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 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.

The behavior of metal nanospheres and nanowires and their bioconjugates in aqueous two-phase systems (ATPS) is described. The ATPS used in this work comprised poly(ethylene glycol) (PEG), dextran, and water or aqueous buffer. Au and Ag nanospheres less than 100 nm in diameter partition between the PEG-rich and dextran-rich phases on the basis of their surface chemistry and can be separated on this basis. Larger Au nanospheres and wires accumulate at the interface between the two aqueous phases. The influence of polymer molecular weight and concentration on interfacial assembly of Au wires is described. DNA-derivatized nanowires at the aqueous/aqueous interface retain the ability to selectively bind to fluorescent complementary DNA. In addition, Au nanoparticles have been bound to Au wires via selective DNA hybridization at the ATPS interface. Transmission electron microscopy and thermal denaturation experiments confirm that DNA-driven assembly is responsible for the formation of the nanosphere/wire assemblies. These results demonstrate the biocompatibility of the two-phase interface and point to future use as scaffolding in biorecognition-driven assembly.

This paper describes the use of layer-by-layer growth of metal−bis(phosphonate) multilayers to produce acentric thin films with second-order nonlinear optical properties. To incorporate such properties, organic “chromophore” molecules containing conjugated π systems situated between electron donor and electron acceptor groups are oriented uniformly within the film such that the bulk structure is noncentrosymmetric. This is accomplished using chromophoric α,ω-bis(phosphonate) molecules that have one terminal phosphonate group “protected” in ester form, whereas the other is a free phosphonic acid that will bind to a metal-primed silicon or glass surface. After deposition of the acid moiety onto metal-primed silicon, the ester groups are hydrolyzed to enable deposition of additional metal and chromophore layers. We report here the results of this approach using the chromophore bis(1-ethyl)3-{N-methyl[(4-[(4-phenylphosphonic acid)azo]phenyl)amino]decyl}phosphonate, or azobenzene molecule I. Results from multilayer studies and monolayer and solution studies are discussed, with emphasis on results from UV−vis spectroscopy, grazing angle X-ray diffraction, and second harmonic generation.

DNA:Au nanosphere bioconjugates have applications in biosensing and in the bottom-up assembly of materials. These bioconjugates can be selectively assembled into three-dimensional aggregates upon addition of complementary DNA oligonucleotides and can be dissociated by heating above a melting transition temperature at which the DNA duplexes are denatured. Herein we describe the impact of polymeric solutes on the thermal denaturation behavior of DNA:Au nanoparticle bioconjugate assemblies. Polymeric solutes can dramatically impact biochemical reactions via macromolecular crowding. Poly(ethylene glycol)s (PEGs) and dextrans of varying molecular weights were used as crowding reagents. While both PEG and dextran increased the stability of DNA:Au aggregates, melting transition temperatures in the presence of PEG were impacted more significantly. Polymer molecular weight was less important than polymer chemistry and weight percent in solution. For a high (15%) weight percent of PEG, aggregation was observed even in the absence of complementary oligonucleotides. These results underscore the importance of polymer chemistry in addition to physical volume exclusion in macromolecular crowding and point to the importance of understanding these effects when designing biorecognition-based nanoparticle assembly schemes in complex matrixes (i.e., any involving polymeric solutes).