Experimental models for dynamic compartmentalization of biomolecules in liquid organelles: Reversible formation and partitioning in aqueous biphasic systems.

Living cells contain numerous subcellular compartments, many of which lack membranous boundaries and are thought to occur due to liquid-liquid phase coexistence. This review will introduce these biological membraneless organelles and discuss simple experimental models based on liquid-liquid phase separation in polymer solutions. When more than one phase is present, solutes such as proteins or nucleic acids can be compartmentalized by partitioning into one of the phases. This could confer benefits to the cell such as enhanced reaction rates or sequestration of toxic molecules. Liquid-like compartments inside living cells are often dynamic, for example, appearing and disappearing in response to stimuli and/or at different points in the cell cycle. We will discuss mechanisms by which phase transitions can be induced in the laboratory and inside living cells, with special emphasis on regulating phase formation by phosphorylation state. This work is motivated by a desire to understand the physical and chemical mechanisms that underlie biological processes and to enable new nonbiological applications.

[1]  C. Keating,et al.  Phase separation as a possible means of nuclear compartmentalization. , 2014, International review of cell and molecular biology.

[2]  Damien Larivière,et al.  An inventory of the bacterial macromolecular components and their spatial organization. , 2011, FEMS microbiology reviews.

[3]  C. Brangwynne,et al.  Inverse Size Scaling of the Nucleolus by a Concentration-Dependent Phase Transition , 2015, Current Biology.

[4]  J. Schlenoff,et al.  Driving Forces for Oppositely Charged Polyion Association in Aqueous Solutions: Enthalpic, Entropic, but Not Electrostatic. , 2016, Journal of the American Chemical Society.

[5]  Jan C. M. van Hest,et al.  Multi-enzyme systems: bringing enzymes together in vitro , 2012 .

[6]  J. Gong,et al.  Catch and release of DNA in coacervate- dispersed gels , 2006 .

[7]  Christine D. Keating,et al.  Complete Budding and Asymmetric Division of Primitive Model Cells To Produce Daughter Vesicles with Different Interior and Membrane Compositions , 2011, Journal of the American Chemical Society.

[8]  Rajni Hatti-Kaul,et al.  Aqueous two-phase systems : methods and protocols , 2000 .

[9]  Juan A Asenjo,et al.  Aqueous two-phase systems for protein separation: a perspective. , 2011, Journal of chromatography. A.

[10]  J. Landsmeer,et al.  Diameter changes of gelatinized coacervate drops of the complex coacervate gelatine‐gum arabic resulting from a change in the pH of, or from the addition of neutral salts to the surrounding medium. I , 2010 .

[11]  Paul S. Russo,et al.  Phase Transitions in the Assembly of MultiValent Signaling Proteins , 2016 .

[12]  L. Johnson,et al.  Structural basis for control by phosphorylation. , 1997, Chemical reviews.

[13]  H. Bohidar,et al.  pH-induced coacervation in complexes of bovine serum albumin and cationic polyelectrolytes. , 2000, Biomacromolecules.

[14]  C. Keating,et al.  Interactions of Macromolecular Crowding Agents and Cosolutes with Small-Molecule Substrates: Effect on Horseradish Peroxidase Activity with Two Different Substrates , 2014, The journal of physical chemistry. B.

[15]  B. Zaslavsky,et al.  Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications , 1994 .

[16]  Christine D. Keating,et al.  Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation , 2014, Langmuir : the ACS journal of surfaces and colloids.

[17]  Diana M. Mitrea,et al.  Coexisting Liquid Phases Underlie Nucleolar Subcompartments , 2016, Cell.

[18]  Carol K. Hall,et al.  Interfacial tension of polyethyleneglycol-dextran-water systems: influence of temperature and polymer molecular weight , 1990 .

[19]  Seema Agarwal,et al.  Polymers with Upper Critical Solution Temperature in Aqueous Solution: Unexpected Properties from Known Building Blocks. , 2013, ACS macro letters.

[20]  Vladimir N Uversky,et al.  Intrinsically disordered proteins as crucial constituents of cellular aqueous two phase systems and coacervates , 2015, FEBS letters.

[21]  B. Sumerlin,et al.  New directions in thermoresponsive polymers. , 2013, Chemical Society reviews.

[22]  J. Hardy,et al.  Structure and technofunctional properties of protein-polysaccharide complexes: a review. , 1998, Critical reviews in food science and nutrition.

[23]  Philip C Bevilacqua,et al.  Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA. , 2016, Langmuir : the ACS journal of surfaces and colloids.

[24]  David S. Williams,et al.  Polymer/nucleotide droplets as bio-inspired functional micro-compartments , 2012 .

[25]  B. Nerli,et al.  Comparison between the thermodynamic features of alpha1-antitrypsin and human albumin partitioning in aqueous two-phase systems of polyethyleneglycol-dextran. , 2001, Biophysical chemistry.

[26]  P. Albertsson,et al.  Partition of Cell Particles and Macromolecules , 1986 .

[27]  M. Antognozzi,et al.  Small-molecule uptake in membrane-free peptide/nucleotide protocells , 2013 .

[28]  C. Brangwynne,et al.  The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics , 2015, Proceedings of the National Academy of Sciences.

[29]  Philip C Bevilacqua,et al.  Bioreactor droplets from liposome-stabilized all-aqueous emulsions , 2014, Nature Communications.

[30]  Marco Y. Hein,et al.  A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation , 2015, Cell.

[31]  Jeffrey D Varner,et al.  Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy. , 2008, Current opinion in biotechnology.

[32]  Seema Agarwal,et al.  Polymers with upper critical solution temperature in aqueous solution. , 2012, Macromolecular rapid communications.

[33]  M. C. Stuart,et al.  Binodal Compositions of Polyelectrolyte Complexes , 2010 .

[34]  S. Mann,et al.  Nanoparticle-based membrane assembly and silicification in coacervate microdroplets as a route to complex colloidosomes. , 2014, Langmuir : the ACS journal of surfaces and colloids.

[35]  Diane J. Burgess,et al.  Microelectrophoretic studies of gelatin and acacia for the prediction of complex coacervation , 1984 .

[36]  Christine D. Keating,et al.  Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological Molecules , 2012, Accounts of chemical research.

[37]  F. Weinbreck,et al.  Complex coacervation of proteins and anionic polysaccharides , 2004 .

[38]  R. Hancock,et al.  Preface. New models of the cell nucleus: crowding, entropic forces, phase separation, and fractals. , 2014, International review of cell and molecular biology.

[39]  James E. Ferrell,et al.  Mechanisms of specificity in protein phosphorylation , 2007, Nature Reviews Molecular Cell Biology.

[40]  V. Tolstoguzov Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. , 2000, International review of cytology.

[41]  A. Komeili,et al.  Compartmentalization and organelle formation in bacteria. , 2014, Current opinion in cell biology.

[42]  Ruedi Aebersold,et al.  Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling , 2013, Cell.

[43]  A. Piruska,et al.  Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate , 2013, Proceedings of the National Academy of Sciences.

[44]  F. Boisvert,et al.  The multifunctional nucleolus , 2007, Nature Reviews Molecular Cell Biology.

[45]  J. Schlenoff,et al.  The Polyelectrolyte Complex/Coacervate Continuum , 2014 .

[46]  A. Hyman,et al.  Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes , 2011, Proceedings of the National Academy of Sciences.

[47]  Timothy D Craggs,et al.  Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. , 2016, Nature chemistry.

[48]  Bin Zhang,et al.  Biogenesis and function of nuclear bodies. , 2011, Trends in genetics : TIG.

[49]  P. Dubin,et al.  Coacervation and precipitation in polysaccharide-protein systems. , 2016, Soft matter.

[50]  C. Keating,et al.  Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. , 2016, Nature chemistry.

[51]  Sarah Rauscher,et al.  Proline and glycine control protein self-organization into elastomeric or amyloid fibrils. , 2006, Structure.

[52]  R. Larson,et al.  pH and Salt Effects on the Associative Phase Separation of Oppositely Charged Polyelectrolytes , 2014 .

[53]  Donald E. Brooks,et al.  Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology , 1986 .

[54]  Bernard P. Binks,et al.  Emulsions stabilised solely by colloidal particles , 2003 .

[55]  Diana M. Mitrea,et al.  Phase separation in biology; functional organization of a higher order , 2016, Cell Communication and Signaling.

[56]  Peter Gregor,et al.  NOPdb: Nucleolar Proteome Database—2008 update , 2008, Nucleic Acids Res..

[57]  A. Lehninger Principles of Biochemistry , 1984 .

[58]  Christine D. Keating,et al.  Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning. , 2013, Journal of materials chemistry. B.

[59]  Christine D. Keating,et al.  Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization. , 2015, Langmuir : the ACS journal of surfaces and colloids.

[60]  L. Leon,et al.  The Effect of Salt on the Complex Coacervation of Vinyl Polyelectrolytes , 2014 .

[61]  Marina Feric,et al.  A nuclear F-actin scaffold stabilizes RNP droplets against gravity in large cells , 2013, Nature Cell Biology.

[62]  Stephen Mann,et al.  Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. , 2011, Nature chemistry.

[63]  Nicolas L. Fawzi,et al.  Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II. , 2015, Molecular cell.

[64]  David S. Williams,et al.  Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets. , 2012, Chemical communications.

[65]  M. Tirrell,et al.  Phase behaviour and complex coacervation of aqueous polypeptide solutions , 2012 .

[66]  Felipe García Quiroz,et al.  Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers , 2015, Nature materials.

[67]  A. Kanagaraj,et al.  Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization , 2015, Cell.

[68]  R. Lipowsky,et al.  Lipid membranes in contact with aqueous phases of polymer solutions , 2012 .

[69]  Stephen Mann,et al.  In vitro gene expression within membrane-free coacervate protocells. , 2015, Chemical communications.

[70]  P. Fletcher,et al.  Water-in-water emulsions based on incompatible polymers and stabilized by triblock copolymers-templated polymersomes. , 2013, Langmuir : the ACS journal of surfaces and colloids.

[71]  Jasper van der Gucht,et al.  Polyelectrolyte complexes: bulk phases and colloidal systems. , 2011, Journal of colloid and interface science.

[72]  Michael H. Schwartz,et al.  Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress , 2015, Cell.

[73]  David A Weitz,et al.  The cell as a material. , 2007, Current opinion in cell biology.

[74]  J. Lukas,et al.  Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose) , 2015, Nature Communications.

[75]  Antonios Armaou,et al.  Coupled Enzyme Reactions Performed in Heterogeneous Reaction Media: Experiments and Modeling for Glucose Oxidase and Horseradish Peroxidase in a PEG/Citrate Aqueous Two-Phase System , 2014, The journal of physical chemistry. B.

[76]  Roy Parker,et al.  Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. , 2015, Molecular cell.

[77]  J. Pablo,et al.  Ternary, Tunable Polyelectrolyte Complex Fluids Driven by Complex Coacervation , 2014 .

[78]  Peter Tompa,et al.  Polymer physics of intracellular phase transitions , 2015, Nature Physics.

[79]  C. Keating,et al.  Budding and asymmetric protein microcompartmentation in giant vesicles containing two aqueous phases. , 2008, Journal of the American Chemical Society.

[80]  A. Hyman,et al.  Liquid-liquid phase separation in biology. , 2014, Annual review of cell and developmental biology.

[81]  S. N. Olsen Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions , 2006 .

[82]  C. Keating,et al.  Dynamic microcompartmentation in synthetic cells , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[83]  R. Lipowsky,et al.  Transition from complete to partial wetting within membrane compartments. , 2008, Journal of the American Chemical Society.

[84]  Clifford P. Brangwynne,et al.  Soft active aggregates: mechanics, dynamics and self-assembly of liquid-like intracellular protein bodies , 2011 .

[85]  Dominique Durand,et al.  Particles trapped at the droplet interface in water-in-water emulsions. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[86]  Timothy D. Craggs,et al.  Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles , 2015, Molecular cell.

[87]  M. C. Stuart,et al.  Interfacial tension between a complex coacervate phase and its coexisting aqueous phase , 2010 .

[88]  Antonios Armaou,et al.  Colocalization and Sequential Enzyme Activity in Aqueous Biphasic Systems: Experiments and Modeling. , 2015, Biophysical journal.

[89]  Julie D Forman-Kay,et al.  Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications , 2016, The Journal of Biological Chemistry.

[90]  R. Dickinson,et al.  Coordinated Dynamics of RNA Splicing Speckles in the Nucleus , 2016, Journal of cellular physiology.

[91]  B. Narasimhan,et al.  Materials‐based strategies for multi‐enzyme immobilization and co‐localization: A review , 2014, Biotechnology and bioengineering.

[92]  Reinhard Lipowsky,et al.  Concentration dependence of the interfacial tension for aqueous two-phase polymer solutions of dextran and polyethylene glycol. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[93]  K. Leong,et al.  DNA-polycation nanospheres as non-viral gene delivery vehicles. , 1998, Journal of controlled release : official journal of the Controlled Release Society.

[94]  G. Johansson,et al.  Partition of proteins in a three-phase system. , 1974, European journal of biochemistry.

[95]  A. Hyman,et al.  Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation , 2009, Science.

[96]  Erin M. Langdon,et al.  RNA Controls PolyQ Protein Phase Transitions. , 2015, Molecular cell.

[97]  M. Tirrell,et al.  Interfacial energy of polypeptide complex coacervates measured via capillary adhesion. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[98]  M. Tirrell,et al.  Phase Behavior and Coacervation of Aqueous Poly(acrylic acid)−Poly(allylamine) Solutions , 2010 .

[99]  C. Keating,et al.  Microcompartmentation in artificial cells: pH-induced conformational changes alter protein localization. , 2010, Langmuir : the ACS journal of surfaces and colloids.

[100]  M. Dundr,et al.  Nuclear bodies: multifunctional companions of the genome. , 2012, Current opinion in cell biology.

[101]  Huan‐Xiang Zhou,et al.  Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. , 2008, Annual review of biophysics.

[102]  Philip C Bevilacqua,et al.  RNA catalysis through compartmentalization. , 2012, Nature chemistry.