Methods for imaging and tracking single molecules conjugated with fluorescent probes, called single-molecule tracking (SMT), are now providing researchers with the unprecedented ability to directly observe molecular behaviors and interactions in living cells. Current SMT methods are achieving almost the ultimate spatial precision and time resolution for tracking single molecules, determined by the currently available dyes. In cells, various molecular interactions and reactions occur as stochastic and probabilistic processes. SMT provides an ideal way to directly track these processes by observing individual molecules at work in living cells, leading to totally new views of the biochemical and molecular processes used by cells whether in signal transduction, gene regulation or formation and disintegration of macromolecular complexes. Here we review SMT methods, summarize the recent results obtained by SMT, including related superresolution microscopy data, and describe the special concerns when SMT applications are shifted from the in vitro paradigms to living cells.

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The behavior of sphingomyelin (SM) in the plasma membrane is unclear. Using new fluorescent SM analogs and single-molecule tracking, Kinoshita et al. find that SM transiently and continually interacts with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 in oligomer size–, cholesterol-, and GPI anchoring–dependent manners.

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol-based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the basic similarities of liquid-ordered (Lo)-phase domains in giant unilamellar vesicles, Lo-phase-like domains formed at lower temperatures in giant plasma membrane vesicles, and detergent-resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains. The literature contains good evidence, showing that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquidlike molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non-raft domains, as defined here, in the PM.

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non-raft phospholipid molecules (0.33 µm2/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3-DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3-DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them.

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano‐ and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self‐assembly under aqueous conditions. However, orthogonal self‐assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single‐stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi‐artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli‐responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions.

Multicomponent supramolecular materials, which comprise plural supramolecular architectures that exhibit distinct self-sorting behaviors, are receiving increasing attention because they can be implemented with sophisticated functions and hierarchical structures, e.g., living cells. Nevertheless, the application of supramolecular system design to engineer self-sorting behaviors among plural supramolecular architectures remains challenging. Herein, we show that the thermal annealing-induced one-pot assembly of multiple single-stranded DNAs and a single semi-artificial glycopeptide (GP) results in the emergent formation of integrative self-sorted supramolecular nanostructures (ssSNs) consisting of a GP supramolecular nanoribbon surrounded by DNA tile-nanotubes. Fluorescence imaging revealed the formation of each supramolecular nanostructure through orthogonal molecular assembling processes. Moreover, the fluorescence recovery after photobleaching (FRAP) disclosed the presence of reversible attractive interactions between the DNA tile, prior to the formation of the tile-nanotube, and the GP supramolecular nanostructures at the mesoscale level, which are crucial for the formation of the integrative ssSNs. Further, we revealed that the integrative ssSNs retain their biostimuli responsiveness so that each supramolecular nanostructure can be selectively degraded. Finally, we successfully constructed a complex soft nanomaterial composed of ternary supramolecular architectures (a GP supramolecular nanostructure, DNA tile-nanotube, and DNA microsphere) based on the present as well as previous findings.