BPS2026 – Membrane proximal actin cytoskeleton and protein crowding synergistically modulate the mesoscale mobility of membrane components in the cell membrane

The plasma membrane is a dynamic interface where cells interact with their environment, organize signaling cascades, and engage in trafficking processes. Critical to these functions is the diffusion of membrane constituents such as proteins and lipids to enable encounters between functional counterparts leading to signaling reactions or building molecular assemblies in the membrane. The membrane is tethered to a cortical actin meshwork and exhibits a high density of peripheral and membrane proteins, contributing to protein crowding in the membrane. The influence of these features has been considered to be key regulators of diffusion at the membrane. In this study, we combined live-cell imaging with a minimal, reconstituted, in vitro system to test how actin fences and protein crowding shape the mesoscale diffusional transport of membrane proteins. Using imaging-fluorescence correlation spectroscopy (ImFCS), we were able to measure the relationship between membrane proximal actin density and molecular mobility of an actin-binding protein and a tracer protein that is incapable of binding actin. We also developed a robust pipeline to measure the density of proteins at the cell membrane using quantitative microscopy, super resolution imaging, and bioinformatic analyses. We then systematically varied actin-based picketing and protein density in our in vitro and in silico model membrane systems, measured protein mobility, and compared it with mobility in cell membranes. We find that while membrane-proximal actin density directly influences actin-binding membrane proteins, protein crowding in the bilayer masks the influence of local actin structures on tracer mobility at the mesoscale. Therefore, cortical actin density and protein crowding together serve to regulate molecular mobility in the plasma membrane, opening up the possibility for tuning of mobility by the local crowder density and architecture of the actin cortex.