High-speed nanoscopic tracking of the position and orientation of a single virus

Abstract

Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings, which are important for cell infection. Here we present a colocalization methodology that combines scattering interferometry and single-molecule fluorescence microscopy to visualize both position and orientation of single quantum dot-labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resolution, we observed sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions separated by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biological nano-objects.

Lipid-Mediated Endocytosis

“…The diffusion of GM1-bound CTX is more than an order of magnitude slower than that of free GM1 in supported membrane bilayers, plasma membrane vesicles, and cellular membranes (Spiegel et al 1984;Kenworthy et al 2004;Burns et al 2005;Lingwood et al 2008;Eggeling et al 2009). The lateral mobility of GSL-bound polyomaviridae is even lower Kukura et al 2009). Why the diffusion of lipid-bound ligands is reduced to such an extent is not clear, especially because the binding of antibody to lipids does not result in a similar reduction (Lee et al 1991).…”

“…In supported membrane bilayers, the viruses are highly mobile, no confinement is observed (Ewers et al 2005;Kukura et al 2009). However, an exception can be observed when the bilayer contains high amounts of GM1 (1 mol %).…”

“…Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004721 adjacent VP1 pentamers (Kukura et al 2009). How virions become immobilized in such a way is not clear.…”

Dynamic label-free imaging of lipid nanodomains

“…In addition to fluorescence-based approaches, ellipsometry and reflection interference contrast microscopy have been used to characterize phase separation in lipid bilayers (16, 17), taking advantage of different bilayer thicknesses and refractive indices caused by varying degrees of cholesterol content and lipid packing. Given sufficient sensitivity and resolution, this approach should hold for arbitrarily small domains.We recently developed interferometric scattering microscopy (iSCAT) (18)(19)(20) and achieved sensitivity to refractive index perturbations down to the level of a single unlabeled protein molecule in solution with millisecond time resolution (21,22). Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels.…”

“…We recently developed interferometric scattering microscopy (iSCAT) (18)(19)(20) and achieved sensitivity to refractive index perturbations down to the level of a single unlabeled protein molecule in solution with millisecond time resolution (21,22). Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels.…”