Cellular membranes are crowded environments densely packed with proteins and lipids that are constantly remodeled by mechanical forces. Steric pressure generated by these forces is thought to play a significant role in key physiological processes, such as shaping endocytic vesicles and cellular protrusions. However, experimental measurements of steric pressure remain challenging, making it difficult to develop a precise understanding of the role of steric forces during membrane remodeling. To address this gap, we developed a biophysical tool to directly measure steric pressure on crowded membrane surfaces. Using fluorescence lifetime microscopy, we characterized the deformability of polyethylene glycol (PEG) as an entropic spring through Förster Resonance Energy Transfer (FRET). In particular, a membrane-bound PEG chain placed between two fluorophores serves as a molecular sensor for crowding on membranes (Fig. A). Modeling our sensor as an entropic spring, we used changes in fluoresce lifetime to estimate the force required for extension (Fig. B). Here we show that the lifetime of our FRET sensor is modulated by protein coverage on reconstituted vesicle membranes <i>in vitro</i>. Specifically, as the membrane surfaces became crowded by the endocytic adaptor protein, epsin1 ENTH, we observed a nonlinear increase in steric pressure with increasing concentration of membrane bound protein (Fig. C). Using fluorescence correlation spectroscopy (FCS) we observed a significant increase in the number of objects diffusing through our laser focal volume as we increased the concentration of bound protein on our vesicles (Fig. D, E). This increase provides a readout of the number of membrane vesiculation events arising from the steric pressure. By combining FCS and FRET, we have developed a tool that can be used to measure steric pressure during membrane remodeling events relevant to endocytic processes in the cell.

FRET-based sensor for measuring steric pressure during membrane remodeling

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.