Plasma Membrane Of Living Cells Research Articles

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Real-time fluorescence imaging with 20 nm axial resolution.

Measuring the nanoscale organization of protein structures near the plasma membrane of live cells is challenging, especially when the structure is dynamic. Here we present the development of a two-wavelength total internal reflection fluorescence method capable of real-time imaging of cellular structure height with nanometre resolution. The method employs a protein of interest tagged with two different fluorophores and imaged to obtain the ratio of emission in the two channels. We use this approach to visualize the nanoscale organization of microtubules and endocytosis of the epidermal growth factor receptor.

A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells

Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin–Darby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane.

STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics.

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.

Investigating the Role of Cellular Lipid Recognition in Trafficking and Viral Particle Formation by Ebola Virus Matrix Protein VP40

Introduction Virulent pathogens hijack host cell machinery during their infection scheme. Commonly, fundamental biochemical processes are discovered through investigation of pathogen infection, especially in the case of pathogen modulation of low abundance lipid species. Ebola virus (EBOV) is a highly virulent, filamentous, lipid-enveloped virus that causes hemorrhagic fever and a fatality rate of 50-90%. The limited understanding of the viral replication in host cells proves difficult for understanding how it spreads. EBOV only has seven genes in its genome, one of which-VP40-is essential for viral egress. VP40 can form virus like particles (VLPs) on its own at the plasma membrane (PM) in live cells and is an excellent model for studying EBOV spread. Objective: We are implementing several assays in live COS-7 cells to decode the PM phospholipid specificity where viral particle formation occurs. Methods: Confocal Laser Scanning Microscopy (CLSM), Spinning Disk and Total Internal Reflection Microscopy (TIRF), VLP Collection. Results: VP40 PM localization can be blocked by elimination of phospholipid substrates through various assays in live COS-7 cells. We have demonstrated that both PS and PI(4,5)P2 are important for VP40 localization and VLP formation at the PM in live cells. We are also investigating the implications on VLP budding as the result of a decrease in PM localization. Conclusions Phospholipid-focused assays in live cells reveal VP40 lipid binding specificity at various cellular sites, other quantitative methods aid in decoding the dynamics and nature of these interactions. Financial Support: NIH AI081077

Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

GPI-Anchored Proteins do not Reside in Ordered Domains in the Live Cell Plasma Membrane

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid and protein environment and vice versa. Studies on model membranes and plasma membrane spheres indicated characteristic preferences of proteins for ordered or disordered lipid phases, however, whether such phases do indeed exist in live cells is still not known. Here, we used a micropatterning approach combined with super-resolution microscopy to quantify the influence of a glycosylphosphatidylinositol-anchored protein (GPI-AP) - a typical marker of ordered lipid phases - on its molecular environment directly in the live cell plasma membrane. Intriguingly, local enrichment and immobilization of a GPI-anchored mGFP - even at highest densities - did not nucleate the formation of ordered membrane regions. At a molecular scale, immobilized mGFP-GPIs essentially behaved as inert obstacles that reduced the mobility of other membrane constituents according to their molecular size and shape. Overall we conclude that GPI-APs do not significantly influence their membrane environment over distances beyond their actual physical size. Further, our results imply that the outer leaflet of the plasma membrane is in a homogenously mixed single phase state under physiological conditions.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Plasma Membrane Nanoplatforms are Dissolved by Oxidized Phospholipids

We developed a method termed TOCCSL (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allows direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins/lipids on a time-scale of seconds. A special photobleaching protocol is used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform is determined by single molecule brightness analysis. As consensus raft marker we chose monomeric GFP linked via a GPI (glycosyl-phosphatidylinositol) anchor to the cell membrane. We found cholesterol-dependent homo-association of mGFP-GPI in the plasma membrane of living CHO cells, thereby demonstrating the existence of small, mobile, stable platforms hosting these probes. This strong homo-association could be released by addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), two oxidized lipids typically present in oxidatively modified low density lipoprotein. We found a clear dose-response relationship of mGPF-GPI nanodomain disintegration upon addition of POVP, correlating with the signal of an apoptosis marker. Applying similar concentrations of lyso-lipid did not dissolve nanodomains. To understand the mechanism behind nanodomain disintegration we inhibited the activation of acid sphingomyelinase (aSMase), which activates several apoptotic signaling pathways via ceramide generation. Inhibition of aSMase by NB-19 before addition of POVPC completely abolished the nanodomain-disintegration effect of oxidized phospholipids, thereby proofing a rather indirect effect of oxidized phospholipids on lipid nanodomains. Additionally we addressed the role of aSMase activity in the release of mGPF-GPI homo-association upon cholesterol extraction - no correlation was found.In conclusion we could determine the mechanism how oxidized lipid species disrupt mGFP-GPI nanodomains in the live plasma membrane.

Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles

The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

AbstractLipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.

Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors.

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.

Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy.

Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.

Direct visualization of solid ordered domains induced by polyene antibiotics in giant unilamellar vesicles

Polyene antibiotics isolated from Streptomyces are frequently used in treatment of mycoses. Confocal fluorescence microscopy has been employed to investigate the influence of polyene macrolide antibiotics nystatin, amphotericin B, and filipin on the phase separation in giant unilamellar vesicles. It has been demonstrated that nystatin produced the solid ordered domains in vesicles made from DOPC/Chol, DOPC/Chol/SM, and POPC while DOPC vesicles remained homogenous in the presence of polyene antibiotics. The ability of various polyenes to produce the solid ordered phase in POPC membranes has been compared. It has been shown that amphotericin B produced phase separation at lower concentration as compared with nystatin and filipin. Filipin was less effective in promotion of gel domains. The observed efficiency of polyene antibiotics to induce phase separation in lipid bilayers correlates with their biological activity. Present findings probably indicate the limitations of using of polyenes as fluorescence membrane probes for determination of strerol-enriched domains in plasma membrane of live cells.

Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli.

SummaryFluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

GPR37 Protein Trafficking to the Plasma Membrane Regulated by Prosaposin and GM1 Gangliosides Promotes Cell Viability

The subcellular distribution of the G protein-coupled receptor GPR37 affects cell viability and is implicated in the pathogenesis of parkinsonism. Intracellular accumulation and aggregation of GPR37 cause cell death, whereas GPR37 located in the plasma membrane provides cell protection. We define here a pathway through which the recently identified natural ligand, prosaposin, promotes plasma membrane association of GPR37. Immunoabsorption of extracellular prosaposin reduced GPR37(tGFP) surface density and decreased cell viability in catecholaminergic N2a cells. We found that GPR37(tGFP) partitioned in GM1 ganglioside-containing lipid rafts in the plasma membrane of live cells. This partitioning required extracellular prosaposin and was disrupted by lipid raft perturbation using methyl-β-cyclodextrin or cholesterol oxidase. Moreover, complex formation between GPR37(tGFP) and the GM1 marker cholera toxin was observed in the plasma membrane. These data show functional association between GPR37, prosaposin, and GM1 in the plasma membrane. These results thus tie together the three previously defined components of the cellular response to insult. Our findings identify a mechanism through which the receptor's natural ligand and GM1 may protect against toxic intracellular GPR37 aggregates observed in parkinsonism.

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.

Photosensitized Damage Inflicted on Plasma Membranes of Live Cells by An Extracellular Generator of Singlet Oxygen—A Linear Dependence of A Lethal Dose on Light Intensity

We describe a study of the influence of a dose rate, i.e. light intensity or photon flux, on the efficiency of induction of a loss of integrity of plasma membranes of live cells in culture. The influence of a photon flux on the size of the light dose, which was capable of causing lethal effects, was measured in an experimental system where singlet oxygen was generated exclusively outside of live cells by ruthenium(II) phenantroline complex. Instantaneous, sensitive detection of a loss of integrity of a plasma membrane was achieved by fluorescence confocal imaging of the entry of this complex into a cell interior. We demonstrate that the size of the lethal dose of light is directly proportional to the intensity of the exciting light. Thus, the probability of a photon of the exciting light inflicting photosensitized damage on plasma membranes diminishes with increasing density of the incident photons.