University of Minnesota
School of Physics & Astronomy

Physics and Astronomy Calendar

Thursday, November 8th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Isaac Angert, graduate student in Jochen Mueller’s lab, School of Physics and Astronomy, University of Minnesota
Subject: Ultra-sensitive detection of plasma membrane binding and nanometer lengths with dual-color fluorescence z-scan in live 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 length displacements between dual-color fluorescently labeled protein species and show that the DC z-scan technique resolves nanometer scales. We utilize DC z-scans to investigate the passage of retroviral assembly proteins through the actin cortex in live cells, and suggest that the dense cortical meshwork may present a barrier to the assembly and budding of retroviruses such as human immunodeficiency virus.

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