Influence of membrane-cortex linkers on the extrusion of membrane tubes

The cell membrane is an inhomogeneous system composed of phospholipids, sterols, carbohydrates, and proteins that can be directly attached to underlying cytoskeleton. The protein linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here, we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations, we find that the force for tube extrusion has a nonlinear dependence on the density of membrane-cortex attachments: at a range of low and intermediate linker densities, the force is not significantly influenced by the presence of the membrane-cortex attachments and resembles that of the bare membrane. For large concentrations of linkers, however, the force substantially increases compared with the bare membrane. In both cases, the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had a negligible effect on the force. Our results shed light on the importance of local protein rearrangements for membrane reshaping at nanoscopic scales

Fluorescence Correlation Spectroscopy to Examine Protein-Lipid Interactions in Membranes

Fluorescence correlation spectroscopy (FCS) is a versatile technique to study membrane dynamics and protein-lipid interactions. It can provide information about diffusion coefficients, concentrations, and molecular interactions of proteins and lipids in the membrane. These parameters allow for the determination of protein partitioning into different lipid environments, the identification of lipid domains, and the detection of lipid-protein complexes on the membrane. During the last decades, FCS studies were successfully performed on model membrane systems as also on living cells, to characterize protein-lipid interactions. Recent developments of the method described here improved quantitative measurements on membranes and decreased the number of potential artifacts. The aim of this chapter is to provide the reader with the necessary information and some practical guidelines to perform FCS studies on artificial and cellular membranes