Effects of crowding on the diffusivity of membrane adhered particles

The lateral diffusion of cell membrane inclusions, such as integral membrane proteins and bound receptors, drives critical biological processes, including the formation of complexes, cell-cell signaling, and membrane trafficking. These diffusive processes are complicated by how concentrated, or "crowded", the inclusions are, which can occupy between 30-50% of the area fraction of the membrane. In this work, we elucidate the effects of increasing concentration of model membrane inclusions in a free-standing artificial cell membrane on inclusion diffusivity and the apparent viscosity of the membrane. By multiple particle tracking of fluorescent microparticles covalently tethered to the bilayer, we show the transition from expected Brownian dynamics, which accurately measure the membrane viscosity, to subdiffusive behavior with decreased diffusion coefficient as the particle area fraction increases from 1% to around 30%, approaching physiological levels of crowding. At high crowding, the onset of non-Gaussian behavior is observed. Using hydrodynamic models relating the 2D diffusion coefficient to the viscosity of a membrane, we determine the apparent viscosity of the bilayer from the particle diffusivity and show an increase in the apparent membrane viscosity with increasing particle area fraction. However, the scaling of this increase is in contrast with the behavior of monolayer inclusion diffusion and bulk suspension rheology. These results demonstrate that physiological levels of model membrane crowding nontrivially alter the dynamics and apparent viscosity of the system, which has implications for understanding membrane protein interactions and particle-membrane transport processes.