Abstract
Protein self-organization is a hallmark of biological systems. Although the physicochemical principles governing protein–protein interactions have long been known, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a new strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit a messenger RNA to the designed micron-scale structures.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
We provide single-cell measurements of YFP and RFP concentrations for all phase diagrams in two Supplementary Excel tables. Other data are available from the authors upon request. Source Data are provided with this paper.
Code availability
Code and custom scripts used in this work are available from the authors upon request. We used the open source package oxDNA (v.2.4) to run the sedimentation simulations.
References
Hyman, A. A., Weber, C. A. & Julicher, F. Liquid–liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014).
Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).
Holehouse, A. S. & Pappu, R. V. Functional implications of intracellular phase transitions. Biochemistry 57, 2415–2423 (2018).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Tatomer, D. C. et al. Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis. J. Cell Biol. 213, 557–570 (2016).
Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Cai, J., Townsend, J. P., Dodson, T. C., Heiney, P. A. & Sweeney, A. M. Eye patches: protein assembly of index-gradient squid lenses. Science 357, 564–569 (2017).
Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N. & Levy, E. D. Proteins evolve on the edge of supramolecular self-assembly. Nature 548, 244–247 (2017).
Murakami, T. et al. ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function. Neuron 88, 678–690 (2015).
Patel, A. et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).
Peskett, T. R. et al. A liquid to solid phase transition underlying pathological huntingtin exon1 aggregation. Mol. Cell 70, 588–601 (2018).
Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
Bracha, D. et al. Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds. Cell 175, 1467–1480 (2018).
Flory, P. J. Principles of Polymer Chemistry (Cornell Univ. Press, 1953).
Smallenburg, F., Leibler, L. & Sciortino, F. Patchy particle model for vitrimers. Phys. Rev. Lett. 111, 188002 (2013).
Bianchi, E., Largo, J., Tartaglia, P., Zaccarelli, E. & Sciortino, F. Phase diagram of patchy colloids: towards empty liquids. Phys. Rev. Lett. 97, 168301 (2006).
Falkenberg, C. V., Blinov, M. L. & Loew, L. M. Pleomorphic ensembles: formation of large clusters composed of weakly interacting multivalent molecules. Biophys. J. 105, 2451–2460 (2013).
Jacobs, W. M. & Frenkel, D. Phase transitions in biological systems with many components. Biophys. J. 112, 683–691 (2017).
Sanders, D. W. et al. Competing protein-RNA interaction networks control multiphase intracellular organization. Cell 181, 306–324 (2020).
Li, W. et al. Dual recognition and the role of specificity-determining residues in colicin E9 DNase–immunity protein interactions. Biochemistry 37, 11771–11779 (1998).
Buxbaum, A. R., Haimovich, G. & Singer, R. H. In the right place at the right time: visualizing and understanding mRNA localization. Nat. Rev. Mol. Cell Biol. 16, 95–109 (2015).
Isaacs, W. B. & Fulton, A. B. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc. Natl Acad. Sci. USA 84, 6174–6178 (1987).
Shiber, A. et al. Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling. Nature 561, 268–272 (2018).
Natan, E. et al. Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins. Nat. Struct. Mol. Biol. 25, 279–288 (2018).
Kramer, G., Shiber, A. & Bukau, B. Mechanisms of cotranslational maturation of newly synthesized proteins. Annu. Rev. Biochem. 88, 337–364 (2019).
Haim-Vilmovsky, L. & Gerst, J. E. m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast. Nat. Protoc. 4, 1274–1284 (2009).
Lui, J. et al. Granules harboring translationally active mRNAs provide a platform for P-body formation following stress. Cell Rep. 9, 944–954 (2014).
Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets. Cell 168, 159–171 (2017).
Dignon, G. L., Zheng, W., Best, R. B., Kim, Y. C. & Mittal, J. Relation between single-molecule properties and phase behavior of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 115, 9929–9934 (2018).
Dignon, G. L., Zheng, W. & Mittal, J. Simulation methods for liquid–liquid phase separation of disordered proteins. Curr. Opin. Chem. Eng. 23, 92–98 (2019).
Yang, P. et al. G3BP1 Is a tunable switch that triggers phase separation to assemble stress granules. Cell 181, 325–345 (2020).
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. PNAS 112, 7189–7194 (2015).
Mackenzie, I. R. et al. TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics. Neuron 95, 808–816 (2017).
White, M. R. et al. C9orf72 Poly(PR) dipeptide repeats disturb biomolecular phase separation and disrupt nucleolar function. Mol. Cell 74, 713–728 (2019).
Banerjee, P. R., Milin, A. N., Moosa, M. M., Onuchic, P. L. & Deniz, A. A. Reentrant phase transition drives dynamic substructure formation in ribonucleoprotein droplets. Angew. Chem. Int. Ed. Engl. 56, 11354–11359 (2017).
Duncan, C. D. S. & Mata, J. Widespread cotranslational formation of protein complexes. PLoS Genet. 7, e1002398 (2011).
Shieh, Y.-W. et al. Operon structure and cotranslational subunit association direct protein assembly in bacteria. Science 350, 678–680 (2015).
Langdon, E. M. & Gladfelter, A. S. A new lens for RNA localization: liquid–liquid phase separation. Annu. Rev. Microbiol. 72, 255–271 (2018).
Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).
Garcia-Seisdedos, H., Villegas, J. A. & Levy, E. D. Infinite assembly of folded proteins in evolution, disease, and engineering. Angew. Chem. Int. Ed. Engl. 58, 5514–5531 (2019).
Shen, H. et al. De novo design of self-assembling helical protein filaments. Science 362, 705–709 (2018).
Abe, S. et al. Crystal engineering of self-assembled porous protein materials in living cells. ACS Nano 11, 2410–2419 (2017).
Reinkemeier, C. D., Girona, G. E. & Lemke, E. A. Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science 363, eaaw2644 (2019).
Lee, M. J. et al. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm. Nat. Chem. Biol. 14, 142–147 (2018).
Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349. (2018).
Chavent, M. et al. How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins. Nat. Commun. 9, 2846 (2018).
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018).
Choi, J.-M., Dar, F. & Pappu, R. V. LASSI: a lattice model for simulating phase transitions of multivalent proteins. PLoS Comput. Biol. 15, e1007028 (2019).
Panasenko, O. O. et al. Co-translational assembly of proteasome subunits in NOT1-containing assemblysomes. Nat. Struct. Mol. Biol. 26, 110–120 (2019).
Mumberg, D., Müller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995).
Klock, H. E. & Lesley, S. A. The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol. Biol. 498, 91–103 (2009).
Voth, W. P., Jiang, Y. W. & Stillman, D. J. New ‘marker swap’ plasmids for converting selectable markers on budding yeast gene disruptions and plasmids. Yeast 20, 985–993 (2003).
Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).
Anand, R., Beach, A., Li, K. & Haber, J. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature 544, 377–380 (2017).
Liu, H. et al. CRISPR–ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation. Bioinformatics 31, 3676–3678 (2015).
Cohen, Y. & Schuldiner, M. Advanced methods for high-throughput microscopy screening of genetically modified yeast libraries. Methods Mol. Biol. 781, 127–159 (2011).
Matalon, O., Steinberg, A., Sass, E., Hausser, J. & Levy, E. D. Reprogramming protein abundance fluctuations in single cells by degradation. Preprint at bioRxiv https://doi.org/10.1101/260695 (2018).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Nandi, S. K., Heidenreich, M., Levy, E. D. & Safran, S. A. Interacting multivalent molecules: affinity and valence impact the extent and symmetry of phase separation. Preprint at https://arxiv.org/abs/1910.11193 (2019).
Acknowledgements
We thank J. Gerst and R. R. Nair (Weizmann Institute) for sharing plasmids of the MS2 system, S. Schwartz (Weizmann Institute) for the bRA89 plasmid, F. Sciortino and H. Hofmann for helpful discussions and suggestions and H. Greenblatt for help with computer systems. E.D.L. acknowledges support by the Israel Science Foundation (no. 1452/18), by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 819318), by the HFSP Career Development Award (award no. CDA00077/2015), by a research grant from A.-M. Boucher and by research grants from the Estelle Funk Foundation, the Estate of Fannie Sherr, the Estate of Albert Delighter, the Merle S. Cahn Foundation, Mrs. Mildred S. Gosden, the Estate of Elizabeth Wachsman and the Arnold Bortman Family Foundation. E.D.L. is an incumbent of the Recanati Career Development Chair of Cancer Research. L.R. acknowledges support from the European Commission (Marie Skłodowska-Curie Fellowship, no. 702298-DELTAS). S.A.S. thanks the BSF and the ISF program and acknowledges the historical generosity of the Perlmann family foundation. S.K.N. acknowledges support from the Koshland foundation and Department of Atomic Energy (DAE), India. E.L. and L.R. thank the Vienna Scientific Cluster (VSC) for computing time.
Author information
Authors and Affiliations
Contributions
M.H., J.M.G. and E.D.L. designed the research and synthetic protein system. M.H. and J.M.G. performed the experiments with help from Y.N. E.L., L.R. and J.K.P.D developed the theoretical framework for modeling the system based on patchy particles. S.K.N. and S.A.S. developed the theoretical framework for modeling the system based on a lattice model. A.S. wrote the image analysis scripts. E.S. carried out electron microscopy experiments. M.H. and E.D.L. wrote the manuscript with input from all authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
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 The components do not form condensates when expressed individually.
Haploid cells expressing only one of the building blocks show a homogenous distribution of fluorescence throughout the cytoplasm. The left-most image shows cells expressing the dimer component lacking the Im2 domain. The next images show cells expressing the variants of the dimer component in the absence of the tetramer component. The right-most image shows cells expressing the tetramer component in the absence of the dimer component. This result was replicated three times.
Extended Data Fig. 2 The synthetic condensates are not membrane-bound.
a, Transmission electron microscopy (TEM) micrograph of fixed and sectioned yeast shows a condensate formed by our minimal system, in the cytoplasm. b, The yellow arrow points to one of several 10 nm gold-labeled anti-GFP antibodies, confirming the identity of the designed compartments. White arrows highlight the lack of membrane surrounding the compartment. c, Scanning electron microscopy micrograph of cells frozen at high-pressure and cryo-fractured reveals the mosaic of amorphic cytoplasm. The region outlined by white carets exhibits a distinct ultrastructure d, Increased magnification of a suspected condensate within the cytoplasm, outlined with white carets. This ultrastructure has no visible membrane. Scale bar 1 µm. We did not carry independent biological replicates of these electron microscopy experiments.
Extended Data Fig. 3 Impact of affinity on the phase diagram of the dimer-tetramer system.
a, We used a lattice model (Supplementary Note, Section 1) of the dimer-tetramer system. In the square lattice, concentration is measured by fractional occupancy of edges and vertices by dimers and tetramers respectively. We calculated the binodal of this system in the plane corresponding to the fractional occupancy of dimer (x-axis) and tetramer (y-axis). Affinity increases in panels from left to right, where μ is the binding energy in units of kT of a linker and one arm of the tetravalent molecule. Higher affinity (larger μ) increases the fraction of the phase-separated region. b, We used mean-field theoretical calculations of patchy particles matching the geometry of the proteins. The binodal is calculated in the plane corresponding to the concentration of dimers (x-axis) and tetramers (y-axis). Affinity (which is linked to the energy and entropy associated with the formation of a bond, see Supplementary Note, Section 1) increases from left to right.
Extended Data Fig. 4 Simulations recapitulate the kinetic trapping effect observed experimentally.
a, Sedimentation molecular dynamics simulation of patchy particles. Several simulations were conducted at equilibrium or out-of-equilibrium while sampling different concentrations of dimer and tetramer. The protein osmotic pressure as a function of density was inferred from each simulation and used to evaluate the phase boundaries. b, The phase diagram of the patchy mixture computed with equilibrium and non-equilibrium simulations (squares and circles, respectively).
Extended Data Fig. 5 In vivo phase diagrams and fluorescence recovery profiles observed with different affinities.
a, In vivo phase diagrams observed for five affinities investigated initially. Concentrations correspond to those of the binding sites (not of the dimer and tetramer complexes). The red line highlights the diagonal, where the concentrations of binding sites of dimer and tetramers are equal. The grey dotted lines show the lower limit of concentrations that can be reliably estimated. b, Fluorescence recovery profiles of photobleached condensates for different interaction affinities between the components. Grey lines show individual experiments, the red line corresponds to the mean recovery and the transparent red area indicates the standard error. The mean recovery after 25 seconds and associated standard error are given for each affinity.
Extended Data Fig. 6 Replicating the measurement of in vivo phase diagrams with four additional affinities.
Phase diagrams measured for nine affinities. Five affinities come from replicating experiments shown in Extended Data Fig. 5, and four are new. Concentrations correspond to those of the binding sites (not of the dimer and tetramer complexes). The red line highlights the diagonal, where the concentrations of binding sites of dimer and tetramers are equal. The grey dotted lines show the lower limit of concentrations that can be reliably estimated. Affinities and mutations are indicated above. The N34V, R38T, double N34V/R38T and triple D33L/N34V/R38T mutants were added later to further investigate the out-of-equilibrium effect. The same number of randomly selected cells were plotted in all panels (n=4000) to allow comparing the density of points across plots.
Extended Data Fig. 7 The mRNA coding for the dimer is released from condensates within minutes after the addition of puromycin.
Cells were treated with a final concentration of 10 mM puromycin and mRNA release from the condensate was followed by time-lapse microscopy.
Supplementary information
Supplementary Information
Supplementary Tables 1–3, Figs. 1–5 and Note.
Supplementary Video 1
Synthetic condensates expressed in yeast cells. New synthetic condensates appear in budding daughter cells and their size grows over time. The video is representative of at least three independent experiments.
Supplementary Video 2
FRAP on condensates. Condensates involving high- (left) and low- (right) affinity binding domains show slower (left) and faster (right) recovery after photobleaching. The video is representative of at least 13 independent experiments.
Supplementary Video 3
Localization of dimer mRNA in condensates. mRNAs coding for the dimer building block localize in condensates in yeast cells. The video is representative of at least three independent experiments.
Supplementary Video 4
Localization of GB1 mRNA. mRNAs coding for GB1, a protein that does not bind condensates, colocalize with condensates. The video is representative of at least three independent experiments.
Supplementary Video 5
C-terminal variant of the binding domain. mRNAs do not localize at condensates when the binding domain is encoded at the C terminus of the dimer. The video is representative of at least three independent experiments.
Supplementary Video 6
Puromycin. mRNAs detach from condensates in yeast cells treated with puromycin. The video is representative of at least three independent experiments.
Supplementary Video 7
Puromycin + cyclohexamide. mRNAs remain localized at condensates in yeast cells treated with puromycin and cycloheximide. The video is representative of at least three independent experiments.
Source data
Source Data Figs. 2g and 3a and Extended Data Fig. 5
Data for the phase diagrams.
Source Data Extended Data Fig. 6
Data for the phase diagrams.
Rights and permissions
About this article
Cite this article
Heidenreich, M., Georgeson, J.M., Locatelli, E. et al. Designer protein assemblies with tunable phase diagrams in living cells. Nat Chem Biol 16, 939–945 (2020). https://doi.org/10.1038/s41589-020-0576-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41589-020-0576-z
This article is cited by
-
Assembling membraneless organelles from de novo designed proteins
Nature Chemistry (2023)
-
Reduced ADP off-rate by the yeast CCT2 double mutation T394P/R510H which causes Leber congenital amaurosis in humans
Communications Biology (2023)
-
Dynamical control enables the formation of demixed biomolecular condensates
Nature Communications (2023)
-
Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
Nature Communications (2023)
-
Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG
Nature Communications (2022)
