Ultra-Sensitive Detection Of Peripheral Membrane Protein Binding To The Plasma Membrane Of Living Cells

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to many cellular functions and disease states, including cell motility, signal transduction, and the budding and entry steps in the life cycle of many viruses. We describe quantification of PM binding by the use of two-photon dual color z-scan (DC z-scan) deconvolution in cells co-expressing a fluorescently labeled protein of interest along with a soluble fluorescent protein with a distinct color that serves as a reference to delineate the cytoplasmic extent. We show that this technique can detect very small amounts of PM binding that are otherwise extremely difficult to discern. Due to the nature of the two-photon excitation, the DC z-scan PM binding assay technique attains very low levels of systematic artifacts, for which we provide observational upper bounds. We further extend the technique to interrogate the passage of retroviral assembly proteins through the actin cortex, and suggest that the dense cortical meshwork may present a barrier to the assembly and budding of retroviruses such as human immunodeficiency virus. This work is supported by grants from the National Institutes of Health (RO1 GM64589, RO1 GM098550, RO1 GM124279).