# Biology:Biomolecular condensate

Formation and examples of membraneless organelles

Biomolecular condensates are a class of non-membrane bound organelles and organelle subdomains. As with other organelles, biomolecular condensates are specialized subunits of the cell. However, unlike many organelles, biomolecular condensate composition is not controlled by a bounding membrane. Instead they can form through a range of different processes, the most well-known of which is phase separation of proteins, RNA and other biopolymers into colloids, liquid crystals, solid crystals or aggregates.

The term 'colloid' was coined by Thomas Graham to describe the behaviour of certain biological macromolecules and inorganic molecules in 1861,[1] while the physics of phase separation was described by Josiah Willard Gibbs in his landmark paper titled On the Equilibrium of Heterogeneous Substances, published in parts between 1875 and 1878.[2]. Influenced by Willard Gibbs, important contributions were also made by Johannes Diderik van der Waals, who in 1890 published a treatise on the Theory of Binary Solutions.[3]

The concept of intracellular condensation as an organizing principle for the compartmentalization of living cells dates back to the end of the 19th century, beginning with William Bate Hardy and Edmund Beecher Wilson who described the cytoplasm (then called 'protoplasm') as a colloid.[4][5] Around the same time, Thomas Harrison Montgomery Jr. described the morphology of the nucleolus, an organelle within the nucleus, which has subsequently been shown to form through intracellular phase separation.[6] WB Hardy linked formation of biological colloids with phase separation in his study of globulins, stating that: "The globulin is dispersed in the solvent as particles which are the colloid particles and which are so large as to form an internal phase",[7] and further contributed to the basic physical description of oil-water phase separation.[8]

Colloidal phase separation as a driving force in cellular organisation appealed strongly to Stephane Leduc, who wrote in his influential 1911 book The Mechanism of Life: "Hence the study of life may be best begun by the study of those physico-chemical phenomena which result from the contact of two different liquids. Biology is thus but a branch of the physico-chemistry of liquids; it includes the study of electrolytic and colloidal solutions, and of the molecular forces brought into play by solution, osmosis, diffusion, cohesion, and crystallization." [9]

The primordial soup theory of the origin of life, proposed by Alexander Oparin in Russian in 1924 (published in English in 1936)[10] and by J.B.S. Haldane in 1929[11], suggested that life was preceded by the formation of what Haldane called a "hot dilute soup" of "colloidal organic substances", and which Oparin referred to as 'coacervates' (after de Jong [12]) - particles composed of two or more colloids which might be protein, lipid or nucleic acid. These ideas strongly influenced the subsequent work of Sidney W. Fox on proteinoid microspheres.

The notion of biological macromolecules as colloids remained dominant until the 1950s[13], when Frederick Sanger determined the amino acid sequence of Insulin[14][15] and Linus Pauling, Robert Corey and Herman Branson correctly proposed the alpha helix and beta sheet as the primary structural motifs in protein secondary structure[16], while Max Perutz analysed the 3D structure of Haemoglobin. These breakthroughs in protein structure determination led to a general focus of biologists on atomic-scale amino acid sequence- and 3D conformation-specific protein-protein interactions of a lock and key model type, usually between defined numbers of interacting subunits within a stoichiometric complex, although colloid chemistry and polymer physics continued unabated to characterise the non-stoichiometric interactions occurring during colloidal, liquid crystal and other phase behaviour of macromolecular polymers, particularly synthetic polymers developed for industrial applications.

Beginning in the 1970s, Tanaka & Benedek identified phase-separation behaviour of gamma-crystallin proteins from cataracts in solution,[17][18][19][20] which Benedek referred to as 'protein condensation'.[21]

In the 1980s and 1990s, Athene Donald's polymer physics lab extensively characterised phase transitions / phase separation of starch granules from plant cells, which behave as liquid crystals.[22][23][24][25][26][27][28][29]

Advances in confocal microscopy at the end of the 20th century identified proteins, RNA or lipids localising to many non-membrane bound cellular compartments within the cytoplasm or nucleus which were variously referred to as 'puncta', 'dots', 'granules', 'bodies', 'assemblies', 'paraspeckles', 'droplets', 'aggregates' or 'factories'. During this time period, the concept of phase separation was re-borrowed from colloidal chemistry & polymer physics and proposed to underlie both cytoplasmic and nuclear compartmentalization.[30][31][32]

Since 2008, further evidence for biomacromolecules undergoing intracellular phase transitions (phase separation) has been observed in many different contexts, both within cells and in reconstituted in vitro experiments.[33][34][35][36][37][38][39][40][41]

The newly coined term "biomolecular condensate"[42] refers to biological polymers (as opposed to synthetic polymers) that undergo self assembly via clustering to increase the local concentration of the assembling components, and is analogous to the physical definition of condensation.[43][42] In physics, condensation typically refers to a gas-liquid phase transition. In biology the term 'condensation' is used much more broadly and can also refer to liquid-liquid, liquid-gel, or liquid-solid phase separation to form colloidal suspensions or liquid crystals, as well as liquid-to-solid phase transitions such as DNA condensation during prophase of the cell cycle or protein condensation of crystallins in cataracts.[44] With this in mind, the term 'biomolecular condensates' was deliberately introduced to reflect this breadth (see below). Since biomolecular condensation generally involves oligomeric or polymeric interactions between an indefinite number of components, it is generally considered distinct from formation of smaller stoichiometric protein complexes with defined numbers of subunits, such as viral capsids or the proteasome - although both are examples of spontaneous self-assembly or self-organisation.

## Examples

Formation and examples of nuclear membraneless compartments
Stress granule dynamics

Many examples of biomolecular condensates have been characterized in the cytoplasm and the nucleus.

Cytoplasmic biomolecular condensates thought to arise by either liquid-liquid, liquid-gel or liquid-solid phase separation:

• Lewy bodies
• Stress granule
• P-body
• Germline P-granules
• Lipid droplets
• Starch granules
• Glycogen granules
• Corneal lens formation and cataracts
• Other cytoplasmic inclusions such as pigment granules or cytoplasmic crystals
• Misfolded protein aggregation such as amyloid fibrils or mutant Haemoglobin S (HbS) fibres in sickle cell disease
• Membrane protein, or membrane-associated protein, clustering at neurological synapses, cell-cell junctions, or other membrane domains.
• Other large multiprotein complexes or supramolecular assemblies in the Wnt signaling pathway.[45][46]

It can also be argued that cytoskeletal filaments form by a polymerisation process similar to phase separation, except ordered into filamentous networks instead of amorphous droplets or granules.

Nuclear biomolecular condensates:

Other nuclear structures including heterochromatin and DNA condensation in condensed mitosis chromosomes form by mechanisms similar to phase separation, so can also be classified as biomolecular condensates.

## Liquid-liquid phase separation

Biomolecular partitioning

The term biomolecular condensates was introduced in the context of intracellular assemblies as a convenient and non-exclusionary term to describe non-stoichiometric assemblies of biomolecules.[42] The choice of language here is specific and important. It has been proposed that many biomolecular condensates form through liquid-liquid phase separation (LLPS), liquid-gel phase separation or liquid-solid phase separation;[48] however, unequivocally demonstrating that a cellular body forms through liquid-liquid phase separation is challenging.[49][50][51][52] Similarly, while much attention has been paid to the formation of LLPS assemblies, different material states (liquid vs. gel vs. solid) are not always easy to distinguish in living cells.[53][54]

The term "biomolecular condensate" directly address both of these challenges by making no assumption regarding either the physical mechanism through which assembly is achieved, nor the material state of the resulting assembly. Consequently, cellular bodies that form through phase separation are a subset of biomolecular condensates, as are those where the physical origins of assembly are unknown. Historically, many cellular non-membrane bound compartments identified microscopically fall under the broad umbrella of biomolecular condensates.

Further, it appears that intrinsically disordered proteins might play a role in distinguishing between liquid phase states, though how they play this role remains unknown.[48] As of 2019, a light activated system had been developed to experimentally induce condensation.[48]

One of the first discovered examples of a highly dynamic liquid-like biomolecular condensate with a clear physiological function were the supramolecular complexes formed by components of the Wnt signaling pathway.[55][56] The Dishevelled (Dsh) protein undergoes clustering in the cytoplasm via its DIX domain, which mediates protein polymerisation, and is important for signal transduction. The Dsh protein functions both in planar polarity and Wnt signalling, where it recruits another supramolecular complex (the Axin complex) to Wnt receptors at the plasma membrane. The formation of these Dishevelled and Axin containing droplets is conserved across metazoans, including in Drosophila, Xenopus, and human cells.

Another example of liquid droplets in cells are the germline P granules in Caenorhabditis elegans.[48][50] These granules separate out from the cytoplasm and form droplets, as oil does from water. Both the granules and the surrounding cytoplasm are liquid in the sense that they flow in response to forces, and two of the granules can coalesce when they come in contact. When (some of) the molecules in the granules are studied (via fluorescence recovery after photobleaching), they are found to rapidly turnover in the droplets, meaning that molecules diffuse into and out of the granules, just as expected in a liquid droplet. The droplets can also grow to be many molecules across (micrometres)[50] Studies of droplets of the Caenorhabditis elegans protein LAF-1 in vitro[57] also show liquid-like behaviour, with an apparent viscosity $\displaystyle{ \eta \sim 10 }$Pa s. This is about a ten thousand times that of water at room temperature, but it is small enough to enable the LAF-1 droplets to flow like a liquid.

## References

1. "Liquid diffusion applied to analysis". Philosophical Transactions of the Royal Society 151: 183–224. December 1861. doi:10.1098/rstl.1861.0011.
2. Gibbs, J. W. (1961), Scientific Papers, Dover, New York
3. How Fluids Unmix: Discoveries by the School of Van der Waals and Kamerlingh Onnes.. Amsterdam. 2002.
4. "The Structure of Protoplasm". Science 10 (237): 33–45. July 1899. doi:10.1126/science.10.237.33. PMID 17829686. Bibcode1899Sci....10...33W.
5.
6. "Comparative cytological studies, with especial regard to the morphology of the nucleolus". Journal of Morphology 15 (1): 265–582. 1898. doi:10.1002/jmor.1050150204.
7. "Colloidal Solution. The Globulins.". Journal of Physiology 33 (4–5): 255–333. 1905. doi:10.1113/jphysiol.1905.sp001126. PMID 16992817.
8. "The tension of composite fluid surfaces and the mechanical stability of films of fluid.". Proceedings of the Royal Society A 86 (591): 610–635. 1912. doi:10.1098/rspa.1912.0053.
9. Leduc, Stephane (1911). "The Mechanism of Life".
10. Haldane, John B. S.. "The Origin of Life".
11. Bungenberg de Jong, H. G., and H. R. Kruyt (1929). "Coacervation (partial miscibility in colloid systems)". Proc Koninklijke Nederlandse Akademie Wetenschappen 32: 849—856
12. Jirgensons, Bruno (1958). "Organic Colloids". Elsevier.
13. Sanger, F. (1958), Nobel lecture: The chemistry of insulin, Nobelprize.org, retrieved 18 October 2010 . Sanger's Nobel lecture was also published in Science: Sanger 1959
14. Pauling, L; Corey, RB (1951). "Configurations of Polypeptide Chains With Favored Orientations Around Single Bonds: Two New Pleated Sheets". Proceedings of the National Academy of Sciences of the United States of America 37 (11): 729–40. doi:10.1073/pnas.37.11.729. PMID 16578412. Bibcode1951PNAS...37..729P.
15. "Observation of protein diffusivity in intact human and bovine lenses with application to cataract.". Investigative Ophthalmology & Visual Science 14 (6): 449–56. Jun 1975. PMID 1132941.
16. "Phase separation of a protein-water mixture in cold cataract in the young rat lens". Science 197 (4307): 1010–1012. Sep 1977. doi:10.1126/science.887936. PMID 887936.
17. "Cytoplasmic phase separation in formation of galactosemic cataract in lenses of young rats". Proceedings of the National Academy of Sciences of the United States of America 76 (9): 4414–4416. Sep 1979. doi:10.1073/pnas.76.9.4414. PMID 16592709.
18. "Binary-liquid phase separation of lens protein solutions". Proceedings of the National Academy of Sciences of the United States of America 88 (13): 5660–4. Jul 1991. doi:10.1073/pnas.88.13.5660. PMID 2062844.
19. "Cataract as a protein condensation disease: the Proctor Lecture". Investigative Ophthalmology & Visual Science 38 (10): 1911–21. September 1997. PMID 9331254.
20. Waigh, T.A.; Gidley, M.J.; Komanshek, B.U.; Donald, A.M. (2000). "The phase transformations in starch during gelatinisation: a liquid crystalline approach". Carbohydrate Research 328 (2). doi:10.1016/s0008-6215(00)00098-7.
21. Jenkins, P.J.; Donald, A.M. (1998). "Gelatinisation of starch: A combined SAXS/WAXS/DSC and SANS study". Carbohydrate Research 308 (1–2): 133. doi:10.1016/S0008-6215(98)00079-2.
22. Jenkins, P. J.; Donald, A.M. (1995). "The influence of amylose on starch granule structure". International Journal of Biological Macromolecules 17 (6): 315–21. doi:10.1016/0141-8130(96)81838-1. PMID 8789332.
23. Jenkins, P.J.; Cameron, R. E.; Donald, A.M. (1993). "A Universal Feature in the Structure of Starch Granules from Different Botanical Sources". Starch - Stärke 45 (12): 417. doi:10.1002/star.19930451202.
24. Donald, A.M.; Windle, A.H.; Brand, H.R. (1993). "Liquid Crystalline Polymers". Physics Today 46 (11): 87. doi:10.1063/1.2809100. Bibcode1993PhT....46k..87D.
25. Windle, A.H.; Donald, A.D. (1992). Liquid crystalline polymers. Cambridge, UK: Cambridge University Press. ISBN 978-0-521-30666-9.
26. Starch: structure and functionality. Cambridge, England: Royal Society of Chemistry. 1997. ISBN 978-0-85404-742-0.
27. The importance of polymer science for biological systems: University of York. Cambridge, England: Royal Society of Chemistry. March 2008. ISBN 978-0-85404-120-6.
28. "Phase separation in cytoplasm, due to macromolecular crowding, is the basis for microcompartmentation". FEBS Letters 361 (2–3): 135–9. March 1995. doi:10.1016/0014-5793(95)00159-7. PMID 7698310.
29. Microcompartmentation and Phase Separation in Cytoplasm. 192 (1 ed.). Academic Press. October 1999.
30. "Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation?". Theoretical Biology & Medical Modelling 4 (15): 15. April 2007. doi:10.1186/1742-4682-4-15. PMID 17430588.
31. "Protein phase behavior in aqueous solutions: crystallization, liquid-liquid phase separation, gels, and aggregates.". Biophysical Journal 94 (2): 570–83. Jan 2008. doi:10.1529/biophysj.107.116152. PMID 18160663.
32. "Germline P granules are liquid droplets that localize by controlled dissolution/condensation". Science 324 (5935): 1729–32. June 2009. doi:10.1126/science.1172046. PMID 19460965. Bibcode2009Sci...324.1729B.
33. "Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin". Nature 547 (7662): 236–240. July 2017. doi:10.1038/nature22822. PMID 28636604. Bibcode2017Natur.547..236L.
34. "Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles". Molecular Cell 57 (5): 936–947. March 2015. doi:10.1016/j.molcel.2015.01.013. PMID 25747659.
35. "A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation". Cell 162 (5): 1066–77. August 2015. doi:10.1016/j.cell.2015.07.047. PMID 26317470.
36. "Coexisting Liquid Phases Underlie Nucleolar Subcompartments". Cell 165 (7): 1686–1697. June 2016. doi:10.1016/j.cell.2016.04.047. PMID 27212236.
37. "Composition dependent phase separation underlies directional flux through the nucleolus" (in en). bioRxiv: 809210. 2019-10-22. doi:10.1101/809210.
38. "Phase transitions in the assembly of multivalent signalling proteins". Nature 483 (7389): 336–40. March 2012. doi:10.1038/nature10879. PMID 22398450. Bibcode2012Natur.483..336L.
39. "Coexisting Liquid Phases Underlie Nucleolar Subcompartments". Cell 165 (7): 1686–1697. June 2016. doi:10.1016/j.cell.2016.04.047. PMID 27212236.
40. "Biomolecular condensates: organizers of cellular biochemistry". Nature Reviews. Molecular Cell Biology 18 (5): 285–298. May 2017. doi:10.1038/nrm.2017.7. PMID 28225081.
41. "Controlling compartmentalization by non-membrane-bound organelles". Philosophical Transactions of the Royal Society B: Biological Sciences 373 (1747): 4666–4684. May 2018. doi:10.1098/rstb.2017.0193. PMID 29632271.
42. "Cataract as a protein condensation disease: the Proctor Lecture". Investigative Ophthalmology & Visual Science 38 (10): 1911–21. September 1997. PMID 9331254.
43. "Wnt/Beta-Catenin Signaling Regulation and a Role for Biomolecular Condensates". Developmental Cell 48 (4): 429–444. February 2019. doi:10.1016/j.devcel.2019.01.025. PMID 30782412.
44. "Multiprotein complexes governing Wnt signal transduction". Current Opinion in Cell Biology 51 (1): 42–49. April 2018. doi:10.1016/j.ceb.2017.10.008. PMID 29153704.
45. "Coexisting Liquid Phases Underlie Nucleolar Subcompartments". Cell 165 (7): 1686–1697. June 2016. doi:10.1016/j.cell.2016.04.047. PMID 27212236.
46. Tang, Lei (February 2019). "Optogenetic tools light up phase separation". Nature Methods 16 (2): 139. doi:10.1038/s41592-019-0310-5. PMID 30700901. (subscription required)
47. "Liquid-liquid phase separation in biology". Annual Review of Cell and Developmental Biology 30 (1): 39–58. 2014-10-11. doi:10.1146/annurev-cellbio-100913-013325. PMID 25288112.
48. "Germline P granules are liquid droplets that localize by controlled dissolution/condensation". Science 324 (5935): 1729–32. June 2009. doi:10.1126/science.1172046. PMID 19460965. Bibcode2009Sci...324.1729B.
49. "Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences". Genes & Development 33 (23–24): 1619–1634. December 2019. doi:10.1101/gad.331520.119. PMID 31594803.
50. "Phase Separation of Intrinsically Disordered Proteins". Methods in Enzymology (Elsevier) 611: 1–30. 2018. doi:10.1016/bs.mie.2018.09.035. ISBN 978-0-12-815649-0. PMID 30471685.
51. "Organization and Function of Non-dynamic Biomolecular Condensates". Trends in Biochemical Sciences 43 (2): 81–94. February 2018. doi:10.1016/j.tibs.2017.11.005. PMID 29258725.
52. "Protein Phase Separation: A New Phase in Cell Biology". Trends in Cell Biology 28 (6): 420–435. June 2018. doi:10.1016/j.tcb.2018.02.004. PMID 29602697.
53. "Wnt/Beta-Catenin Signaling Regulation and a Role for Biomolecular Condensates". Developmental Cell 48 (4): 429–444. February 2019. doi:10.1016/j.devcel.2019.01.025. PMID 30782412.
54. "Multiprotein complexes governing Wnt signal transduction". Current Opinion in Cell Biology 51 (1): 42–49. April 2018. doi:10.1016/j.ceb.2017.10.008. PMID 29153704.
55. "The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics". Proceedings of the National Academy of Sciences of the United States of America 112 (23): 7189–94. June 2015. doi:10.1073/pnas.1504822112. PMID 26015579. Bibcode2015PNAS..112.7189E.