Abstract
Cells compartmentalize their intracellular environment to orchestrate countless simultaneous biochemical processes. Many intracellular tasks rely on membrane-less organelles, multicomponent condensates that assemble by liquid–liquid phase separation. A decade of intensive research has provided a basic understanding of the biomolecular driving forces underlying the form and function of such organelles. Here we review the technologies enabling these developments, along with approaches to designing spatiotemporally actuated organelles based on multivalent low-affinity interactions. With these recent advances, it is now becoming possible both to modulate the properties of native condensates and to engineer entirely new structures, with the potential for widespread biomedical and biotechnological applications.
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
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
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).
Berry, J., Weber, S. C., Vaidya, N., Haataja, M. & Brangwynne, C. P. RNA transcription modulates phase transition-driven nuclear body assembly. Proc. Natl Acad. Sci. USA 112, E5237–E5245 (2015).
Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature 547, 241–245 (2017).
Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).
Shin, Y. et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell 175, 1481–1491.e13 (2018).
Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412–415 (2018).
Flory, P. J. Principles of Polymer Chemistry (Cornell University Press, 1953).
Rubinstein, M. & Colby, R. H. Polymer Physics (Oxford University Press, 2003).
Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
Brangwynne, C. P., Tompa, P. & Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 11, 899–904 (2015).
Putnam, A., Cassani, M., Smith, J. & Seydoux, G. A gel phase promotes condensation of liquid P granules in Caenorhabditis elegans embryos. Nat. Struct. Mol. Biol. 26, 220–226 (2019).
Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).
Rhee, H. W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).
Markmiller, S. et al. Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 172, 590–604.e13 (2018).
Ramanathan, M. et al. RNA-protein interaction detection in living cells. Nat. Methods 15, 207–212 (2018).
Huang, R., Han, M., Meng, L. & Chen, X. Transcriptome-wide discovery of coding and noncoding RNA-binding proteins. Proc. Natl Acad. Sci. USA 115, E3879–E3887 (2018).
Schmidtmann, E., Anton, T., Rombaut, P., Herzog, F. & Leonhardt, H. Determination of local chromatin composition by CasID. Nucleus 7, 476–484 (2016).
Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9-APEX-mediated proximity labeling. Nat. Methods 15, 437–439 (2018).
Gao, X. D. et al. C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2. Nat. Methods 15, 433–436 (2018).
Quinodoz, S. A. et al. Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell 174, 744–757.e24 (2018).
Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
Wang, J. T. et al. Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans. eLife 3, e04591 (2014).
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).
West, J. A. et al. Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization. J. Cell Biol. 214, 817–830 (2016).
Fei, J. et al. Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution. J. Cell. Sci. 130, 4180–4192 (2017).
Asano, S., Engel, B. D. & Baumeister, W. In situ cryo-electron tomography: a post-reductionist approach to structural biology. J. Mol. Biol. 428, 332–343 (2016). 2 Pt A.
Freeman Rosenzweig, E. S. et al. The eukaryotic CO2-concentrating organelle is liquid-like and exhibits dynamic reorganization. Cell 171, 148–162.e19 (2017).
Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349.e20 (2018).
Guo, Q. et al. In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment. Cell 172, 696–705.e12 (2018).
Ishikawa-Ankerhold, H. C., Ankerhold, R. & Drummen, G. P. C. Advanced fluorescence microscopy techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17, 4047–4132 (2012).
Mueller, F., Mazza, D., Stasevich, T. J. & McNally, J. G. FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know? Curr. Opin. Cell Biol. 22, 403–411 (2010).
Taylor, N. O., Wei, M., Stone, H. A. & Brangwynne, C. P. Quantifying phase-separated protein condensates using fluorescence recovery after photobleaching. Biophys. J. 117, 1285–1300 (2019).
Weidtkamp-Peters, S. et al. Dynamics of component exchange at PML nuclear bodies. J. Cell Sci. 121, 2731–2743 (2008).
Magde, D., Elson, E. & Webb, W. W. Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705–708 (1972).
Wei, M.-T. et al. Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat. Chem. 9, 1118–1125 (2017).
Bracha, D. et al. Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds. Cell 175, 1467–1480.e13 (2018).
Pliss, A. et al. Cycles of protein condensation and discharge in nuclear organelles studied by fluorescence lifetime imaging. Nat. Commun. 10, 455 (2019).
Kaminski Schierle, G. S. et al. A FRET sensor for non-invasive imaging of amyloid formation in vivo. ChemPhysChem 12, 673–680 (2011).
Laine, R. F. et al. Fast fluorescence lifetime imaging reveals the aggregation processes of α-synuclein and polyglutamine in aging Caenorhabditis elegans. ACS Chem. Biol. 14, 1628–1636 (2019).
Gall, J. G., Bellini, M., Wu, Z. & Murphy, C. Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. Mol. Biol. Cell 10, 4385–4402 (1999).
Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).
Brangwynne, C. P., Mitchison, T. J. & Hyman, A. A. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl Acad. Sci. USA 108, 4334–4339 (2011).
Feric, M. & Brangwynne, C. P. A nuclear F-actin scaffold stabilizes ribonucleoprotein droplets against gravity in large cells. Nat. Cell Biol. 15, 1253–1259 (2013).
Eggers, J., Lister, J. R. & Stone, H. A. Coalescence of liquid drops. J. Fluid Mech. 401, 293–310 (1999).
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).
Caragine, C. M., Haley, S. C. & Zidovska, A. Surface fluctuations and coalescence of nucleolar droplets in the human cell nucleus. Phys. Rev. Lett. 121, 148101 (2018).
Hubstenberger, A., Noble, S. L., Cameron, C. & Evans, T. C. Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development. Dev. Cell 27, 161–173 (2013).
Patel, A. et al. ATP as a biological hydrotrope. Science 356, 753–756 (2017).
Wirtz, D. Particle-tracking microrheology of living cells: principles and applications. Annu. Rev. Biophys. 38, 301–326 (2009).
Mason, T. G. & Weitz, D. A. Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids. Phys. Rev. Lett. 74, 1250–1253 (1995).
Taylor, N. et al. Biophysical characterization of organelle-based RNA/protein liquid phases using microfluidics. Soft Matter 12, 9142–9150 (2016).
Jawerth, L. M. et al. Salt-dependent rheology and surface tension of protein condensates using optical traps. Phys. Rev. Lett. 121, 258101 (2018).
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).
Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452 (2003).
Cohen, G. B., Ren, R. & Baltimore, D. Modular binding domains in signal transduction proteins. Cell 80, 237–248 (1995).
Schwarz-Romond, T., Merrifield, C., Nichols, B. J. & Bienz, M. The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles. J. Cell Sci. 118, 5269–5277 (2005).
Burke, K. A., Janke, A. M., Rhine, C. L. & Fawzi, N. L. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II.
Monahan, Z. et al. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 36, 2951–2967 (2017).
Brady, J. P. et al. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc. Natl Acad. Sci. USA 114, E8194–E8203 (2017).
Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).
Wang, J. et al. A Molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699.e16 (2018).
Riback, J. A. et al. Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168, 1028–1040.e19 (2017).
Das, R. K. & Pappu, R. V. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl Acad. Sci. USA 110, 13392–13397 (2013).
Martin, E. W. & Mittag, T. Relationship of sequence and phase separation in protein low-complexity regions. Biochemistry 57, 2478–2487 (2018).
Simon, J. R., Carroll, N. J., Rubinstein, M., Chilkoti, A. & López, G. P. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nat. Chem. 9, 509–515 (2017).
Roberts, S., Dzuricky, M. & Chilkoti, A. Elastin-like polypeptides as models of intrinsically disordered proteins. FEBS Lett. 589, 2477–2486 (2015). 19 Pt A.
Urry, D. W. Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers †. J. Phys. Chem. B 101, 11007–11028 (1997).
Urry, D. W. The change in Gibbs free energy for hydrophobic association: Derivation and evaluation by means of inverse temperature transitions. Chem. Phys. Lett. 399, 177–183 (2004).
Quiroz, F. G. & Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 14, 1164–1171 (2015).
Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).
Schuster, B. S. et al. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 9, 2985 (2018).
Nott, T. J., Craggs, T. D. & Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 8, 569–575 (2016).
Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets. Cell 168, 159–171.e14 (2017).
Más, P., Devlin, P. F., Panda, S. & Kay, S. A. Functional interaction of phytochrome B and cryptochrome 2. Nature 408, 207–211 (2000).
Zhang, P. et al. Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology. eLife 8, e39578 (2019).
Wei, M.-T., Chang, Y.-C., Shimobayashi, S. F., Shin, Y. & Brangwynne, C. P. Nucleated transcriptional condensates amplify gene expression. Preprint at https://www.biorxiv.org/content/10.1101/737387 (2019).
Liao, Y.-C. et al. RNA granules hitchhike on lysosomes for long-distance transport, using annexin A11 as a molecular tether. SSRN Electron. J. https://doi.org/10.2139/ssrn.3312723 (2019).
Mann, J. R. et al. RNA binding antagonizes neurotoxic phase transitions of TDP-43. Neuron 102, 321–338.e8 (2019).
Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).
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).
Dine, E., Gil, A. A., Uribe, G., Brangwynne, C. P. & Toettcher, J. E. Protein phase separation provides long-term memory of transient spatial stimuli. Cell Syst. 6, 655–663.e5 (2018).
Nakamura, H. et al. Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions. Nat. Mater. 17, 79–89 (2018).
Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).
Zwicker, D., Seyboldt, R., Weber, C. A., Hyman, A. A. & Jülicher, F. Growth and division of active droplets provides a model for protocells. Nat. Phys. 13, 408–413 (2017).
Strulson, C. A., Molden, R. C., Keating, C. D. & Bevilacqua, P. C. RNA catalysis through compartmentalization. Nat. Chem. 4, 941–946 (2012).
Drobot, B. et al. Compartmentalised RNA catalysis in membrane-free coacervate protocells. Nat. Commun. 9, 3643 (2018).
Wang, H. et al. CRISPR-mediated programmable 3D genome positioning and nuclear organization. Cell 175, 1405–1417.e14 (2018).
Rege, M. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Preprint at https://www.biorxiv.org/content/10.1101/349340 (2018).
Hammer, S. K. & Avalos, J. L. Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832 (2017).
Castellana, M. et al. Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat. Biotechnol. 32, 1011–1018 (2014).
An, S., Kumar, R., Sheets, E. D. & Benkovic, S. J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science 320, 103–106 (2008).
Zhao, E. M. et al. Light-based control of metabolic flux through assembly of synthetic organelles. Nat. Chem. Biol. 15, 589–597 (2019).
Woodruff, J. B. et al. The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin. Cell 169, 1066–1077.e10 (2017).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Reinkemeier, C. D., Girona, G. E. & Lemke, E. A. Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science 363, eaaw2644 (2019).
McDaniel, J. R., Callahan, D. J. & Chilkoti, A. Drug delivery to solid tumors by elastin-like polypeptides. Adv. Drug Deliv. Rev. 62, 1456–1467 (2010).
Mullard, A. Biomolecular condensates pique drug discovery curiosity. Nat. Rev. Drug Discov. https://doi.org/10.1038/d41573-019-00069-w (2019).
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).
Boke, E. et al. Amyloid-like self-assembly of a cellular compartment. Cell 166, 637–650 (2016).
Mateju, D. et al. An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function. EMBO J. 36, 1669–1687 (2017).
Jastrow, H. Dr. Jastrow’s Electron Microscopic Atlas http://www.drjastrow.de/WAI/EM/EMAtlas.html (accessed 23 October 2019).
Rubinstein, M. & Colby, R. H. Polymer Physics (Oxford University Press, 2003).
Berry, J., Brangwynne, C. P. & Haataja, M. Physical principles of intracellular organization via active and passive phase transitions. Rep. Prog. Phys. 81, 046601 (2018).
Acknowledgements
We thank members of the Brangwynne laboratory for comments on the manuscript. This work was supported by the Howard Hughes Medical Institute and by grants from the NIH 4D Nucleome Program (U01 DA040601), the Princeton Center for Complex Materials, and an NSF-supported MRSEC (DMR 1420541). D.B. acknowledges support through a Cross-Disciplinary Postdoctoral Fellowship from the Human Frontiers Science Program. M.T.W. is supported by the National Science Foundation Graduate Research Fellowship Program under grant no. DGE-1656466.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors have filed patents on technologies discussed, including the Corelet, CasDrop and optoDroplet systems.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
About this article
Cite this article
Bracha, D., Walls, M.T. & Brangwynne, C.P. Probing and engineering liquid-phase organelles. Nat Biotechnol 37, 1435–1445 (2019). https://doi.org/10.1038/s41587-019-0341-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41587-019-0341-6
This article is cited by
-
High-throughput and proteome-wide discovery of endogenous biomolecular condensates
Nature Chemistry (2024)
-
Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility
Nature Chemistry (2024)
-
An aberrant fused in sarcoma liquid droplet of amyotrophic lateral sclerosis pathological variant, R495X, accelerates liquid–solid phase transition
Scientific Reports (2024)
-
RNA structure promotes liquid-to-solid phase transition of short RNAs in neuronal dysfunction
Communications Biology (2024)
-
Dipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis
Nature Communications (2024)