Living Cell Membranes Research Articles

Effect of fatty acids on the permeability barrier of model and biological membranes

Because of the amphipathicity and conical molecular shape of fatty acids, they can efficiently incorporate into lipid membranes and disturb membrane integrity, chain packing, and lateral pressure profile. These phenomena affect both model membranes as well as biological membranes. We investigated the feasibility of exploiting fatty acids as permeability enhancers in drug delivery systems for enhancing drug release from liposomal carriers and drug uptake by target cells. Saturated fatty acids, with acyl chain length from C8 to C20, were tested using model drug delivery liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the breast cancer MCF-7 cell line as a model cell. A calcein release assay demonstrated reduction in the membrane permeability barrier of the DPPC liposomes, proportionally to the length of the fatty acid. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) experiments revealed that C12 to C20 fatty acids can stabilize DPPC liposomal bilayers and induce the formation of large structures, probably due to liposome aggregation and bilayer morphological changes. On the other hand, the short fatty acids C8 and C10 tend to destabilize the bilayers and only moderately cause the formation of large structures. The effect of fatty acids on DPPC liposomes was not completely transferrable to the MCF-7 cell line. Using cytotoxicity assays, the cells were found to be relatively insensitive to the fatty acids at apoptotic sub-millimolar concentrations. Increasing the fatty acid concentration to few millimolar substantially reduced the viability of the cells, most likely via the induction of necrosis and cell lysis. A bioluminescence living-cell-based luciferase assay showed that saturated fatty acids in sub-cytotoxic concentrations cannot reduce the permeability barrier of cell membranes. Our results confirm that the membrane perturbing effect of fatty acids on model membranes cannot simply be carried over to biological membranes of live cells.

Synthesis, lipid membrane incorporation, and ion permeability testing of carbon nanotube porins.

Carbon nanotube porins (CNTPs) are 10- to 20-nm-long segments of lipid-stabilized single-walled carbon nanotubes (CNTs) that can be inserted into phospholipid membranes to form nanometer-scale-diameter pores that approximate the geometry and many key transport characteristics of biological membrane channels. We describe protocols for CNTP synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles, and for validation of CNTP incorporation into a lipid membrane using a proton permeability assay. In addition, we describe protocols for measuring conductance of individual CNTPs in planar lipid bilayers and plasma membranes of live cells. The protocol for the preparation and testing of the CNTPs in vesicle systems takes 3 d, and single CNTP conductance measurements take 2-5 h. The CNTPs produced by this cutting protocol remain stable and active for at least 10-12 weeks.

Nanometer-Scale Permeabilization and Osmotic Swelling Induced by 5-ns Pulsed Electric Fields.

High-intensity nanosecond pulsed electric fields (nsPEFs) permeabilize cell membranes. Although progress has been made toward an understanding of the mechanism of nsPEF-induced membrane poration, the dependence of pore size and distribution on pulse duration, strength, number, and repetition rate remains poorly defined experimentally. In this paper, we characterize the size of nsPEF-induced pores in living cell membranes by isosmotically replacing the solutes in pulsing media with polyethylene glycols and sugars before exposing Jurkat T lymphoblasts to 5ns, 10MV/m electric pulses. Pore size was evaluated by analyzing cell volume changes resulting from the permeation of osmolytes through the plasma membrane. We find that pores created by 5ns pulses have a diameter between 0.7 and 0.9nm at pulse counts up to 100 with a repetition rate of 1kHz. For larger number of pulses, either the pore diameter or the number of pores created, or both, increase with increasing pulse counts. But the prevention of cell swelling by PEG 1000 even after 2000 pulses suggests that 5ns, 10MV/m pulses cannot produce pores with a diameter larger than 1.9nm.

On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.

Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy

Microscopy based on the interferometric detection of light scattered from nanoparticles (iSCAT) was introduced in our laboratory more than a decade ago. In this work, we present various capabilities of iSCAT for biological studies by discussing a selection of our recent results. In particular, we show tracking of lipid molecules in supported lipid bilayers (SLBs), tracking of gold nanoparticles with diameters as small as 5 nm and at frame rates close to 1 MHz, 3D tracking of Tat peptide-coated nanoparticles on giant unilamellar vesicles (GUVs), imaging the formation of lipid bilayers, sensing single unlabelled proteins and tracking their motion under electric fields, as well as challenges of studying live cell membranes. These studies set the ground for future quantitative research on dynamic biophysical processes at the nanometer scale.

Nanoparticles in Bioimaging

This Special Issue of Nanomaterials is dedicated to the application of nanoparticulate materials in biological imaging.[...].

Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

Probing Cd2+-Stressed Live Cell Membrane Permeability with Various Redox Mediators in Scanning Electrochemical Microscopy

Heavy metal cytotoxicity has become a mainstay in scientific research due to the vast range of detrimental effects on living organisms. Cd2+ in particular can activate a wide range of physiological pathways that can cause significant increases in oxidative stress, cancer formation, or cellular death. This is partly due to its ability to bioaccumulate in the body with a long half-life for excretion. Here, we used hydrophilic redox mediators, ferrocenecarboxylate, 1′1-ferrocene dicarboxylate and hexaamineruthenium, as molecular probes in scanning electrochemical microscopy (SECM) to determine the membrane permeability in single live human bladder cancer (T24) cells affected by Cd2+. Hydrophilic and charged sensing agents should be impermeable to the cells. However, when T24 cells were treated with 25 μM Cd2+ (t ≤ 6 h), the membrane permeability to these sensing agents can be induced to approximately 7.0 × 10–5 m/s. For acute mM Cd2+ exposure, the membrane permeability can be induced to 2.0 × 10–4 m/s within...

Specific cell surface labeling of GPCRs using split GFP.

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.

Accurate High Speed Imaging of Single Protein Diffusion within the Live Cell Membrane

The diffusive motion of proteins and lipids within the cellular membrane is complex and nuanced owing to the diverse and heterogeneous nature of its composition and structure. Insight into the diffusive motion of such proteins yields important information on their behavior and biological function. Conventional means to investigate diffusion uses a fluorescent label, but the intrinsic limitations of fluorescence such as photobleaching and saturation ultimately restrict what we may observe. These limitations strongly restrict both the timescale over which the protein may be observed and the accuracy to which they can be localized in space. Fluorescence microscopy additionally suffers from poor axial resolution and so provides almost no information as to the out-of-plane movement of the protein.Free from these constraints, interferometric imaging of scattered light (iSCAT microscopy) provides three-dimensional information on the motion of a single protein labeled with a gold nanoparticle. Here, we demonstrate single particle tracking of individual epidermal growth factor receptor (EGFR) proteins within the live HeLa cell membrane. We localize the diffusing protein to sub-nanometer accuracy with microsecond temporal resolution. The interferometric nature of the detection additionally provides information on the subtle out-of-plane movement of the protein to nanometer accuracy. We are therefore able to track with high spatio-temporal resolution the diffusion of single EGFR proteins in three-dimensions as they navigate a live HeLa cell membrane. This yields unheralded information about the minutia of diffusion within the cell membrane and demonstrates the potential of iSCAT microscopy in cell membrane imaging.

Probing the Dimerization Affinity of Visual Opsins

Visual opsins are the light sensing proteins at the center of human vision. Rhodopsin is the pigment for low-light vision, and three cone opsins serve as the pigments for daytime image-forming vision. Opsin proteins are part of the G protein-coupled receptor (GPCR) family, and rhodopsin has served as something like a prototype for structural studies of GPCRs. Rhodopsin has also been observed to form dimeric complexes in vitro, in tissue, and in cell cultures, including recent work from our lab using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Here we report on our recent investigation of cone opsin dimerization in a heterologous expression system. PIE-FCCS is used to resolve the relative population of dimeric complexes over a range of concentrations in live cell membranes. Surprisingly, the homodimerization affinities of the cone opsins are not identical to rhodopsin and there are significant differences in the dimerization affinity of the three cone opsins. This raises important questions about the physiological role of opsin dimerization, which has wide-ranging implications for GPCR dimerization in general. To investigate these questions we explored the differential dimerization affinity with mutational studies, in which we identify key residues responsible for dimerization. To connect the dimerization affinity to its functional consequences we also measured the spectral shifts in the absorption profiles of the wild-type and mutant cone opsins regenerated with the retinal chromophore. The results of our work provide critical insight into the molecular basis of color vision. They also will inform the ongoing study of GPCR dimerization in cell signaling.

Difffusion Dynamics of AChR Receptors on Live Muscle Cell Membrane

The dynamic structure of a cell membrane allows it to become an effective platform for various biological functions, such as signal transduction, molecule transportation and endocytosis. We report here a single-molecular tracking experiment on a quantum-dot-labeled transmembrane protein, acetylcholine receptor (AChR), in cultured Xenopus muscle cells. We carried out a complete statistical analysis on a large set of AChR trajectories with more than 500 cells examined. Various drug treatments were used to perturb F-actin and scaffold proteins and examine their roles in regulating the motion of the AChRs. The diffusion dynamics of AChRs was characterized by three quantities: the mean-square displacement 〈Δr2(τ)〉, the probability density function P(Dx) of instantaneous displacement Dx(τ) and the probability distribution f(δ) of instantaneous diffusion coefficient δ. After a careful analysis, we conclude that (1) AChRs show a hindered motion by the surrounding membrane molecules at short time and become diffusive at long time. (2) The mobile AChRs have a broad distribution in diffusion coefficient δ with a long exponential tail, which is universal and independent of different sample conditions. (3) The exponential distribution f(δ) leads to an exponential distribution P(Dx). Our measurements of membrane diffusion based on a large number of single molecular trajectories provide a complete statistical description of dynamic heterogeneity on live cell membrane. By combining all the experimental results available, we propose a dynamic picket-fence model of membrane organization involving slow active remodeling of the underlying cortical actin network to explain the observed non-Gaussian statistics and dynamic heterogeneity. (Work was supported by the Research Grants Council of Hong Kong SAR.)

Probing Single-Molecule Ion Channel Conformational Dynamics in Living Cells

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels that are crucial for cell functions, neuronal signaling, and brain functions. We have developed a new combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging for probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificail lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. We will focus our discussion on the new development and results of real-time imaging of the dynamics of gramicidin, colicin, and NMDA receptor ion channels in lipid bilayers and living cells. We have probed NMDA (N-Methyl-D-Aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. Based on our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.

A Photogate Microscopy to Track Single Molecules in Crowded Environments

Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. Here, we developed PhotoGate, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. The method enables tracking of single particles at high spatial and temporal resolution until they photobleach or dissociate from their stationary target. This technical ability opens new possibilities for tracking single molecules in live cells at concentrations two orders of magnitude higher than the diffraction limit without photoactivation. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. Furthermore, we directly measured binding and the dissociation rate of single APPL1 molecules from early endosomes in the crowded environment of the cytoplasm. We have also applied PhotoGate to intraflagellar transport and observed the dynamics of cargo disaasembly and reassembly at the ciliary tip. Thus, PhotoGate reveals spatial and temporal features of signal transduction events at the cell surface and on intracellular compartments with single molecule resolution.

Live Quantification of Changes to Membrane Cytoskeleton due to Restricted Access to Laminin or Substrate Stiffness

Physical (stiffness and dimensionality) and chemical (cell adhesion promoting molecules) properties of cellular environment are thought to take key roles in tumorigenesis. Mammary epithelial cells undergo mesenchymal transition during invasive progression of breast cancer. We modeled basal membrane with different gel stiffness and altering laminin concentrations. Our results indicate carcinoma cells become invasive in stiff tissues in response of limited access to laminin at basement membrane. Better understanding of the transition dynamics requires single cell level live quantification of membrane cytoskeleton changes for different sizes of observation areas simultaneously, in relevant timescales. We facilitate bimFCS technique, a non-perturbing optical method giving opportunity to acquire data over tens of minutes with less than a minute temporal resolution in analysis in a time-course manner. Our method lets quantitative analysis of (1) the diffusion speed of these proteins in live cell membranes while freely moving between meshwork fences, (2) their strength of interaction with the cytoskeleton and (3) the average separation of fences.

From Single-Molecule Spectroscopy to Super-Resolution Microscopy: Super-Resolution Optical Fluctuation Imaging and Metal-Induced Energy Transfer

The presentation gives and overviews of two super-resolution fluorescence microscopy techniques are presented which are inspired by single-molecule spectroscopy. The first is Superresolution Optical Fluctuation Imaging (SOFI), which converts temporal intensity fluctuations of emitters into an enhanced spatial resolution of an image. Besides providing higher resolution, SOFI is also very efficient in suppressing any background or auto-fluorescence, increases image contrast, and allows for recording three-dimensional images (z-sectioning) with a wide field microscope. The fundamental principles behind SOFI as well as several applications for super-resolved, three-dimensional cell imaging will be presented.The second technique is Metal-Induced Energy-Transfer Imaging. When placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). We call this effect metal-induced energy transfer or MIET. The MIET-coupling between an excited emitter and a metal film is strongly dependent on the emitter's distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ∼3 nm. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques which enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of using single-molecule localization microscopy for structural studies of biomolecules and biomolecular complexes.

Improved Methods for Preparing Asymmetric Vesicles using Methyl-Alpha-Cyclodextrin

Many living cell membranes display lipid asymmetry, with a distinct difference between the phospholipids of the inner and outer leaflets of the lipid bilayer. Although artificial lipid vesicles have long been an important tool to study biological membranes, they are limited due to their lack of lipid asymmetry. We have recently developed methods to prepare asymmetric lipid vesicles using cyclodextrins, and improved methods using alpha-cyclodextrins. These hexasaccharide rings can promote the exchange of lipids between vesicles, but do not transport sterols. This allows the efficient and selective replacement of the outer leaflet lipids of lipid vesicles while at the same time tightly controlling sterol content. As a result, asymmetric vesicles can now be constructed using a range of lipids which closely mimic the natural asymmetry and content of mammalian plasma membranes. Here we show that methyl-alpha-cyclodextrin is an efficient tool for constructing asymmetric vesicles with asymmetric phospholipid distribution closely resembling that of natural mammalian plasma membranes. Progress in using methyl-alpha-cyclodextrin to prepare “inside-out” asymmetric vesicles, in which the lipids that normally face the cytosolic side of plasma membranes (PE and PS) are in the outer leaflet while those normally on the extra-cellular leaflet (SM and PC) are in the inner leaflet, and in developing improved assays for lipid asymmetry, will also be described.

Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10-80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Taken together, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.