Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles

Abstract

The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a better understanding of biomolecular processes, such as the recruitment of receptors at interfaces, at the molecular level. Here we report a model system based on supported lipid bilayers (SLBs) for the investigation of the clustering of receptors at their interface. Biotinylated SLBs, used as cell membrane mimics, were functionalized with streptavidin (SAv), used here as receptor. Subsequently, biotinylated small (SUVs) and giant (GUVs) unilamellar vesicles were bound to the SAv-functionalized SLBs by multivalent interactions and found to induce the recruitment of both SAv on the SLB surface and the biotin moieties in the vesicles. The recruitment of receptors was investigated with quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed the identification of the biotin and SAv densities necessary to obtain receptor recruitment. At approx. 0.6% of biotin in the vesicles, a transition between dense and low vesicle packing was observed, which coincided with the transitions between recruitment in the vesicles vs. recruitment in the SLB and between full and partial use of the biotin moieties in the vesicle. Direct optical visualization of the clustering at the interface of individual GUVs with the SLB platform was achieved with fluorescence microscopy, showing recruitment of SAv at the contact area as well as the deformation of the vesicles upon binding. Different vesicle binding regimes were observed for lower and higher biotin densities in the vesicles and at the SLBs. A more quantitative analysis of the molecular parameters implied in the interaction, indicated that approx.10% of the vesicle area constitutes the contact area. Moreover, the SUV binding and recruitment appeared to be fast on the analysis time scale, whereas the binding of GUVs is slower due to the larger SLB area over which SAv recruitment needs to occur. The mechanisms revealed in this study may provide insight in biological processes in which recruitment occurs. † Electronic supplementary information (ESI) available: DLS, FRAP and QCM measurements, uorescence microscopy images and calculation details. See

Ligand-receptor-mediated attachment of lipid vesicles to a supported lipid bilayer

“…To link the general conclusions above to real systems, one can look through a recent QCM-D study of attachment of biotinylated small (SUVs, ≃ 100 nm in diameter) and giant (GUVs, ≃ 15-20 m in diameter) unilamellar DOPC vesicles to a biotinylated SLB functionalized with streptavidin (Di Iorio et al 2020). One of the advantages of this choice of species is that the biotin-streptavidin interaction has long been used for biomolecule immobilization, and its strength is well established [ K d ≃ 10 −14 M (Di Iorio et al 2020) or ΔG = 18.3 kcal/mol (Weber et al 1992)]. In the experiment, the fraction of biotinylated lipids, f, in an SLB was varied from 0.002 to 0.02, whereas in vesicles it was from 0.001 to 0.02.…”

“…Thus, this analysis also indicates that can be much larger than 25 k B T . Concerning GUVs, their deformation of attached was observed explicitly by using fluorescence microscopy (Di Iorio et al 2020), but the discussion of the corresponding results is beyond our goals.…”

“…One of the advantages of this choice of species is that the biotin-streptavidin interaction has long been used for biomolecule immobilization, and its strength is well established [ M (Di Iorio et al. 2020 ) or kcal/mol (Weber et al. 1992 )].…”

Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease

“…SUVs were prepared as reported previously [ 20 , 21 ]. Lipids were first dissolved in chloroform and mixed in the desired molar ratio in a glass vial (25 mg/mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 2 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (DOPE-biotin)).…”

A Dynamic, Supramolecular View on the Multivalent Interaction between Influenza Virus and Host Cell

“…[28] Because bound receptors need to unbind before they can move away from the particle, the contact area acts as a sink for diffusing receptors, leading to a higher local density of receptors inside the contact area and a lower density outside (Figure 3b). [28,29] This clustering of receptors strongly increases the number of interactions for already bound particles as well as decreasing the number of available receptors for yet unbound particles. [28,30] Depending on the density of receptors and ligands, recruitment may either increase or decrease the binding.…”