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

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

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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.

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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.

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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.

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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

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  1. Ru Li
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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.

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

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

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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.

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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.

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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.

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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

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