Multivalent interactions, i.e., the formation of multiple, non-covalent bonds in parallel, are typical for a multitude of biological processes. They are observed, for example, in the attachment of infectious agents (e.g., viruses and bacteria) to the membrane of their host cells, a dynamic process that eventually leads to internalization and infection. As an individual interaction is typically weak, each bond is maintained only transiently within a multivalent interaction. As a consequence, multivalent interactions are highly dynamic and a quantification of their binding strength and valency (i.e., the number of individual interactions collectively engaged within a multivalent interaction) is experimentally demanding, which limits our knowledge about important biological processes, such as the penetration of biological barriers (e.g., cell membranes or mucus) by viruses and bacteria.

In this talk, a dynamic virus binding assay will be introduced, in which the transient attachment and release of viruses to supported lipid bilayers (SLBs; serving as a model cell membrane) is tracked using total internal reflection fluorescence (TIRF) microscopy. Applied to influenza A viruses (IAVs; strain X31), it will be shown that tracking the motion of SLB-engaged IAVs enables to estimate IAV valency values on the single-virus level,^1,2 which provides access to key properties of the multivalent virus-membrane interaction: the attachment rate, the distribution of the (apparent) valency values as well as the valency-dependent off-rate distribution. It will be demonstrated that addition of virus binding inhibitors alters these properties, providing information on the inhibitor’s mode-of-action using binding as functional readout.³

Two extensions of this assay are then discussed: First, it will be shown that performing such measurements within a microfluidic channel significantly improves the accuracy and temporal resolution^4,5 and allows to quantify the motion of SLB-engaged infectious agents being subject to shear forces. It will be demonstrated that applying the fluctuation-dissipation theorem to the deterministic and random components of the observed motion allows for extracting the shear force acting on single nanoparticles and infection agents, respectively. Second, an extension towards actively moving infectious agents, such as the bacterium Salmonella enterica serovar Typhimurium (S. Tm.), will be demonstrated. It will be shown that key properties of S. Tm.’s motility (i.e., its transversal diffusion coefficient and propulsion force) can be extracted, provided that the anisotropic shape of these bacteria is correctly taken into account. Finally, first steps of extending this methodology to track the 3D motion of S. Tm. in complex environments (e.g., epithelial cells being covered by a protective mucus layer) will be discussed.

References

1. M. Müller, S. Block et al. (2019) Nano Letters; DOI: 10.1021/acs.nanolett.8b04969.

2. S. Block et al. (2016) Nano Letters; DOI: 10.1021/acs.nanolett.6b01511.

3. M. Wallert, S. Block et al. (2020) Small; DOI: 10.1002/smll.202004635.

4. S. Block et al. (2016) Nature Communications; DOI: 10.1038/ncomms12956.

5. Y. Kerkhoff, S. Block et al. (2023) Small; DOI: 10.1002/smll.202206713.