Models of biological cells: Cytomimetic chemistry

Chemists have always learned by synthesis.  Historically, chemists prepared metallic alloys and later organic molecules, varying the synthetic procedure to better understand the properties of the resulting materials.  We carry on this tradition by using the tools of molecular self-assembly and the behavior of thermodynamically nonideal polymer solutions to construct primitive models of biological cells and cell-like environments.  These systems then become test beds for evaluating hypotheses about how cells work and how the earliest cells may have evolved. 

Motivations for this work range from basic to applied science; by asking how intracellular structure leads to function we hope to learn not only about how cells function but also to find routes to preparing new, nonbiological materials for desired functions.  Examples of the latter include potential medical applications in drug delivery or inorganic/organic composite materials with improved optical or mechanical properties. 

Experimental models for subcellular microcompartmentalization.  We are developing synthetic cytoplasm and nucleoplasm based on aqueous polymer solutions that mimic the crowded, compartmentalized internal environment of living cells. Microcompartmentalization of the aqueous interior is accomplished in our model cells by phase separation of the polymer solutions.  Macromolecules (proteins, nucleic acids, carbohydrates) are present in living cells at levels well above those required for phase separation in synthetic polymer solutions. Phase separation within living cells has been hypothesized as an explanation for the inhomogeneous distribution of many intracellular components not bound by membranes. 

Our experiments support the hypothesis that aqueous phase separation is one viable mechanism for, and could contribute to, microcompartmentation in living cells. Further, they provide an experimental model system in which the mechanisms and functional significance of microcompartmentation – regardless of its cause – can be investigated. Current areas of interest include asymmetric division of artificial cells, local control over enzymatic reactions to control mineralization, and new routes to controlled encapsulation of macromolecular solutes within artificial cells. On a more basic level, this research aims to understand the degree to which very simple self-assembled systems can display apparently complex behaviors reminiscent of living cells and what the similarities and differences between these models and biological cells can tell us about both early steps in cellular evolution and the workings of modern cells. 

Reactions in biomimetic complex mediaThe intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. What are the consequences of this heterogeneous reaction media on the outcome of enzyme reactions? Under some conditions, reaction rates can be increased by compartmentalization into one phase, where the local concentration is determined by both the partitioning coefficient and the relative compartment volume. Reactions in biphasic media can be more complex, however, if partitioning does not result in favorable local concentrations of all necessary reaction participants. Media-specific effects due to crowding and chemical interactions can also complicate kinetics, for example leading to activity losses when substrate molecules are unavailable for reaction. Our efforts to improve understanding of biochemical reactions in multiphase, crowded media will help shed light on possible biological roles for intracellular “liquid organelles” and could lead to biotechnological advances. Our work on reactions in phase-separated media includes collaborations with the Bevilacqua lab on RNA catalysis and origin of life work, and the Armaou lab for computational modeling of multistep reactions in these media, and the Mansy lab for in vitro transcription/translation.

Water-in-water emulsions. A key challenge in artificial cells and bioreactors is maintaining a favourable internal environment while allowing substrate entry and product departure. We are developing semipermeable, size-controlled bioreactors with aqueous, macromolecularly crowded interiors based on liposome stabilization of all-aqueous emulsions. Droplets are on the order of ten microns in diameter (similar in size to biological cells). Inter-droplet repulsion provides electrostatic stabilization of the emulsion, with droplet coalescence prevented even for submonolayer interfacial coatings of negatively-charged lipid vesicles. RNA and DNA can enter and exit these bioreactors by diffusion, with final concentrations dictated by partitioning.

Representative Publications:

Polyamine/nucleotide coacervates provide strong compartmentalization of Mg2+, nucleotides, and RNA. Frankel, E. A.; Bevilacqua, P. C.; Keating, C. D. Langmuir 201632, 2041-2049.

Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Aumiller, Jr., W. M.; Keating, C. D. Nature Chemistry 20168, 129-137.

Colocalization and Sequential Enzyme Activity in Aqueous Biphasic Systems: Experiments and Modeling Davis, B. W.; Aumiller, Jr., W. M.; Hashemian, N.; An, S.; Armaou, A.; Keating, C. D. Biophysical Journal 2015109, 2182-2194.

Aqueous emulsion droplets stabilized by lipid vesicles as microcompartments for biomimetic mineralization. Cacace, D. N.; Rowland, A. T.; Stapleton, J. J.; Dewey, D. C. and Keating, C. D. Langmuir 201531, 11329-11338. 

Bioreactor droplets from liposome-stabilized all-aqueous emulsions.  Dewey, D. C.; Strulson, C. A.; Cacace, D. N.; Bevilacqua, P. C.; Keating, C. D. Nature Commun20145, 4670 (doi: 10.1038/ ncomms5670).

Interactions of macromolecular crowding agents and cosolutes with small molecule substrates: Effect on horseradish peroxidase activity with two different substrates. Aumiller Jr., W. M.; Davis, B. W.; Hatzakis, E.; Keating,, C. D. J. Phys. Chem. B 2014 (in press and available online at DOI: 10.1021/jp506594f).

Aqueous multi-phase systems within water-in-oil emulsion droplets for the construction of genetically encoded cellular mimics.  Torre, P.; Keating, C. D.; Mansy, S. S. Langmuir 201430, 5695-5699.

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.  Aumiller Jr., W. M.; Davis, B. W.; Hashemian, N.; Maranas, C. D.; Armaou, A.; Keating, C. D. Journal of Physical Chemistry B 2014118, 2506-2517.

Aqueous phase separation as a possible route to compartmentalization of biological molecules. Keating, C. D. Accounts of Chemical Research 2012, 45, 2114-2124.

RNA catalysis through compartmentalization. Strulson, C. A.; Molden, R. C.; Keating, C. D.; Bevilacqua, P. C. Nature Chem20124, 941-946.

Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane
composition. Andes-Koback, M.; Keating, C. D. J. Am. Chem. Soc. 2011, 133, 9545-9555.

Thanks to our Sponsors:

DOE Biomolecular Materials supports our work in biomimetic mineralization.

The National Science Foundation MCB Division supports our work on reversible compartmentalization in model cells.

NASA Exobiology funds our work in possible roles for aqueous phase separation on the early-earth, from a "RNA World" perspective.  This is a collaboration with Prof. Phil Bevilacqua.

               Spri© Chris Keating 2013