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.
Liquid-liquid phase separation as a model system for intracellular membraneless organelles.
Representative Publications:
Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles. Choi, S.; Bevilacqua, P. C.; Keating, C. D. Nature Chemistry 2022 (https://doi.org/10.1038/s41557-022-00980-7).
Formation of multi-phase complex coacervates and partitioning of biomolecules within them. Mountain, G. A. and Keating, C. D. Biomacromolecules 2020, 21, 630-640.
Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization. Marianelli, A. M.; Miller, B. M.; Keating, C. D. Soft Matter 2018, 14, 368-378.
Experimental models for dynamic compartmentalization of biomolecules in liquid organelles: Reversible formation and partitioning in aqueous biphasic systems. Aumiller Jr., W. M.; Keating, C. D. Advances in Colloid and Interface Science 2017, 239, 75-87.
RNA-based coacervates as a model for membraneless organelles: Formation, properties, and interfacial liposome assembly. Aumiller Jr., W. M.; Pir Cakmak, F.; Davis, B. W.; Keating, C. D. Langmuir 2016, 32, 10042-10053.
Phosphorylation-mediated RNA/peptide complex coacervation: a model for intracellular liquid organelles. Aumiller Jr., W. M. and Keating, C. D. Nature Chemistry 2016, 8, 129-137.
Subcellular microcompartmentalization in synthetic cells. 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 is an important physical mechanism for the inhomogeneous distribution of many intracellular components not bound by membranes.
Our experiments provide experimental model systems in which the mechanisms and functional significance of compartmentalization via liquid-liquid phase coexistence 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.
Representative Publications:
Aqueous phase separation as a possible route to compartmentalization of biological molecules. Keating, C. D. Accounts of Chemical Research 2012, 45, 2114-2124.
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. Journal of the American Chemical Society 2011, 133, 9545-9555.
Microcompartmentation in artificial cells: pH-induced conformational changes alter protein localization. Dominak, L. M.; Gundermann, E. L.; Keating, C. D. Langmuir 2010, 26, 5697-5705.
Positioning lipid membrane domains in giant vesicles by micro-organization of aqueous cytoplasm mimic. Cans, A-S.; Andes-Koback, M.; Keating, C. D. Journal of the American Chemical Society 2008, 130, 7400-7406.
Dynamic microcompartmentation within synthetic cells. Long, M. S.; Jones, C.; Helfrich, M. R.; Mangeney-Slavin, L. K.; Keating, C. D. Proceedings of the National Academy of Science, USA, 2005, 102, 5920-5925.
Aqueous phase separation in giant vesicles. M. R. Helfrich, L. K. Mangeney-Slavin, M. S. Long, K. Y. Djoko, C. D. Keating Journal of the American Chemical Society 2002, 124, 13374-13375.
Reactions in biomimetic complex media. The 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.
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 introduced 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.
Bioinspired mineralization in microreactors pre-organized by liquid-liquid phase coexistence.
Representative Publications:
Bioinspired mineralizing microenvironments generated by liquid-liquid phase coexistence. Rowland, A. T.; Cacace, D. N.; Pulati, N.; Gulley, M. L.; Keating, C. D. Chemistry of Materials 2019, 31, 10243-10255.
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 2015, 31, 11329-11338.
Multiphase water-in-oil emulsion droplets for cell-free transcription–translation. Torre, P.; Keating, C. D.; Mansy, S. S. Langmuir 2014, 30, 5695-5699.
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 2015, 109, 2182-2194.
Encapsulation of compartmentalized cytoplasm mimic within lipid membrane by microfluidics. Sobrinos-Sanguino, M.; Silvia Zorrilla, S.; Keating, C. D.; Monterroso, B.; Rivas, G. Chemical Communications 2017, 53, 4775-4778.
Bioreactor droplets from liposome-stabilized all-aqueous emulsions. Dewey, D. C.; Strulson, C. A.; Cacace, D. N.; Bevilacqua, P. C.; Keating, C. D. Nature Communications 2014, 5, 4670 (doi: 10.1038/ ncomms5670).
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.