Receptor Mobility Research Articles

Proton-triggered rearrangement of the AMPA receptor N-terminal domains impacts receptor kinetics and synaptic localization.

AMPA glutamate receptors (AMPARs) are ion channel tetramers that mediate the majority of fast excitatory synaptic transmission. They are composed of four subunits (GluA1-GluA4); the GluA2 subunit dominates AMPAR function throughout the forebrain. Its extracellular N-terminal domain (NTD) determines receptor localization at the synapse, ensuring reliable synaptic transmission and plasticity. This synaptic anchoring function requires a compact NTD tier, stabilized by a GluA2-specific NTD interface. Here we show that low pH conditions, which accompany synaptic activity, rupture this interface. All-atom molecular dynamics simulations reveal that protonation of an interfacial histidine residue (H208) centrally contributes to NTD rearrangement. Moreover, in stark contrast to their canonical compact arrangement at neutral pH, GluA2 cryo-electron microscopy structures exhibit a wide spectrum of NTD conformations under acidic conditions. We show that the consequences of this pH-dependent conformational control are twofold: rupture of the NTD tier slows recovery from desensitized states and increases receptor mobility at mouse hippocampal synapses. Therefore, a proton-triggered NTD switch will shape both AMPAR location and kinetics, thereby impacting synaptic signal transmission.

Prior Fc receptor activation primes macrophages for increased sensitivity to IgG via long-term and short-term mechanisms.

Macrophages measure the "eat-me" signal immunoglobulin G (IgG) to identify targets for phagocytosis. We tested whether prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc receptor. To temporally control Fc receptor activation, we engineered an Fc receptor that is activated by the light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that subthreshold Fc receptor activation primes mouse bone-marrow-derived macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced subthreshold Fc receptor activation eat more IgG-bound human cancer cells. Increased phagocytosis occurs by two discrete mechanisms-a short- and long-term priming. Long-term priming requires new protein synthesis and Erk activity. Short-term priming does not require new protein synthesis and correlates with an increase in Fc receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

Structural and Functional Characteristics of the Oral Cavity Organs and Tissues in Workers of the Petrochemical Industry of the Republic of Uzbekistan.

Purpose: The aim of the study was to determine the dental status, level, and structure of dental diseases, as well as to identify some clinical and functional deviations in the organs and tissues of the oral cavity among workers in certain industrial enterprises. Methodology: A comprehensive dental examination was conducted on 1600 individuals, including 420 from the Fergana Oil Refinery Plant (FNPP - main group (MG)-1), 425 from the Almalyk Mining and Metallurgical Plant (AGMK - MG-2), and 605 from the Navoi Chemical Plant (NavCP - MG-3), as well as 150 patients as a control group (CG) who visited a dental clinic. Tooth sensitivity (TS), pain and discriminatory sensitivity (PS and DS) of the oral mucosa, taste perception threshold, and functional mobility of the taste receptors on the tongue, enamel resistance to caries, microhardness (MH) of enamel and dentin, and the condition of periodontal tissues and pH of mixed saliva were determined. The data obtained were processed using statistical methods. Findings: It was determined that the levels and relative weights of the main dental diseases among workers in the studied industries remain quite high; carious and non-carious lesions of the tooth hard tissues, deformation and defects of teeth and dental rows (18.5%), periodontal tissue diseases (74.7%), diseases of the oral mucosa - 38.7%; the need for prosthetics (58.4%) and other pathologies. Additionally, almost all clinical and functional indicators and local non-specific reactivity of the tissues of the oral cavity in workers were disrupted. Unique Contribution to Theory, Policy and Practice: The presence of the main dental diseases among those surveyed from the studied groups is quite high, with noted reductions in the excitability threshold of teeth, around tooth tissues and the oral mucosa, disruption of PS and DS of the oral mucosa, as well as deterioration of the condition of tooth hard tissues. Based on the obtained results, the authors suggest continuing in-depth scientific research with the aim of developing effective methods for the diagnosis and prevention of dental diseases among workers in these industries.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Unraveling fast G-protein coupled receptor mobility and binding

Unraveling fast G-protein coupled receptor mobility and binding

NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting

Endothelial cells express neuropilin 1 (NRP1), endoglin (ENG) and vascular endothelial growth factor receptor 2 (VEGFR2), which regulate VEGF-A-mediated vascular development and angiogenesis. However, the link between complex formation among these receptors with VEGF-A-induced signaling and biology is yet unclear. Here, we quantify surface receptor interactions by IgG-mediated immobilization of one receptor, and fluorescence recovery after photobleaching (FRAP) measurements of the mobility of another coexpressed receptor. We observe stable ENG/NRP1, ENG/VEGFR2, and NRP1/VEGFR2 complexes, which are enhanced by VEGF-A. ENG augments NRP1/VEGFR2 interactions, suggesting formation of tripartite complexes bridged by ENG. Effects on signaling are measured in murine embryonic endothelial cells expressing (MEEC+/+) or lacking (MEEC-/-) ENG, along with NRP1 and/or ENG overexpression or knockdown. We find that optimal VEGF-A-mediated phosphorylation of VEGFR2 and Erk1/2 requires ENG and NRP1. ENG or NRP1 increase VEGF-A-induced sprouting, becoming optimal in cells expressing all three receptors, and both processes are inhibited by a MEK1/2 inhibitor. We propose a model where the maximal potency of VEGF-A involves a tripartite complex where ENG bridges VEGFR2 and NRP1, providing an attractive therapeutic target for modulation of VEGF-A signaling and biological responses.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case–control study

A lack of biomarkers that detect drug-induced liver injury (DILI) accurately continues to hinder early- and late-stage drug development and remains a challenge in clinical practice. The Innovative Medicines Initiative’s TransBioLine consortium comprising academic and industry partners is developing a prospective repository of deeply phenotyped cases and controls with biological samples during liver injury progression to facilitate biomarker discovery, evaluation, validation and qualification.In a nested case–control design, patients who meet one of these criteria, alanine transaminase (ALT) ≥ 5 × the upper limit of normal (ULN), alkaline phosphatase ≥ 2 × ULN or ALT ≥ 3 ULN with total bilirubin > 2 × ULN, are enrolled. After completed clinical investigations, Roussel Uclaf Causality Assessment and expert panel review are used to adjudicate episodes as DILI or alternative liver diseases (acute non-DILI controls). Two blood samples are taken: at recruitment and follow-up. Sample size is as follows: 300 cases of DILI and 130 acute non-DILI controls. Additional cross-sectional cohorts (1 visit) are as follows: Healthy volunteers (n = 120), controls with chronic alcohol-related or non-alcoholic fatty liver disease (n = 100 each) and patients with psoriasis or rheumatoid arthritis (n = 100, 50 treated with methotrexate) are enrolled. Candidate biomarkers prioritised for evaluation include osteopontin, glutamate dehydrogenase, cytokeratin-18 (full length and caspase cleaved), macrophage-colony-stimulating factor 1 receptor and high mobility group protein B1 as well as bile acids, sphingolipids and microRNAs. The TransBioLine project is enabling biomarker discovery and validation that could improve detection, diagnostic accuracy and prognostication of DILI in premarketing clinical trials and for clinical healthcare application.

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

A new simulation framework to evaluate the suitability of eDNA for marine and aquatic Environmental Impact Assessments

AbstractA model was developed to forecast and compare changes in species presence assessed with either eDNA or traditional observations. We use it to explore how ecosystem conditions could affect the suitability of eDNA for Environmental Impact Assessment. First, a deterministic model simulated the dynamics of the impacted population (called “receptor” in EIA) and their shed DNA fragment concentrations. Second, random distributions of receptor organisms and eDNA fragment quantities at steady state were simulated within the impacted spatial domain (called “project area”). Then, simple random samplings were performed for both the receptor and eDNA. Third, post‐sampling processes (eDNA extraction, amplification, and analysis) were simulated to estimate the taxon detection probability. Fourth, we simulated an impact by modifying the growth, mortality, and mobility (null, passive, and active) parameters of the receptor taxon. eDNA detection probability curves were then estimated for a range of environmental sample volumes by fitting a Weibull cumulative distribution function. A F‐like statistic compared detection curves before and after impact. Statistically significant differences were detected with eDNA in impact scenarios where receptor taxon growth rate decreased and receptor mobility was null or passive. In scenarios where the project area accumulates DNA shed from multiple categories of the same taxon (e.g., from dead organisms if mortality increased or when individuals can cross project area boundaries), it is difficult to assess impact. Our study shows that results obtained from eDNA sampling will not always agree with an impact classically assessed on a receptor population. One reason is that sources of the total eDNA pool are not identified. The modeling highlights the need: to do preliminary testing of sample sizes, to develop new approaches that will identify sources from the pool of extracted DNA, and to improve descriptions of the ecogeochemical processes required to forecast shed DNA reactivity.

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors.

G-protein-coupled receptor (GPCR) density at the cell surface is thought to regulate receptor function. Spatially resolved measurements of local-density effects on GPCRs are needed but technically limited by density heterogeneity and mobility of membrane receptors. We now develop a deep-learning (DL)-enhanced diffusion imaging assay that can measure local-density effects on ligand-receptor interactions in the plasma membrane of live cells. In this method, the DL algorithm allows the transformation of 100 ms exposure images to density maps that report receptor numbers over any specified region with ∼95% accuracy by 1 s exposure images as ground truth. With the density maps, a diffusion assay is further established for spatially resolved measurements of receptor diffusion coefficient as well as to express relationships between receptor diffusivity and local density. By this assay, we scrutinize local-density effects on chemokine receptor CXCR4 interactions with various ligands, which reveals that an agonist prefers to act with CXCR4 at low density while an inverse agonist dominates at high density. This work suggests a new insight into density-dependent receptor regulation as well as provides an unprecedented assay that can be applicable to a wide variety of receptors in live cells.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Effect of St. John’s wort extract Ze 117 on the lateral mobility of β1-adrenergic receptors in C6 cells

Depression has been associated with altered signal transduction of serotonergic, dopaminergic and adrenergic neurotransmitter systems in the brain. Signaling relies on receptor-ligand interactions and subsequent regulatory processes, but also on lateral receptor mobility. The aim of this study was to investigate the effect of the St. John’s wort extract Ze 117 on the lateral mobility of SNAP-tagged β1-adrenergic receptors (β1AR) in the plasma membrane of C6 cells under both, non-stimulating and isoprenaline-stimulating conditions. Single particle tracking (SPT) was used, whereby the registered trajectories were evaluated by variational Bayesian treatment of a hidden Markov model (vbSPT) and packing coefficient (Pc) analysis with respect to diffusion coefficients, receptor state occupancies and confinement. Three different diffusion states were identified, differing in their diffusion coefficients. Treatment with Ze 117 [25 µg/ml] decreased the mobility of the β1AR, which was manifested by a relative increase in the slow-diffusing state S1 (0.21–0.30) compared to control and by an increase in receptor confinement (79.4–68.1 nm). After isoprenaline stimulation of control cells, the slow-diffusing state was more pronounced, whereas confinement was not affected. In summary, SPT has been shown to be a powerful method to analyze lateral receptor mobility. Furthermore, the present study identified a correlation between Ze 117 treatment and β1AR mobility.

Spinal Cord Synaptic Plasticity by GlyRβ Release from Receptor Fields and Syndapin I-Dependent Uptake.

Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We imaged receptor fields at ultrahigh-resolution at freeze-fractured membranes, tracked surface and internalized glycine receptors (GlyR), and studied differential regulations of GlyRβ interactions with the scaffold protein gephyrin and the F-BAR domain protein syndapin I and thereby reveal key principles of this process. S403 phosphorylation of GlyRβ, known to be triggered by synaptic signaling, caused a decoupling from gephyrin scaffolds but simultaneously promoted association of syndapin I with GlyRβ. In line, kainate treatments used to trigger rearrangements of glycine receptors in murine syndapin I KO spinal cords (mixed sex) showed even more severe receptor field fragmentation than already observed in untreated syndapin I KO spinal cords. Syndapin I deficiency furthermore resulted in more dispersed receptors and increased receptor mobility, also pointing out an important contribution of syndapin I to the organization of GlyRβ fields. Strikingly, syndapin I KO also led to a complete disruption of kainate-induced GlyRβ internalization. Accompanying quantitative ultrahigh-resolution studies in dissociated spinal cord neurons proved that the defects in GlyR internalization observed in syndapin I KO spinal cords are neuron-intrinsic defects caused by syndapin I deficiency. Together, our results unveiled important mechanisms organizing and altering glycine receptor fields during both steady state and particularly also as a consequence of kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy and plasticity of glycinergic synapses in the spinal cord.SIGNIFICANCE STATEMENT Initial observations suggested that also glycinergic synapses, key for spinal cord and brainstem functions, may display some form of synaptic plasticity. Imaging receptor fields at ultrahigh-resolution at freeze-fractured membranes, tracking surface and internalized glycine receptors (GlyR) and studying regulations of GlyRβ interactions, we here reveal key principles of these kainate-inducible adaptations. A switch from gephyrin-mediated receptor scaffolding to syndapin I-mediated GlyRβ scaffolding and internalization allows for modulating synaptic receptor availability. In line, kainate-induced GlyRβ internalization was completely disrupted and GlyRβ receptor fields were distorted by syndapin I KO. These results unveiled important mechanisms during both steady-state and kainate-induced alterations of synaptic GlyR fields, principles underlying synaptic efficacy and plasticity of synapses in the spinal cord.

St. John's wort extract STW3-VI affects the binding behaviour, signal transduction and lateral mobility of 5-HT2A receptors observed by single molecule detection and the use of a novel fluorescence-labeled ligand

Background5-HT2A receptors play an important role in the development and therapy of depression. St. John's wort (SJW) extracts are used in the treatment of depression. The effects of pre-treatment with SJW extract on the behaviour of 5-HT2A receptors in the plasma membrane have not been well described at the molecular level. MethodsIn this work, the influence of the SJW extract STW3-VI on 5-HT2A receptor internalization and calcium response under stimulating conditions are determined using different reporter cell lines. Influences on 5-HT2A receptor binding as well as on the lateral diffusion behaviour of receptor-ligand complexes (RLC) in the plasma membrane are investigated by fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) in real time on living cells. Therefore, a selective fluorescently labeled ligand was first developed. ResultsSTW3-VI pre-treatment inhibited both the 5-HT2A receptor internalization and the calcium response after agonistic stimulation with (±)-TCB-2, which suggests an inhibitory effect. Receptor binding studies using FCS surprisingly showed a dose-dependent increase in 5-HT2A receptor binding under STW3-VI. However, it is noticeable that there was a clear redistribution towards faster diffusing RLC, who possibly contribute less to signal transduction. The diffusive states of 5-HT2A receptors identified by SPT changed also under STW3-VI. Remarkably, there was a redistribution from faster diffusing to immobile RLC, while at the same time the transition probabilities for a receptor to change the diffusive state decreased clearly compared to control cells. This particular lateral diffusion behaviour likely characterizes 5-HT2A receptors with impaired signal transduction. ConclusionThe present data indicate a correlation between receptor status and lateral mobility, with STW3-VI apparently having an inhibitory effect on 5-HT2A receptor-mediated signaling in the cell models used here. Single-molecule techniques such as FCS and SPT provide important data that contribute to a better understanding of therapeutically relevant drug-target interactions and downstream regulatory processes.

Sphingomyelin Depletion Inhibits CXCR4 Dynamics and CXCL12-Mediated Directed Cell Migration in Human T Cells.

Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior.

MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

Unraveling the hidden temporal range of fast \u03b22-adrenergic receptor mobility by time-resolved fluorescence

G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β2-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.

Receptor tyrosine kinase MET ligand-interaction classified via machine learning from single-particle tracking data.

Internalin B–mediated activation of the membrane-bound receptor tyrosine kinase MET is accompanied by a change in receptor mobility. Conversely, it should be possible to infer from receptor mobility whether a cell has been treated with internalin B. Here, we propose a method based on hidden Markov modeling and explainable artificial intelligence that machine-learns the key differences in MET mobility between internalin B–treated and –untreated cells from single-particle tracking data. Our method assigns receptor mobility to three diffusion modes (immobile, slow, and fast). It discriminates between internalin B–treated and –untreated cells with a balanced accuracy of >99% and identifies three parameters that are most affected by internalin B treatment: a decrease in the mobility of slow molecules (1) and a depopulation of the fast mode (2) caused by an increased transition of fast molecules to the slow mode (3). Our approach is based entirely on free software and is readily applicable to the analysis of other membrane receptors.

Significance of Receptor Mobility in Multivalent Binding on Lipid Membranes

AbstractNumerous key biological processes rely on the concept of multivalency, where ligands achieve stable binding only upon engaging multiple receptors. These processes, like viral entry or immune synapse formation, occur on the diffusive cellular membrane. One crucial, yet underexplored aspect of multivalent binding is the mobility of coupled receptors. Here, we discuss the consequences of mobility in multivalent processes from four perspectives: (I) The facilitation of receptor recruitment by the multivalent ligand due to their diffusivity prior to binding. (II) The effects of receptor preassembly, which allows their local accumulation. (III) The consequences of changes in mobility upon the formation of receptor/ligand complex. (IV) The changes in the diffusivity of lipid environment surrounding engaged receptors. We demonstrate how understanding mobility is essential for fully unravelling the principles of multivalent membrane processes, leading to further development in studies on receptor interactions, and guide the design of new generations of multivalent ligands.