An experimental model for cytoplasmic organization is presented. We demonstrate dynamic control over protein distribution within synthetic cells comprising a lipid bilayer membrane surrounding an aqueous polymer solution. This polymer solution generally exists as two immiscible aqueous phases. Protein partitioning between these phases leads to microcompartmentation, or heterogeneous protein distribution within the ''cell'' interior. This model cytoplasm can be reversibly converted to a single phase by slight changes in temperature or osmolarity, such that local protein concentrations can be manipulated within the vesicle interior.aqueous phase separation ͉ intracellular organization ͉ vesicle T he interior of living cells is a crowded milieu of macromolecules, cytoskeletal filaments, and organelles. Even in cytoplasmic regions not separated by obvious barriers such as lipid membranes, differences in local composition are common. This phenomenon, referred to as microcompartmentation, is thought to have profound implications for cell function (1, 2). Understanding its role in living cells has been complicated by the lack of an experimental model system in which hypotheses could be tested. Even the mechanism(s) by which microcompartmentation is maintained remain unclear. Several possibilities have been proposed, including specific targeting and processes driven by macromolecular crowding, such as multiprotein complex formation, binding to intracellular surfaces, or phase separation (3). Aqueous phase separation occurs readily in bulk solutions of macromolecules even at much lower weight percents than are present in living cells (2). Thus, the question has been posed as to whether cytoplasm can exist without undergoing phase separation (4). Phase separation, and the accompanying partition of solutes between phases, could account for microcompartmentation of macromolecules, metabolites, and ions. Thus far, the complexity of living cells has precluded direct testing of the phase separation hypothesis. † We have encapsulated a poly(ethylene glycol) (PEG)͞dextran aqueous two-phase system (ATPS) within lipid vesicles to construct synthetic cells capable of dynamic protein and nucleic acid microcompartmentation. Substantial local variations in protein concentration can be maintained in the absence of intervening membranous barriers within these ATPS-containing vesicles. Our synthetic cytoplasm is promising as an experimental model for intracellular organization in general and demonstrates that aqueous phase separation is a viable mechanism for microcompartmentation.This work represents a bottom-up approach to understanding cell biology, in contrast to the top-down approach often adopted in biochemistry and perhaps best exemplified by efforts to generate the ''minimal cell'' through gene disruption in already simple organisms (6). Experimental model systems such as this one enable us to begin to test hypotheses in cell biology such as that of cytoplasmic phase separation. An analogy is lipid bilayer models of cell membrane...
We report the effect of external osmolarity on giant lipid vesicles containing an aqueous two-phase system (ATPS GVs). The ATPS, which is comprised of poly(ethyleneglycol) [PEG], dextran, and water, serves as a primitive model of the macromolecularly crowded environment of the cytoplasm. Coexisting PEG-rich and dextran-rich aqueous phases provide chemically dissimilar microenvironments, enabling local differences in protein concentration to be maintained within single ATPS GVs. The degree of biomolecule microcompartmentation can be increased by exposing the ATPS GVs to a hypertonic external solution, which draws water out of the vesicles, concentrating the polymers. Enrichment of a protein, soybean agglutinin, in the dextran-rich phase improves from 2.3-fold to 10-fold with an increase in external osmolarity from 100 to 200 mmol/kg. In some cases, budding occurs, with the bud(s) formed by partial expulsion of one of the two polymer-rich aqueous phases. Budding results in asymmetry in the internal polymer and biomolecule composition, giving rise to polarity in these primitive model cells. Budding is observed with increasing frequency as external ionic strength increases, when membrane elasticity permits, and can be reversed by decreasing external osmolarity. We note that the random symmetry-breaking induced by simple osmotic shrinkage resulted in polarity in both the structure and internal protein distribution in these primitive model cells. Budding in ATPS-containing GVs thus offers an experimental model system for investigating the effects of biochemical asymmetry on the length scale of single cells.
We report the synthesis and initial characterization of approximately 10 mum diameter lipid vesicles that contain two distinct aqueous phases. The aqueous two-phase system is a dextran/poly(ethylene glycol) solution that exhibits temperature-dependent phase behavior. Vesicles were prepared above the phase transition temperature of the polymer solution. Upon cooling to room temperature, the polymer solution phase separated both within the vesicles and in the bulk solution. The location of poly(ethylene glycol)-rich and dextran-rich phases was determined by fluorescence microscopy. These structures are exciting in that they enable for the first time the interior volume of liposomes to be structured.
We describe the effect of bioconjugation to colloidal Au nanoparticles on protein partitioning in poly(ethylene glycol) (PEG)/dextran aqueous two-phase systems (ATPS). Horseradish peroxidase (HRP) was conjugated to colloidal Au nanoparticles by direct adsorption. Although HRP alone had very little phase preference, HRP/Au nanoparticle conjugates typically partitioned to the PEG-rich phase, up to a factor of 150:1 for conjugates of 15-nm colloidal Au. Other protein/Au nanoparticle conjugates exhibited partitioning of greater than 2000:1 to the dextran-rich phase, as compared with approximately 5:1 for the free protein. The degree of partitioning was dependent on polymer concentration and molecular weight, nanoparticle diameter, and in some instances, nanoparticle concentration in the ATPS. The substantial improvements in protein partitioning achievable by conjugation to Au nanoparticles appear to result largely from increased surface area of the conjugates and require neither chemical modification of the proteins or polymers with affinity ligands, increased polymer concentrations, nor addition of high concentrations of salts. Adsorption to colloidal particles thus provides an attractive route for increased partitioning of enzymes and other proteins in ATPS. Furthermore, these results point to ATPS partitioning as a powerful means of purification for biomolecule/nanoparticle conjugates, which are increasingly used in diagnostics and materials applications.
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