Transient, nano-scale, liquid-like molecular assemblies coming of age.

This review examines the dynamic mechanisms underlying cellular signaling, communication, and adhesion via transient, nano-scale, liquid-like molecular assemblies on the plasma membrane (PM). Traditional views posit that stable, solid-like molecular complexes perform these functions. However, advanced imaging reveals that many signaling and scaffolding proteins only briefly reside in these molecular complexes and that micron-scale protein assemblies on the PM, including cell adhesion structures and synapses, are likely made of archipelagoes of nanoliquid protein islands.

Three-dimensional fruit growth analysis clarifies developmental mechanisms underlying complex shape diversity in persimmon fruit.

The determination of fruit size and shape are of considerable interest in horticulture and developmental biology. Fruit typically exhibits three-dimensional structures characterized by geometric features that are dependent on the genotype. Although minor developmental variations have been recognized, few studies have fully visualized and measured these variations throughout fruit growth.

Synapsin condensation controls synaptic vesicle sequestering and dynamics.

Neuronal transmission relies on the regulated secretion of neurotransmitters, which are packed in synaptic vesicles (SVs). Hundreds of SVs accumulate at synaptic boutons. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity.

A Survey of Models of Cell Membranes: Toward a New Understanding of Membrane Organization.

The cell membrane, the boundary that separates living cells from their environment, has been the subject of study for over a century. The fluid-mosaic model of Singer and Nicolson in 1972 proposed the plasma membrane as a two-dimensional fluid composed of lipids and proteins. Fifty years hence, advances in biophysical and biochemical tools, particularly optical imaging techniques, have allowed for a better understanding of the physical nature, organization, and composition of cell membranes.

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized.

Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified.

Cholesterol- and actin-centered view of the plasma membrane: updating the Singer-Nicolson fluid mosaic model to commemorate its 50th anniversary.

Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer-Nicolson fluid mosaic model. For others, due to the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM.

Refinement of Singer-Nicolson fluid-mosaic model by microscopy imaging: Lipid rafts and actin-induced membrane compartmentalization.

This year celebrates the 50th anniversary of the Singer-Nicolson fluid mosaic model for biological membranes. The next level of sophistication we have achieved for understanding plasma membrane (PM) structures, dynamics, and functions during these 50 years includes the PM interactions with cortical actin filaments and the partial demixing of membrane constituent molecules in the PM, particularly raft domains. Here, first, we summarize our current knowledge of these two structures and emphasize that they are interrelated.

Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions.

Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry.

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV.

Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75 play a critical role in neuronal survival and their functions are altered in Alzheimer's disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17β-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques.

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.

Defining raft domains in the plasma membrane.

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 fundamental similarities of liquid-ordered (Lo)-phase domains in giant unilamellar vesicles, Lo-phase-like domains formed at lower temperatures in giant PM vesicles, and detergent-resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol.

AMPA receptors in the synapse turnover by monomer diffusion.

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1-4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition.

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane.

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44.

Dual-FRET imaging of IP and Ca revealed Ca-induced IP production maintains long lasting Ca oscillations in fertilized mouse eggs.

In most species, fertilization induces Ca transients in the egg. In mammals, the Ca rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP generated by PLCζ induces Ca release from the intracellular Ca store through IP receptor, termed IP-induced Ca release. Here, we developed new fluorescent IP sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.

Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes.

Ganglioside s are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods.

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.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al.

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy.

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters".

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.

Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains.

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes.

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging.

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules.

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.