concentrations were 100 nM. Time-lapse imaging with an interval of 0.5 hours was performed with IncuCyte ZOOM. Representative movies after treatment are shown.

Supplementary Video 3

SJSA-1 cells were treated with SIM3-CNB(Alexa594-red) and SUM3-DNB(Alexa647-green). After adding protein, time-lapse imaging was performed immediately with SIM/STOM-TRIF microscope. A representative movie after treatment is shown. The protein concentrations were 100 nM. Scale bar, 1 µm.

Supplementary Video 4

Cell survival analysis of SJSA-1 cells under different treatments. The protein concentrations were 100 nM. Time-lapse imaging with an interval of 0.5 hours was performed with IncuCyte ZOOM. Representative movies after treatment are shown.

Supplementary Video 5

Cell survival analysis of Colo-205 cells under different treatments. The protein concentrations were 100 nM. Time-lapse imaging with an interval of 1.5 hours was performed with IncuCyte ZOOM. Representative movies after treatment are shown.

Source data

Source Data Fig. 1

Statistical Source Data.

Source Data Fig. 2

Statistical Source Data.

Source Data Fig. 3

Statistical Source Data.

Source Data Fig. 4

Statistical Source Data.

Source Data Fig. 5

Statistical Source Data.

Source Data Extended Data Fig. 1

Statistical Source Data.

Source Data Extended Data Fig. 1

Western Blots.

Source Data Extended Data Fig. 2

Statistical Source Data.

Source Data Extended Data Fig. 2

Western Blots.

Source Data Extended Data Fig. 3

Statistical Source Data.

Source Data Extended Data Fig. 4

Statistical Source Data.

Source Data Extended Data Fig. 5

Statistical Source Data.

Source Data Extended Data Fig. 6

Statistical Source Data.

Source Data Extended Data Fig. 7

Statistical Source Data.

Source Data Extended Data Fig. 7

Western Blots.

Source Data Extended Data Fig. 8

Statistical Source Data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, R., Li, T., Lu, G. et al. Programming cell-surface signaling by phase-separation-controlled compartmentalization. Nat Chem Biol 18, 1351–1360 (2022). https://doi.org/10.1038/s41589-022-01192-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-022-01192-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing