GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold–induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Gαi2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein–protein and lipid–lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP3 production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Gαi2. Because both Gαi2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Gαi2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster

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. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model

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

Domain Growth Kinetics in a Cell-sized Liposome

We investigated the kinetics of domain growth on liposomes consisting of a ternary mixture (unsaturated phospholipid, saturated phospholipid, and cholesterol) by temperature jump. The domain growth process was monitored by fluorescence microscopy, where the growth was mediated by the fusion of domains through the collision. It was found that an average domain size r develops with time t as r ~ t^0.15, indicating that the power is around a half of the theoretical expectation deduced from a model of Brownian motion on a 2-dimensional membrane. We discuss the mechanism of the experimental scaling behavior by considering the elasticity of the membrane

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

細胞膜上の分子がバレエの群舞のように見えてきた： 1蛍光分子の感度で、究極速度で撮像できるカメラを開発. 京都大学プレスリリース. 2023-06-06.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. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640 × 640 pixel view-field

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. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48 ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 ms and 12 ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa

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

Influences of Excluded Volume of Molecules on Signaling Processes on Biomembrane

We investigate the influences of the excluded volume of molecules on biochemical reaction processes on 2-dimensional surfaces using a model of signal transduction processes on biomembranes. We perform simulations of the 2-dimensional cell-based model, which describes the reactions and diffusion of the receptors, signaling proteins, target proteins, and crowders on the cell membrane. The signaling proteins are activated by receptors, and these activated signaling proteins activate target proteins that bind autonomously from the cytoplasm to the membrane, and unbind from the membrane if activated. If the target proteins bind frequently, the volume fraction of molecules on the membrane becomes so large that the excluded volume of the molecules for the reaction and diffusion dynamics cannot be negligible. We find that such excluded volume effects of the molecules induce non-trivial variations of the signal flow, defined as the activation frequency of target proteins, as follows. With an increase in the binding rate of target proteins, the signal flow varies by i) monotonically increasing; ii) increasing then decreasing in a bell-shaped curve; or iii) increasing, decreasing, then increasing in an S-shaped curve. We further demonstrate that the excluded volume of molecules influences the hierarchical molecular distributions throughout the reaction processes. In particular, when the system exhibits a large signal flow, the signaling proteins tend to surround the receptors to form receptor-signaling protein clusters, and the target proteins tend to become distributed around such clusters. To explain these phenomena, we analyze the stochastic model of the local motions of molecules around the receptor.Comment: 31 pages, 10 figure

Confining Domains Lead to Reaction Bursts: Reaction Kinetics in the Plasma Membrane

Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity