Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models

The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking

Whether class-A G-protein coupled receptors (GPCRs) exist and work as monomers or dimers has drawn extensive attention. A class-A GPCR dopamine D2 receptor (D2R) is involved in many physiological and pathological processes and diseases, indicating its critical role in proper functioning of neuronal circuits. In particular, D2R homodimers might play key roles in schizophrenia development and amphetamine-induced psychosis. Here, using single-molecule imaging, we directly tracked single D2R molecules in the plasma membrane at a physiological temperature of 37 degrees C, and unequivocally determined that D2R forms transient dimers with a lifetime of 68 ms in its resting state. Agonist addition prolonged the dimer lifetime by a factor of ~1.5, suggesting the possibility that transient dimers might be involved in signaling

Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function

Single-fluorescent-molecule imaging tracking (SMT) is becoming an important tool to study living cells. However, photobleaching and photoblinking (hereafter referred to as photobleaching/photoblinking) of the probe molecules strongly hamper SMT studies of living cells, making it difficult to observe in vivo molecular events and to evaluate their lifetimes (e.g., off rates). The methods used to suppress photobleaching/photoblinking in vitro are difficult to apply to living cells because of their toxicities. Here using 13 organic fluorophores we found that, by combining low concentrations of dissolved oxygen with a reducing-plus-oxidizing system, photobleaching/photoblinking could be strongly suppressed with only minor effects on cells, which enabled SMT for as long as 12,000 frames (~7 min at video rate, as compared to the general 10-s-order durations) with ~22-nm single-molecule localization precisions. SMT of integrins revealed that they underwent temporary (<80-s) immobilizations within the focal adhesion region, which were responsible for the mechanical linkage of the actin cytoskeleton to the extracellular matrix

High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions and thus serve as a key signal transduction platform

Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown. Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein’s higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein’s slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters. Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein molecules would strongly enhance the spreading of misfolded prion protein

Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging.

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed

Single-molecule imaging revealed dynamic GPCR dimerization

Single fluorescent-molecule video imaging and tracking in living cells are revolutionizing our understanding of molecular interactions in the plasma membrane and intracellular membrane systems. They have revealed that molecular interactions occur surprisingly dynamically on much shorter time scales (≪1s) than those expected from the results by conventional techniques, such as pull-down assays (minutes to hours). Single-molecule imaging has unequivocally showed that G-protein-coupled receptors (GPCRs) undergo dynamic equilibrium between monomers and dimers, by enabling the determination of the 2D monomer–dimer equilibrium constant, the dimer dissociation rate constant (typically ∼10s−1), and the formation rate constant. Within one second, GPCRs typically undergo several cycles of monomer and homo-dimer formation with different partners