Abstract
With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such as surface plasmon resonance.
Highlights
Single-particle tracking (SPT) is a versatile technique for studying protein-protein binding interactions occurring at surfaces, the binding of viruses to host cell membrane receptors [1,2,3,4,5]
The viral receptor can be loaded onto a flat substrate by tethering receptors to polymers attached covalently to the substrate [6], adsorbing lipid vesicles containing the receptor lipid or protein [7], or forming supported lipid bilayers (SLBs) containing membrane receptors [4,5, 8,9,10]
The SLB option is advantageous because 1) the receptor type and surface density can be carefully controlled through bilayer preparation steps, 2) receptors are properly orientated in the membrane [11], 3) viral membrane fusion kinetics can be studied using the same assay [8, 12,13,14,15], and 4) mobile lipids allow the virus to recruit receptors and form multivalent bonds
Summary
Single-particle tracking (SPT) is a versatile technique for studying protein-protein binding interactions occurring at surfaces, the binding of viruses to host cell membrane receptors [1,2,3,4,5]. The SLB option is advantageous because 1) the receptor type and surface density can be carefully controlled through bilayer preparation steps, 2) receptors are properly orientated in the membrane [11], 3) viral membrane fusion kinetics can be studied using the same assay [8, 12,13,14,15], and 4) mobile lipids allow the virus to recruit receptors and form multivalent bonds. The SPT-SLB assay contains several technical challenges with experimental design, image processing, and binding kinetic data analysis that limit its adaptation as a standard analytical tool. To increase the utility of SPT-SLB assays, we explain the cause of and demonstrate solutions to these issues as we study of influenza virus binding to several types of α2,3 sialic acid (SA) glycolipids
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