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Designer membraneless organelles sequester native factors for control of cell behavior

Nature Chemical Biology volume 17, pages 998–1007 (2021)Cite this article

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Abstract

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

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Fig. 1: Robust cargo recruitment to synthetic condensates via protein–protein interaction domains.
Fig. 2: Control of cellular behavior through targeted insulation of native enzymes in synthetic organelles.
Fig. 3: Control of cell proliferation by induced target sequestration.
Fig. 4: Optical and thermal client release for reversible cell cycle control.
Fig. 5: CRISPR-tagged endogenous clients enrich within synthetic condensates expressed in mammalian cells.

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Data availability

All data supporting the findings of this study are included in the published article and its supplementary information files. Original data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Kumar for sharing yeast strains, the E. Bi lab for yeast plasmids and technical support, B. Xia and H. Ahmed for cloning, H. Ramage for U2OS cell lines, the M. Shapiro lab for TlpA plasmids, the D. Hammer lab for critical reading of the manuscript and A. Stout and the Penn CDB Microscopy Core for imaging and support. This cellular-engineering study was supported by a National Institute of Biomedical Imaging and Bioengineering R01 grant EB028320 (M.C.G.). Biochemical characterization of disordered proteins was partly funded by a National Science Foundation (NSF) iSuperseed grant DMR1720530 (M.C.G.). Synthesis of optochemical dimerizers was supported by NSF grant CHE-1404836 (A.D.). Conceptual development of condensates as decision hubs and investigator salary support was supported, in part, by a Department of Energy BES Biomolecular Materials grant DE-SC0007063 (M.C.G.).

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA

    Mikael V. Garabedian, Jorge B. Dabdoub, Michelle Tong, Reese M. Caldwell & Matthew C. Good

  2. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Wentao Wang, William Benman & Matthew C. Good

  3. Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ, USA

    Benjamin S. Schuster

  4. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Alexander Deiters

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Contributions

M.V.G., J.B.D., W.W., R.M.C., W.B. and M.C.G. conceptualized the project and designed the experiments. J.B.D., M.T. and W.B. performed initial characterization of scaffold valency on condensate formation and client recruitment in yeast. M.V.G. performed cloning, strain generation and imaging for yeast experiments throughout the article. W.W. generated mammalian knock-in cell lines, imaged them and characterized client relocalization. B.S.S. contributed imaging data from yeast experiments included in Extended Data Fig. 1. A.D. contributed synthesized dimerizers. M.V.G., W.W. and M.C.G. analyzed data. M.V.G. and M.C.G. wrote the manuscript.

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Correspondence to Matthew C. Good.

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Peer review information Nature Chemical Biology thanks Ulrich Krauss 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 Properties of in vivo synthetic condensates.

a, Temperature dependence of condensate assembly as a function of scaffold RGG domain valency. Representative images of yeast cells expressing galactose induced GFP tagged scaffold with 1, 2, or 3 RGG domains at different temperatures. b, Heat map: quantitation of turbidity data of purified proteins from Schuster et al., 2018. c, Heat map: number of condensates per cell as a function of temperature and RGG domain valency. d, Fluorescence recovery after photobleaching (FRAP) of condensates formed by (RGG)3 scaffold; n = 10 condensates. Shaded area, 95% CI. e, Fluorescence loss in photobleaching (FLIP) of condensates formed by (RGG)3 scaffold. n = 13 cells. f, Steady state cytoplasmic scaffold concentration outside of condensates (Ccyto) as a function of cellular concentration (Ccell) for 30 cells per scaffold type. Dashed line, slope of 1. g, Average enrichment of scaffold protein in condensates for SZ1-(RGG)3 or TsCC(A)-(RGG)3. n = 164 and 97 condensates respectively. Error bars, s.d. h, Representative images of exogenously expressed mScarlet-SZ2 and mScarlet-TsCC(B) diffusely distributed in cytosol in the absence of condensates. i, Representative images of cells expressing FRB-(RGG)3 scaffold and mScarlet-FKBP as a client. The client is diffuse in the cytosol before the addition of Rap and concentrated in condensates after Rap addition. j, Quantitation of the fraction of client protein as in i localized to condensates over time after Rap addition. n = 15 cells. Shaded area 95% CI.

Extended Data Fig. 2 Condensate expression relocalizes tagged native clients and regulates cell growth.

a, Representative images of tagged, natively expressed Cdc24 show its cortical localization. b, Representative images of tagged natively expressed Cdc5 show its punctate localization to spindle pole body. c, Images of the same Cdc24-mScarlet-TsCC(B) cell before and after induced expression of TsCC(A)-(RGG)3 scaffold for 6 hr, show loss of cortical Cdc24 signal and partitioning to synthetic condensate. d, Client recruitment to condensates specifically depends on CC tag interaction; Cdc24 does not interact with (RGG)3 condensates that lack the interaction tag. e, Left, scheme: cortical Cdc24 is relocalized from cortex to synthetic condensates after induction of scaffold. Right, Average cortical Cdc24- mScarlet-TsCC(B) signal before and after TsCC(A)-(RGG)3 scaffold expression (6 hr). n = 20 cells before and after hours of galactose induction. Significance calculated by unpaired, two-tailed, t-test (****, p < 0.0001). f, Kinetics of loss of cortical Cdc24-mCherry-TsCC(B) signal (red) concomitant with cellular accumulation of expressed TsCC(A)-(RGG)3 scaffold (green) upon induction with galactose for 20 cells over 6 hours. Shaded area, s.d. g, Cell proliferation: measurements of cell density (OD600) over time for indicated Cdc24 strains in liquid media containing galactose. h, Growth assay for Cdc24 strains: five-fold serial dilution of indicated strains grown on solid- media containing glucose or galactose. i, Average cell area of mother cells only increases upon TsCC(A)-(RGG)3 expression in Cdc24-mScarlet-TsCC(B) cells, consistent with cell cycle arrest in G1. j, Growth assay for Cdc5 strains: five-fold serial dilution of indicated strains grown on solid-media containing glucose or galactose. In all cases, growth defect depends on presence of tagged client and expression of scaffold to form condensates. Phenotype is not observed with only native client tagging or only scaffold expression.

Source data

Extended Data Fig. 3 Reversible control of cell proliferation-arrest state.

a, Representative images of Cdc24-mScarlet-TsCC(B) cells expressing TsCC(A)-(RGG)3 scaffold at the indicated temperatures for 14 hours. Thermally responsive coiled-coil pair dissociate upon heating to 37 or 42 °C, releasing client to promote cell polarity and reversing the cell cycle arrest associated with Cdc24 sequestration to condensates. b, Representative images of Cdc24-mScarlet-TsCC(B) in the presence of TsCC(A)-PhoCl2f-(RGG)3 before and after illumination with 405 nm light. c, Schematic of client release strategy: Cdc24 is tagged with PhoCl-TsCC(B). 405 nm light results in PhoCl cleavage and client release. d, Percentage of cells expressing Cdc24-mScarlet-TsCC(B) arrested (unbudded cells) over time after scaffold induction +/− illumination. n = 4048 cells in total pooled from three trials. e, Prediction: cycling of cell state between budded-arrested-budded-arrested. f, Representative images of cells at the indicated time points. Wildtype levels of budding at time 0 h. Cells incubated in galactose at 25 °C from 0-6 h timepoints to induce condensate formation, blocking budding, then heated from 6 h to 8 h timepoints, promoting polarization and budding and cooled back to 25 °C and arrested by 12 h.

Extended Data Fig. 4 Sequestration to synthetic condensates in mammalian cells.

a, Schematic of CRISPR tagging approach to endogenous loci in mammalian cells and expression of the scaffold by a CMV promoter. b, PCR validation of CRISPR tagging in mammalian cell lines. Only tagged strains show a PCR product of the expected size as indicated. c, Representative images of tagged ERK1 robustly partitions to synthetic condensates formed by expression of TsCC(A)-(RGG)3 scaffold. d, Representative images of tagged Par6 localized to the cell cortex in the absence of scaffold expression (left) and to condensate structures when scaffold is expressed (right). e, Quantitation of cortical Par6-mCherry-TsCC(B) in the absence and presence of condensates with cognate coiled coil. n = 10 cells for each condition. Error bars, s.d. Significance calculated by unpaired, two-tailed t-test. (**, p < 0.01).

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Video 1. Rapamaycin-induced client recruitment to preformed condensates in cells; green, FRB–(RGG)3–GFP-tagged scaffold; red, exogenous client, mScarlet–TsCC(B).

Supplementary Video 2

Video 2. Inducible expression of scaffold disrupts native cortical localization of Cdc24, sequestering it to newly formed synthetic condensates; green, TsCC(A)–(RGG)3–GFP-tagged scaffold; red, native client, Cdc24–mScarlet–TsCC(B).

Supplementary Video 3

Video 3. Induced condensate sequestration of endogenous client, Cdc24, following rapamycin addition; green, FRB–(RGG)3–GFP-tagged scaffold; red, native client, Cdc24–mScarlet–FKBP.

Source data

Source Data Fig. 3

Unprocessed images of spot assay plates.

Source Data Extended Data Fig. 2

Unprocessed images of spot assay plates.

Source Data Extended Data Fig. 4

Unprocessed gel image.

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Garabedian, M.V., Wang, W., Dabdoub, J.B. et al. Designer membraneless organelles sequester native factors for control of cell behavior. Nat Chem Biol 17, 998–1007 (2021). https://doi.org/10.1038/s41589-021-00840-4

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