Microfluidic setup for quantification of multivalent interactions and massive parallelization of single-molecule force spectroscopy: Unraveling virus-receptor interactions

• Applicant: Dr. Stephan Block
• Subject Area: Biophysics
• Term: from 2017 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 364654521

Multivalent interactions are observed in a multitude of biological processes like the attachment of pathogens (e.g., viruses) to cells. A quantitative understanding of such processes requires the quantification of the underlying multivalent interaction on the single-interaction level, which is a yet unresolved issue. To address this challenge, we established in the previous funding period two assays based on microfluidics and total internal reflection fluorescence (TIRF) microscopy, which enabled us to probe weak and multivalent (virus-membrane) interactions with single-molecule resolution and high data throughput. We demonstrated that the mobility of membrane-bound viruses provides a measure for the virus valency (i.e., the number of single interactions engaged within a multivalent virus-membrane interaction), which enabled to determine key binding properties, such as the rate of virus attachment, a qualitative measure for the distribution of the virus valency as well as the valency-dependent rate of virus release from the membrane. Although these investigations yielded new fundamental insights on the properties of virus-membrane interactions, an accurate quantification of virus valency was not yet possible. This limitation is caused by a lack of experimental data on the impact of the valency value on the mobility of particles linked to a supported lipid bilayer (SLB). This aspect of membrane hydrodynamics is theoretically very well established, but was so far not experimentally confirmed. In the final period of this project, we aim to use recently established methods and systems to perform dedicated mobility studies, which will enable to quantitatively determine the valency-mobility relationship of SLB-bound nanoparticles (proteins and viruses). To this end, we will combine microfluidics, TIRF-based single-molecule localization microscopy, and single particle tracking (SPT) to probe the motion of the subunit B of cholera toxin (CTxB), of virus-like particles of the polyomavirus SV40 and of influenza A viruses in absence and presence of an applied shear force. A dual labeling approach, in which receptors as well as nanoparticles are fluorescently labeled, will enable to simultaneously determine the valency (by stepwise bleaching) and mobility (by SPT) of individual SLB-bound nanoparticles. Application of a well-defined hydrodynamic shear force will enable to induce release of individual receptors (engaged in a multivalent interaction) and hence to quantify the off-rate of receptors in dependence of the valency.Taken together, both approaches will provide novel information about hydrodynamics of supported membranes and allow for a quantitative determination of the valency of multivalent interactions. Although applied here mainly to viruses, the presented methodology can, in principle, be extended to any type of multivalent interaction that is formed at interfaces (i.e., to infectious agents in general).

• DFG Programme: Independent Junior Research Groups