High Macromolecular Crowding in Liposomes from Microfluidics.

The intracellular environment is crowded with macromolecules that influence biochemical equilibria and biomacromolecule diffusion. The incorporation of such crowding in synthetic cells would be needed to mimic the biochemistry of living cells. However, only a few methods provide crowded artificial cells, moreover providing cells with either heterogeneous size and composition or containing a significant oil fraction. Therefore, a method that generates monodisperse liposomes with minimal oil content and tunable macromolecular crowding using polydimethylsiloxane (PDMS)-based microfluidics is presented. Lipid stabilized water-in-oil-in-water emulsions that are stable for at least several months and with a high macromolecular crowder concentration that can be controlled with the external osmolality are formed. A crucial feature is that the oil phase can be removed using high flow conditions at any point after production, providing the highly crowded liposomes. Genetically encoded macromolecular crowding sensors show that the high level of macromolecular crowding in the emulsions is fully retained throughout the generation of minimal-oil lipid bilayers. This modular and robust platform will serve the study of biochemistry under physiologically relevant crowding conditions.

The more the merrier: effects of macromolecular crowding on the structure and dynamics of biological membranes.

Proteins are essential and abundant components of cellular membranes. Being densely packed within the limited surface area, proteins fulfil essential tasks for life, which include transport, signalling and maintenance of cellular homeostasis. The high protein density promotes nonspecific interactions, which affect the dynamics of the membrane-associated processes, but also contribute to higher levels of membrane organization. Here, we provide a comprehensive summary of the most recent findings of diverse effects resulting from high protein densities in both living membranes and reconstituted systems and display why the crowding phenomenon should be considered and assessed when studying cellular pathways. Biochemical, biophysical and computational studies reveal effects of crowding on the translational mobility of proteins and lipids, oligomerization and clustering of integral membrane proteins, and also folding and aggregation of proteins at the lipid membrane interface. The effects of crowding pervade to larger length scales, where interfacial and transmembrane crowding shapes the lipid membrane. Finally, we discuss the design and development of fluorescence-based sensors for macromolecular crowding and the perspectives to use those in application to cellular membranes and suggest some emerging topics in studying crowding at biological interfaces.