Living cells contain millions of enzymes and proteins, which carry out multiple reactions simultaneously. To optimize these processes, cells compartmentalize reactions in membraneless liquid condensates. Certain features of cellular condensates can be explained by principles of liquid-liquid phase separation studied in material science. However, biological condensates exist in the inherently out of equilibrium environment of a living cell, being driven by force-generating microscopic processes. These cellular conditions are fundamentally different than the equilibrium conditions of liquid-liquid phase separation studied in materials science and physics. How condensates function in the active riotous environment of a cell is essential for understanding of cellular functions, as well as to the onset of neurodegenerative diseases. Currently, we lack model systems that enable rigorous studies of these processes. Living cells are too complex for quantitative analysis, while reconstituted equilibrium condensates fail to capture the non-equilibrium environment of biological cells. To bridge this gap, we reconstituted a DNA based membraneless condensates in an active environment that mimics the conditions of a living cell. We combine condensates with a reconstituted network of cytoskeletal filaments and molecular motors, and study how the mechanical interactions change the phase behavior and dynamics of membraneless structures. Studying these composite materials elucidates the fundamental physics rules that govern the behavior of liquid-liquid phase separation away from equilibrium while providing insight into the mechanism of condensate phase separation in cellular environments.

At small length-scales, capillary effects are significant, and thus the mechanics of soft material interfaces may be dominated by solid surface stresses or liquid surface tensions. The balance between surface and bulk properties is described by an elasto-capillary length-scale, in which equilibrium interfacial energies are constant. However, at small length-scales in biological materials, including living cells and tissues, interfacial energies are not constant but are actively regulated and driven far from equilibrium. Thus, the balance between surface and bulk properties depends upon the distance from equilibrium. Here, we model the spreading (wetting) of living cell aggregates as ‘active droplets’, with a non-equilibrium surface tension that depends upon internal stress generated by the actomyosin cytoskeleton. Depending upon the extent of activity, droplet surface properties adapt to the mechanics of their surroundings. The impact of this adaptation challenges contemporary models of interfacial mechanics, including extensively used models of contact mechanics and wetting.

Life of biological molecules spans time and length scales relevant at atomic to cellular time and length scales. Hence, novel molecular modeling approaches are required to be inherently multi-scale. Here we describe multiple methodologies developed in our laboratory: rapid discrete molecular dynamics simulation algorithm, protein design and structural refinement tools. Using these methodologies, we describe therapeutic strategies to combat this HIV and cancer, as well as design novel approaches for controlling proteins in living cells and organisms.