POWRE: Towards Functional Model Cells: Incorporating Internal Structure

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The goal of this work is to synthesize cytomimetic assemblies with internal complexity approaching that of their biological counterparts. Although scientists from many disciplines have attempted to create synthetic replicas of living cells, only the very simplest of cell structures and functions have been mimicked in vitro. To date, most model cells have been liposomes. These can be excellent models of the plasma membrane, but they lack intracellular organization. Living cells exhibit internal ordering which can be divided into two broad categories based on the presence or absence of a surrounding membrane separating the structure from the cytosol. The exploratory work supported by this POWRE award is aimed at generating experimental systems in which each of these classes of intracellular organization is modeled. It is not difficult to create giant vesicles having smaller internal vesicles - in fact, it can be hard not to, when attempting to synthesize large unilamellar vesicles. This work will move beyond previous studies by designing model organelles (inner vesicles) which differ in composition from the model plasma membrane (outer vesicle). This will be a significant step towards approximating the level of sophistication found in living cells. Internal vesicles will be incorporated within giant unilamellar vesicles (GUVs) by including small vesicles in the aqueous phase during swelling of giant unilamellar vesicles. Alternately, preformed vesicles will be encapsulated within large liposomes by microinjection. Encapsulation of "organelles" will be verified (and in some cases followed) by video-enhanced optical microscopy. Transfer of molecules between "organelles" and the outer "cell" will be followed by fluorescence microscopy and flow cytometry. A second approach to model the internal structure of cells is via macromolecular crowding. Macromolecular crowding has been postulated to control the association of intracellular components through phase segregation, which occurs more readily in the presence of high concentrations of noninteracting macromolecules due to volume exclusion. The effects of "crowding" on the contents of single GUVs will be investigated first with colloidal particles (e.g. latex microspheres and metal nanorods or virus particles), and then with biological macromolecules (e.g. albumin and tubulin or actin). High concentrations of volume excluders will be encapsulated within GUVs to generate static crowding conditions and initiate phase segregation of anisotropic molecules or particles. To control the "crowding" pressure during an experiment, GUV volume will be altered, e.g. via control of external osmolarity. Organization of internal particles and macromolecules will be followed primarily by quantitative polarized light microscopy. The process of phase-separation into ordered phases will be tracked by monitoring changes (increases) in birefringence. Other methods for observation of internal ordering that will be used include reflected light microscopy (for 200 nm metallic nanorods), fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). This POWRE award will allow Dr. Keating to perform preliminary studies to establish the feasibility of these approaches and to begin to make observations. The longer term development of this project will, if fully successful, lead to a change in the way scientists think about cell models, and will greatly advance understanding of molecular self-assembly in living cells, and will establish this research as an independent line of investigation for Dr. Keating.