UC Merced

Giant unilamellar vesicles (GUVs) are spherical structures composed of an aqueous compartment enclosed by a bilayer membrane. They are often seen as a simplified analog of a cell membrane and can be utilized as minimal cell models for studying cellular systems due to their cell-like sizes and capacity to mediate membrane interactions. A paper-based diffusive loading technique termed, OSM-PAPYRUS, is shown to assemble GUVs in physiologically relevant salt solutions with gentle loading of proteins. Characterization of this loading process reveals cell-like variation of encapsulated protein concentrations and a gamma distribution often cited for protein distributions in the cell. This ability to mimic cellular variability in vitro reveals potential in bridging the gap between in vitro and in vivo experimentation. The highly controlled environment of in vitro experiments can be combined with cell-like volumes, phospholipid bilayer, and cellular variation in GUVs. A practical application is explored, encapsulating the post-translation oscillator (PTO) of the cyanobacteria circadian clock system which shows membrane interactions in vivo. The results showed that cellular variation and membrane binding significantly hampers the fidelity of the clock, in contrast to bulk experiments where concentration did not matter once a critical concentration is met. An increase in concentration to cellular levels helps counteract the effect of variation. Modeling the clock reaction using expected distributions and variation of encapsulated proteins, corroborated with the hypothesis that intercellular variation and membrane binding were responsible for trends in the experimental data. The experimental data and model showed that the PTO by itself was not capable of achieving the near 100% fidelity observed in the native cyanobacteria, instead, other cellular components, like SasA and CikA or transcriptional-translational feedback loop (TTFL) would be necessary to achieve in vivo clock fidelity. The GUV model demonstrated advantages over in vivo studies, particularly in the isolation of the PTO, which allowed for the determination that large period variations seen in vivo cannot be produced by the PTO even under cell-like variability and volumes. This demonstrates the ability of GUV in vitro models to obtain context on behaviors not appreciated by either previous bulk in vitro or in vivo studies.