Tracking Single Molecules in Biomembranes: Is Seeing Always Believing?

Abstract

The spatial organization of molecules in cell membranes and their dynamic interactions play a central role in regulating cell functions. Single-particle tracking (SPT), a technique in which single molecules are imaged and tracked in real time, has led to breakthrough discoveries regarding these spatiotemporal complexities of cell membranes. There are, however, emerging concerns about factors that might produce misleading interpretations of SPT results. Here, we briefly review the application of SPT to understanding the nanoscale heterogeneities of plasma membranes, with a focus on the unique challenges, pitfalls, and limitations that confront the use of nanoparticles as imaging probes for tracking the dynamics of single molecules in cell membranes.

Innate immune receptor clustering and its role in immune regulation

“…Recent studies using super-resolution microscopy have provided evidence supporting the existence of lipid-raft-like membrane domains (Eggeling et al, 2009;Owen et al, 2012a,b), but results have been conflicting (Frisz et al, 2013;Lee et al, 2015;Sevcsik et al, 2015). The super-resolution microscopic techniques have their unique complications (Huang et al, 2009;MacDonald et al, 2015;Yu et al, 2019). A more detailed discussion on the lipid raft hypothesis can be found elsewhere (Levental et al, 2020;Levental and Veatch, 2016;Sevcsik and Schutz, 2016;Sezgin et al, 2017).…”

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition

“…5 Characterizing nanoparticle diffusivity at such a short distance to an interface is therefore crucial for in-depth understanding of fundamental hydrodynamical effects, which are relevant in a multitude of biological processes, including viral infection, 6 exosome-controlled intracellular communication, 7 nanoparticle-assisted drug delivery, 8 as well as when nanoparticles are used as labels for molecules residing in laterally fluid cell membranes. 9 Since the hydrodynamics around hydrophilic interfaces often is well-described by the no-slip boundary condition, 10 this boundary condition is typically employed for biological interfaces, as they often consist of lipid bilayers with hydrophilic headgroups facing the surrounding fluid. Previous studies using a dynamic surface force apparatus to determine the boundary conditions of supported lipid bilayers (SLBs) indicate that the gel-phase DPPC (dipalmitoylphosphatidyl-choline) bilayers appear to fulfill this condition, 11,12 whereas a slip length of 6 ± 0.5 nm has been observed for fluid DOPC (dioleoylphosphatidylcholine) bilayers.…”

“…Since the motion of tethered nanoparticles is largely determined by the diffusivity of the linker, 19 it is common to neglect the potential influence on mobility of the nanoparticle itself. 9 It was recently shown, however, that in the limit of single or few tethers between gold nanoparticles and an SLB, the nanoparticle size had an appreciable influence on the resulting mobility down to particle diameters as small as 10 to 20 nm. 20 This suggests in turn that quantification of the size-dependent mobility of nanoparticles bound to an SLB could offer a novel opportunity to gain new insights about the hydrodynamic boundary conditions and confined nanoparticle diffusion near planar interfaces of both experimental and theoretical importance, especially since the current theoretical representations of confined diffusion contain several approximations that are in need of experimental validation.…”

“…In the case of nanoparticles near biological interfaces, nanoparticle diffusivity can be used to estimate both the nature of the interfacial interactions and nanoparticle size. − However, confined nanoparticle diffusion is significantly influenced by hydrodynamical boundary conditions in general and especially when the distance between the nanoparticle and an interface is shorter than the size of the particle, , which naturally occurs during the initial interaction between biological nanoparticles and cellular membranes . Characterizing nanoparticle diffusivity at such a short distance to an interface is therefore crucial for in-depth understanding of fundamental hydrodynamical effects, which are relevant in a multitude of biological processes, including viral infection, exosome-controlled intracellular communication, nanoparticle-assisted drug delivery, as well as when nanoparticles are used as labels for molecules residing in laterally fluid cell membranes …”

“…In the context of nanoparticles near fluid lipid bilayers, the distance between a nanoparticle and an SLB is often constrained due to the formation of ligand–receptor pairs or, in other words, molecular tethers, making it comparable to the length scale of the potential slip. Since the motion of tethered nanoparticles is largely determined by the diffusivity of the linker, it is common to neglect the potential influence on mobility of the nanoparticle itself . It was recently shown, however, that in the limit of single or few tethers between gold nanoparticles and an SLB, the nanoparticle size had an appreciable influence on the resulting mobility down to particle diameters as small as 10 to 20 nm .…”