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Stepping up CAR T cell sensitivity: Chimeric antigen receptors (CARs) were designed to mimic activated endogenous T cell receptors (TCRs) and induce a cytotoxic response to tumor antigen. However, CARs require more antigen than TCRs for activation, thereby causing treatment exclusion or failure and relapse in many patients. To design CARs with a lower activation threshold, Salter et al. compared antigen-induced pathway signaling in human T cells expressing a current CAR construct to that in cells with endogenous TCRs. The analysis informed the design of new CARs that more robustly activated a T cell co-receptor and an adaptor, resulting in CAR T cells that more effectively responded to low antigen density tumor cells in culture and decreased tumor burden and mortality in mice. Chimeric antigen receptor (CAR)–modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins—such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT—were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling. [ABSTRACT FROM AUTHOR]

Lateral organization in the plane of the plasma membrane is an important driver of biological processes. The past dozen years have seen increasing experimental support for the notion that lipid organization plays an important role in modulating this heterogeneity. Various biophysical mechanisms rooted in the concept of liquid–liquid phase separation have been proposed to explain diverse experimental observations of heterogeneity in model and cell membranes with distinct but overlapping applicability. In this review, we focus on the evidence for and the consequences of the hypothesis that the plasma membrane is poised near an equilibrium miscibility critical point. Critical phenomena explain certain features of the heterogeneity observed in cells and model systems but also go beyond heterogeneity to predict other interesting phenomena, including responses to perturbations in membrane composition. [ABSTRACT FROM AUTHOR]

Domain diffusion is studied as a function of composition and temperature and is measured how diffusion depends on domain size. Mechanisms of domain diffusion are consistent with membrane-dominated drag in viscous [L.sub.o] phases and bulk-dominated drag for less various [L.sub.[alpha]] phases.

The effects of fluorescence probes on the miscibility transition in multilamellar ternary vesicles are studied by using [super 2]H NMR. The results have shown that the miscibility transition in lipid membranes is sensitive to the presence of trace components, a property that can be used in biological processes.

Ion channels are embedded in the plasma membrane, a compositionally diverse two-dimensional liquid that has the potential to exert profound influence on their function. Recent experiments suggest that this membrane is poised close to an Ising critical point, below which cell-derived plasma membrane vesicles phase separate into coexisting liquid phases. Related critical points have long been the focus of study in simplified physical systems, but their potential roles in biological function have been underexplored. Here we apply both exact and stochastic techniques to the lattice Ising model to study several ramifications of proximity to criticality for idealized lattice channels, whose function is coupled through boundary interactions to critical fluctuations of membrane composition. Because of diverging susceptibilities of system properties to thermodynamic parameters near a critical point, such a lattice channel's activity becomes strongly influenced by perturbations that affect the critical temperature of the underlying Ising model. In addition, its kinetics acquire a range of time scales from its surrounding membrane, naturally leading to non-Markovian dynamics. Our model may help to unify existing experimental results relating the effects of small-molecule perturbations on membrane properties and ion channel function. We also suggest ways in which the role of this mechanism in regulating real ion channels and other membranebound proteins could be tested in the future. [ABSTRACT FROM AUTHOR]

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs. [ABSTRACT FROM AUTHOR]

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes. [ABSTRACT FROM AUTHOR]

Since its initial formalization nearly 20 years ago, the concept of lipid rafts has generated a tremendous amount of attention and interest and nearly as much controversy. The controversy is perhaps surprising because the notion itself is intuitive: compartmentalization in time and space is a ubiquitous theme at all scales of biology, and therefore, the partitioning of cellular membranes into lateral subdivision should be expected. Nevertheless, the physicochemical principles responsible for compartmentalization and the molecular mechanisms by which they are functionalized remain nearly as mysterious today as they were two decades ago. Herein, we review recent literature on this topic with a specific focus on the major open questions in the field including: (1) what are the best tools to assay raft behavior in living membranes? (2) what is the function of the complex lipidome of mammalian cells with respect to membrane organization? (3) what are the mechanisms that drive raft formation and determine their properties? (4) how can rafts be modulated? (5) how is membrane compartmentalization integrated into cellular signaling? Despite decades of intensive research, this compelling field remains full of fundamental questions. [ABSTRACT FROM AUTHOR]

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients. [ABSTRACT FROM AUTHOR]

Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions. [ABSTRACT FROM AUTHOR]

HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-( 4,5)-bisphosphate [PI(4,5)P2]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2 with unsaturated or saturated acyl chains affect membrane binding of PHPLCδ1 and Gag. Both unsaturated dioleoyl-PI(4,5)P2 [DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2 [DP-PI(4,5)P2] successfully recruited PHPLCδ1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2 but not DP-PI(4,5)P2 recruited Gag to GUVs, indicating that PI(4,5)P2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLCδ1 regardless of PI(4,5)P2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2 had DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2 still promoted recruitment of Gag, but not PHPLCδ1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2 acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLCδ1 . Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P2 containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2 acyl chain saturation is determined directly by the matrix-PI(4,5)P2 interaction, rather than indirectly by a myristate-dependent mechanism. IMPORTANCE Binding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P2. Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P2-interacting domains represented by PHPLCδ1 . However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P2 with an unsaturated acyl chain recruited PHPLCδ1 and Gag similarly, whereas PI(4,5)P2 with saturated acyl chains either recruited PHPLCδ1 but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P2 acyl chains differentially modulate membrane binding of PI(4,5)P2-binding proteins. Since Gag membrane binding is essential for progeny virion production, the PI(4,5)P2 acyl chain property may serve as a potential target for anti-HIV therapeutic strategies. [ABSTRACT FROM AUTHOR]

HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants. [ABSTRACT FROM AUTHOR]

Recent experiments suggest that membranes of living cells are tuned close to a miscibility critical point in the two-dimensional Ising universality class. We propose that one role for this proximity to criticality in live cells is to provide a conduit for relatively long-range critical Casimir forces. Using techniques from conformal field theory we calculate potentials of mean force between membrane bound inclusions mediated by their local interactions with the composition order parameter. We verify these calculations using Monte Carlo simulations where we also compare critical and off-critical results. Our findings suggest that membrane bound proteins experience weak yet long-range forces mediated by critical composition fluctuations in the plasma membranes of living cells. [ABSTRACT FROM AUTHOR]

Antigen-mediated cross-linking of IgE bound to its receptor,FcεRI,initiates a transmembrane signaling cascade that results in mast cellactivation in the allergic response. Using immunogold labeling ofintact RBL mast cells and scanning electron microscopy (SEM), we visualizemolecular reorganization of IgE-FcεRI and early signaling proteinson both leaflets of the plasma membrane, without the need for rippedoff membrane sheets. As quantified by pair correlation analysis, weobserve dramatic changes in the nanoscale distribution of IgE-FcεRIafter binding of multivalent antigen to stimulate transmembrane signaling,and this is accompanied by similar clustering of Lyn and Syk tyrosinekinases, and adaptor protein LAT. We find that Lyn co-redistributeswith IgE-FcεRI into clusters that cross-correlate throughout20 min of stimulation. Inhibition of tyrosine kinase activity reducesthe numbers of both IgE-FcεRI and Lyn in stimulated clusters.Coupling of these proteins is also decreased when membrane cholesterolis reduced either before or after antigen addition. These resultsprovide evidence for involvement of FcεRI phosphorylation andcholesterol-dependent membrane structure in the interactions thataccompany IgE-mediated activation of RBL mast cells. More generally,this SEM view of intact cell surfaces provides new insights into thenanoscale organization of receptor-mediated signaling complexes inthe plasma membrane. [ABSTRACT FROM AUTHOR]

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcϵRI) on the plasma membranes of chemically fixed RBL- 2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcϵRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcϵRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcϵRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgEFcϵRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. [ABSTRACT FROM AUTHOR]

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies. [ABSTRACT FROM AUTHOR]

Abstract: Scaling laws associated with critical points have the power to greatly simplify our description of complex biophysical systems. We first review basic concepts and equations associated with critical phenomena for the general reader. We then apply these concepts to the specific biophysical system of lipid membranes. We recently reported that lipid membranes can contain composition fluctuations that behave in a manner consistent with the two-dimensional Ising universality class. Near the membrane''s critical point, these fluctuations are micron-sized, clearly observable by fluorescence microscopy. At higher temperatures, above the critical point, we expect to find submicron fluctuations. In separate work, we have reported that plasma membranes isolated directly from cells exhibit the same Ising behavior as model membranes do. We review other models describing submicron lateral inhomogeneity in membranes, including microemulsions, nanodomains, and mean field critical fluctuations, and we describe experimental tests that may distinguish these models. [Copyright &y& Elsevier]

Abstract: Macroscopic coexisting liquid phases are readily observed in certain model membranes containing cholesterol and at least two other lipid components. Recent fluorescence microscopy and deuterium NMR work indicates that submicron fluctuations are also found, in vesicles with near-critical lipid compositions. In principle, the magnitude of critical fluctuations can be controlled by changing temperature, or through other means of shifting the phase boundary such as including impurities or cross-linking components. Critical fluctuations are dynamic submicron domains in model membranes, and provide a plausible physical mechanism to produce putative ‘raft’ domains in cholesterol-rich biomembranes. [Copyright &y& Elsevier]

Abstract: Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes. [Copyright &y& Elsevier]

The article presents information on the lipids in the influenza viral envelope. the article defines the term "lipids" as small, very mobile biomolecules that depend on their structure and interactions to carry out their biological functions. The nature and functions of lipids are also given attention.

Abstract: In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcεRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE–FcεRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells. [Copyright &y& Elsevier]

[Display omitted] Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells. [ABSTRACT FROM AUTHOR]

Tetherin/BST-2 (here called tetherin) is an antiviral protein that restricts release of diverse enveloped viruses from infected cells through physically tethering virus envelope and host plasma membrane. For HIV-1, specific recruitment of tetherin to assembly sites has been observed as its colocalization with the viral structural protein Gag or its accumulation in virus particles. Because of its broad range of targets, we hypothesized that tetherin is recruited through conserved features shared among various enveloped viruses, such as lipid raft association, membrane curvature, or ESCRT dependence. We observed that reduction of cellular cholesterol does not block tetherin anti-HIV-1 function, excluding an essential role for lipid rafts. In contrast, mutations in the capsid domain of Gag, which inhibit induction of membrane curvature, prevented tetherin-Gag colocalization detectable by con focal microscopy. Disruption of Gag-ESCRT interactions also inhibited tetherin-Gag colocalization when disruption was accomplished via amino acid substitutions in late domain motifs, expression of a dominant-negative TsglOl derivative, or small interfering RNA (siRNA)-mediated depletion of TsglOl or Alix. However, further analyses of these conditions by quantitative superresolution localization microscopy revealed that Gag-tetherin coclustering is significantly reduced but persists at intermediate levels. Notably, this residual tetherin recruitment was still sufficient for the full restriction of HIV-1 release. Unlike the late domain mutants, the capsid mutants defective in inducing membrane curvature showed little or no coclustering with tetherin in superresolution analyses. These results support a model in which both Gag-induced membrane curvature and Gag-ESCRT interactions promote tetherin recruitment, but the recruitment level achieved by the former is sufficient for full restriction. [ABSTRACT FROM AUTHOR]

Determinants regulating sorting of host transmembrane proteins at sites of enveloped virus assembly on the plasma membrane (PM) remain poorly understood. Here, we demonstrate for the first time that PM acidic phospholipid PIP2 regulates such sorting into an enveloped virus, HIV-1. Incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles is known to have profound effects on viral spread; however, the mechanisms promoting their incorporation were unknown. We found that depletion of cellular PIP2 blocks the incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles. Expansion microscopy revealed that PIP2 depletion diminishes nanoscale co-clustering between viral structural protein Gag and the three transmembrane proteins at PM and that Gag induces PIP2 enrichment around itself. CD43, PSGL-1, and CD44 also increased local PIP2 density, revealing their PIP2 affinity. Altogether, these results support a new mechanism where local enrichment of an acidic phospholipid drives co-clustering between viral structural and cellular transmembrane proteins, thereby modulating the content, and hence the fate, of progeny virus particles., Competing Interests: Competing interests The authors declare no competing interests.

Both membranes and biopolymers can individually separate into coexisting liquid phases. Here we explore biopolymer prewetting at membranes, a phase transition that emerges when these two thermodynamic systems are coupled. In reconstitution, we couple short poly-L-Lysine and poly-L-Glutamic Acid polyelectrolytes to membranes of saturated lipids, unsaturated lipids, and cholesterol, and detect coexisting prewet and dry surface phases well outside of the region of coexistence for each individual system. Notability, polyelectrolyte prewetting is highly sensitive to membrane lipid composition, occurring at 10 fold lower polymer concentration in a membrane close to its phase transition compared to one without a phase transition. In cells, protein prewetting is achieved using an optogenetic tool that enables titration of condensing proteins and tethering to the plasma membrane inner leaflet. Here we show that protein prewetting occurs for conditions well outside those where proteins condense in the cytoplasm, and that the stability of prewet domains is sensitive to perturbations of plasma membrane composition and structure. Our work presents an example of how thermodynamic phase transitions can impact cellular structure outside their individual coexistence regions, suggesting new possible roles for phase-separation-prone systems in cell biology.

Several groups have recently reported evidence for the emergence of domains in cell plasma membranes when membrane proteins are organized by ligand binding or assembly of membrane proximal scaffolds. These domains recruit and retain components that favor the liquid-ordered phase, adding to a decades-old literature interrogating the contribution of membrane phase separation in plasma membrane organization and function. Here we propose that both past and present observations are consistent with a model in which membranes have a high compositional susceptibility, arising from their thermodynamic state in a single phase that is close to a miscibility phase transition. This rigorous framework naturally allows for both transient structure in the form of composition fluctuations and long-lived structure in the form of induced domains. In this way, the biological tuning of plasma membrane composition enables a responsive compositional landscape that facilitates and augments cellular biochemistry vital to plasma membrane functions., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of nonideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (μ c ) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, referenced to crystalline cholesterol. μ c is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase separated mixture. It is simply related to concentration under conditions of ideal mixing, but is far from ideal for the majority of lipid mixtures investigated here. Measurements of μ c can vary with phospholipid composition by 1.5 k B T at constant cholesterol mole fraction implying a more than fivefold change in its availability for binding receptors and other reactions. Experimental measurements are fit to thermodynamic models including cholesterol-DPPC complexes or pairwise interactions between lipid species to provide intuition about the magnitude of interactions. These findings reinforce that μ c depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single molecule imaging in live cells enables the study of protein interactions and dynamics as they participate in signaling processes. When combined with fluorophores that stochastically transition between fluorescent and reversible dark states, as in super-resolution localization imaging, labeled molecules can be visualized in single cells over time. This improvement in sampling enables the study of extended cellular responses at the resolution of single molecule localization. This chapter provides optimized experimental and analytical methods used to quantify protein interactions and dynamics within the membranes of adhered live cells. Importantly, the use of pair-correlation functions resolved in both space and time allows researchers to probe interactions between proteins on biologically relevant distance and timescales, even though fluorescence localization methods typically require long times to assemble well-sampled reconstructed images. We describe an application of this approach to measure protein interactions in B cell receptor signaling and include sample analysis code for post-processing of imaging data. These methods are quantitative, sensitive, and broadly applicable to a range of signaling systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single-molecule localization microscopy techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here, we present an efficient and robust method for estimating drift, using a simple peak-finding algorithm based on mean shifts that is effective for single-molecule localization microscopy in two or three dimensions.