Microscopic simulation of membrane molecule diffusion on corralled membrane surfaces

The current understanding of how receptors diffuse and cluster in the plasma membrane is limited. Data from single-particle tracking and laser tweezer experiments have suggested that membrane molecule diffusion is affected by the presence of barriers dividing the membrane into corrals. Here, we have developed a stochastic spatial model to simulate the effect of corrals on the diffusion of molecules in the plasma membrane. The results of this simulation confirm that a fence barrier (the ratio of the transition probability for diffusion across a boundary to that within a corral) on the order of 103–104 recreates the experimentally measured difference in diffusivity between artificial and natural plasma membranes. An expression for the macroscopic diffusivity of receptors on corralled membranes is derived to analyze the effects of the corral parameters on diffusion rate. We also examine whether the lattice model is an appropriate description of the plasma membrane and look at three different sets of boundary conditions that describe diffusion over the barriers and whether diffusion events on the plasma membrane may occur with a physically relevant length scale. Finally, we show that to observe anomalous (two-timescale) diffusion, one needs high temporal (microsecond) resolution along with sufficiently long (more than milliseconds) trajectories

Probing microscopic origins of confined subdiffusion by first-passage observables

Subdiffusive motion of tracer particles in complex crowded environments, such as biological cells, has been shown to be widepsread. This deviation from brownian motion is usually characterized by a sublinear time dependence of the mean square displacement (MSD). However, subdiffusive behavior can stem from different microscopic scenarios, which can not be identified solely by the MSD data. In this paper we present a theoretical framework which permits to calculate analytically first-passage observables (mean first-passage times, splitting probabilities and occupation times distributions) in disordered media in any dimensions. This analysis is applied to two representative microscopic models of subdiffusion: continuous-time random walks with heavy tailed waiting times, and diffusion on fractals. Our results show that first-passage observables provide tools to unambiguously discriminate between the two possible microscopic scenarios of subdiffusion. Moreover we suggest experiments based on first-passage observables which could help in determining the origin of subdiffusion in complex media such as living cells, and discuss the implications of anomalous transport to reaction kinetics in cells.Comment: 21 pages, 3 figures. Submitted versio

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

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

Membrane shape as a reporter for applied forces

Recent advances have enabled 3-dimensional reconstructions of biological structures in vivo, ranging in size and complexity from single proteins to multicellular structures. In particular, tomography and confocal microscopy have been exploited to capture detailed 3-dimensional conformations of membranes in cellular processes ranging from viral budding and organelle maintenance to phagocytosis. Despite the wealth of membrane structures available, there is as yet no generic, quantitative method for their interpretation. We propose that by modeling these observed biomembrane shapes as fluid lipid bilayers in mechanical equilibrium, the externally applied forces as well as the pressure, tension, and spontaneous curvature can be computed directly from the shape alone. To illustrate the potential power of this technique, we apply an axial force with optical tweezers to vesicles and explicitly demonstrate that the applied force is equal to the force computed from the membrane conformation

Papers please: Predictive factors of national and international attitudes toward immunity and vaccination passports. Online representative surveys

BACKGROUND: In response to the COVID-19 pandemic, countries are introducing digital passports that allow citizens to return to normal activities if they were previously infected with (immunity passport) or vaccinated against (vaccination passport) SARS-CoV-2. To be effective, policy decision-makers must know whether these passports will be widely accepted by the public and under what conditions. This study focuses attention on immunity passports, as these may prove useful in countries both with and without an existing COVID-19 vaccination program; however, our general findings also extend to vaccination passports. OBJECTIVE: We aimed to assess attitudes toward the introduction of immunity passports in six countries, and determine what social, personal, and contextual factors predicted their support. METHODS: We collected 13,678 participants through online representative sampling across six countries—Australia, Japan, Taiwan, Germany, Spain, and the United Kingdom—during April to May of the 2020 COVID-19 pandemic, and assessed attitudes and support for the introduction of immunity passports. RESULTS: Immunity passport support was moderate to low, being the highest in Germany (775/1507 participants, 51.43%) and the United Kingdom (759/1484, 51.15%); followed by Taiwan (2841/5989, 47.44%), Australia (963/2086, 46.16%), and Spain (693/1491, 46.48%); and was the lowest in Japan (241/1081, 22.94%). Bayesian generalized linear mixed effects modeling was used to assess predictive factors for immunity passport support across countries. International results showed neoliberal worldviews (odds ratio [OR] 1.17, 95% CI 1.13-1.22), personal concern (OR 1.07, 95% CI 1.00-1.16), perceived virus severity (OR 1.07, 95% CI 1.01-1.14), the fairness of immunity passports (OR 2.51, 95% CI 2.36-2.66), liking immunity passports (OR 2.77, 95% CI 2.61-2.94), and a willingness to become infected to gain an immunity passport (OR 1.6, 95% CI 1.51-1.68) were all predictive factors of immunity passport support. By contrast, gender (woman; OR 0.9, 95% CI 0.82-0.98), immunity passport concern (OR 0.61, 95% CI 0.57-0.65), and risk of harm to society (OR 0.71, 95% CI 0.67-0.76) predicted a decrease in support for immunity passports. Minor differences in predictive factors were found between countries and results were modeled separately to provide national accounts of these data. CONCLUSIONS: Our research suggests that support for immunity passports is predicted by the personal benefits and societal risks they confer. These findings generalized across six countries and may also prove informative for the introduction of vaccination passports, helping policymakers to introduce effective COVID-19 passport policies in these six countries and around the world

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

Curvature-coupling dependence of membrane protein diffusion coefficients

We consider the lateral diffusion of a protein interacting with the curvature of the membrane. The interaction energy is minimized if the particle is at a membrane position with a certain curvature that agrees with the spontaneous curvature of the particle. We employ stochastic simulations that take into account both the thermal fluctuations of the membrane and the diffusive behavior of the particle. In this study we neglect the influence of the particle on the membrane dynamics, thus the membrane dynamics agrees with that of a freely fluctuating membrane. Overall, we find that this curvature-coupling substantially enhances the diffusion coefficient. We compare the ratio of the projected or measured diffusion coefficient and the free intramembrane diffusion coefficient, which is a parameter of the simulations, with analytical results that rely on several approximations. We find that the simulations always lead to a somewhat smaller diffusion coefficient than our analytical approach. A detailed study of the correlations of the forces acting on the particle indicates that the diffusing inclusion tries to follow favorable positions on the membrane, such that forces along the trajectory are on average smaller than they would be for random particle positions.Comment: 16 pages, 8 figure

The molecular epidemiology of human immunodeficiency virus type 1 in six cities in Britain and Ireland

The authors sequenced the p17 coding regions of the gag gene from 211 patients infected either through injecting drug use (IDU) or by sexual intercourse between men from six cities in Scotland, N. England, N. Ireland, and the Republic of Ireland. All sequences were of subtype 5. Phylogenetic analysis revealed substantial heterogeneity in the sequences from homosexual men. In contrast, sequence from over 80% of IDUs formed a relatively tight cluster, distinct both from those of published isolates and of the gay men. There was no large-scale clustering of sequences by city in either risk group, although a number of close associations between pairs of individuals were observed. From the known date of the HIV-1 epidemic among IDUs in Edinburgh, the rate of sequence divergence at synonymous sites is estimated to be about 0.8%. On this basis it has been estimated that the date of divergence of the sequences among homosexual men to be about 1975, which may correspond to the origin of the B subtype epidemic

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