A Quantitative Live-Cell Superresolution Imaging Framework for Measuring the Mobility of Single Molecules at Sites of Virus Assembly
Molecular and Cellular Biophysics Program, Department of Biological Sciences, University of Denver, Denver, CO 80210, USA
Author to whom correspondence should be addressed.
Pathogens 2020, 9(11), 972; https://doi.org/10.3390/pathogens9110972
Received: 23 September 2020 / Revised: 13 November 2020 / Accepted: 18 November 2020 / Published: 21 November 2020
(This article belongs to the Special Issue Deciphering Viral-Host Interactions with Single Molecule and Superresolution Imaging Approaches)
The insurgence of superresolution microscopy into the fields of virology and microbiology has begun to enable the mapping of molecular assemblies critical for host–pathogen interfaces that organize on a scale below the resolution limit of the light microscope. It is, however, challenging to completely understand the molecular interactions between host and pathogen from strictly time-invariant observations. Herein, we describe a method using simultaneous dual-color superresolution microscopy to gain both structural and dynamic information about HIV-1 assembly. Specifically, we demonstrate the reconstruction of single virus assembly sites using live-cell photo-activated localization microscopy (PALM) while concurrently assessing the sub-viral mobility of the HIV-1 envelope glycoprotein during interaction with the viral lattice. We propose that our method is broadly applicable to elucidating pathogen and host protein–protein interactions through quantification of the dynamics of these proteins at the nanoscale.