Single molecule studies of molecular diffusion in cellular membranes: determining membrane structure

Since the advent of single particle/molecule microscopies, researchers have applied these techniques to understanding the fluid membranes of cells. By observing diffusion of membrane proteins and lipids in live cell membranes of eukaryotic cells, it has been found that membranes contain a mosaic of fluid compartments. Such structure may be instrumental in understanding key characteristics of the membrane. Recent single molecule observations on prokaryotic cell membranes will also be discussed.     

Cell viability and probe-cell membrane interactions of XR1 glial cells imaged by atomic force microscopy.

As atomic force microscopy (AFM) imaging of live specimens becomes more commonplace, at least two important questions arise: 1) do live specimens remain viable during and after AFM, and 2) is there transfer of membrane components from the cell to the AFM probe during probe-membrane interactions? We imaged live XR1 glial cells in culture by single- or dual-pass contact or tapping-mode AFM, examined cell viability at various postimaging times, and report that AFM-imaged live XR1 cells remained viable up to 48 h postimaging and that cell death rates did not increase. To determine if nonlethal, transient interactions between the AFM probe and cell membrane led to transfer of XR1 cell membrane phospholipid components on the probe, we treated the scanned probes with the lipid-binding fluorophore FM 1-43. Confocal microscopy revealed that phospholipid membrane components did accumulate on the probe, and to a generally greater extent during contact-mode imaging than during tapping-mode imaging. Moreover, membrane accumulations on the probe were greater when live XR1 cells were damaged or perturbed, yet membrane did not accumulate in fluorescently detectable quantities during repeated "force curves" during control experiments. Taken together, our data indicate that although AFM imaging of live cells in culture does not affect long-term cell viability, there are substantial probe-membrane interactions that lead to transfer of membrane components to the probe.     

Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/μm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.     

Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine.

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a fluorescent probe for lipid peroxidation in live cells. DPPP was successfully incorporated into U937 cells. Incorporation of DPPP into the cell membrane was confirmed by fluorescence microscopy. Reaction of DPPP with hydroperoxides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between increase in fluorescence intensity and the amount of methyl linoleate hydroperoxide applied to the cell was observed. DPPP gave little effect on cell proliferation, cell viability or cell morphology for at least 3 d. DPPP oxide, fluorescent product of DPPP, was quite stable in the membrane of living cells for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluorescence. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H(2)O(2), AAPH, and DEM to compare the formation of hydoperoxides in the membrane and cytosol, distinct patterns of peroxide formation were observed. These results indicate that fluorescent probe DPPP is eligible for estimation of lipid peroxidation proceeding in the membrane of live cells, and use of this probe is especially advantageous in long-term peroxidation of the cell.     

Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments

In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.     

Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.     

Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived 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 or lipids on a time scale of seconds. A special photobleaching protocol was 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 was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.     

Live cell measurements of interaction forces and binding kinetics between Discoidin Domain Receptor 1 (DDR1) and collagen I with atomic force microscopy

BackgroundDiscoidin Domain Receptors (DDRs) are membrane-tethered proteins of the receptor tyrosine kinase family, which signal in response to collagen. DDR expression is associated with human diseases, including fibrosis and cancer. The role of DDRs in human pathogenesis is mediated by dysregulated receptor function in response to the collagenous milieu. Thus, understanding DDR-collagen interactions is important for developing novel therapeutic strategies against DDRs.MethodsWe developed a biophysical method to isolate and measure specific interactions between DDR1 and collagen in live cells at the single molecule level using atomic force microscopy. This new method is capable of providing density and kinetics of membrane receptors in live cells.ResultsWe isolated DDR1-collagen interactions and quantified the association and dissociation rates of the DDR1-collagen I complex. We estimated separate binding probabilities of collagen I to DDR and integrin, and by combining kinetic and binding probability data, we were able to estimate the density of receptors in two cancer cell types. We also tested the viability of a DDR1 blocking antibody and determined its efficacy in suppressing DDR1-collagen binding.ConclusionsThe new method shows promise in quantifying receptor-ligand kinetics and receptor density on live cells.General significanceThe new approach is applicable to other receptor-ligand systems and allows the determination of critical parameters at the single cell/single molecule level – in particular, the direct determination of kinetic and density differences of receptors in different cell types. This capability should prove to be useful in cancer research and drug design.     

Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy

This experimental design presents a single molecule approach based on fluorescence correlation spectroscopy (FCS) for the quantification of outer membrane proteins which are receptors to an aptamer specifically designed to target the surface receptors of live Salmonella typhimurium. By using correlation analysis, we also show that it is possible to determine the associated binding kinetics of these aptamers on live single cells. Aptamers are specific oligonucleotides designed to recognize conserved sequences that bind to receptors with high affinity, and therefore can be integrated into selective biosensor platforms. In our experiments, aptamers were constructed to bind to outer membrane proteins of S. typhimurium and were assessed for specificity against Escherichia coli. By fluorescently labeling aptamer probes and applying FCS, we were able to study the diffusion dynamics of bound and unbound aptamers and compare them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at single molecule sensitivity. The dissociation constants for these aptamer probes calculated from autocorrelation data were 0.1285 and 0.3772 nM and the respective receptor densities were 42.27 receptors per µm² and 49.82 receptors per µm². This study provides ample evidence that the number of surface receptors is sufficient for binding and that both aptamers have a high‐binding affinity and can therefore be used in detection processes. The methods developed here are unique and can be generalized to examine surface binding kinetics and receptor quantification in live bacteria at single molecule sensitivity levels. The impact of this study is broad because our approach can provide a methodology for biosensor construction and calculation of live single cell receptor‐ligand kinetics in a variety of environmental and biological applications. Bioeng. 2011; 108:1222–1227. © 2010 Wiley Periodicals, Inc.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Single nanoparticle photothermal tracking (SNaPT) of 5-nm gold beads in live cells

Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.     

Localization Microscopy (SPDM) Reveals Clustered Formations of P-Glycoprotein in a Human Blood-Brain Barrier Model

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.     

Near-field fluorescence microscopy

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Modulation of cargo release from dense core granules by size and actin network

During regulated fusion of secretory granules with the plasma membrane, a fusion pore first opens and then dilates. The dilating pore allows cargo proteins from the dense core to be released into the extracellular space. Using real-time evanescent field fluorescence microscopy of live PC12 cells, it was determined how rapidly proteins of different sizes escape from single granules after fusion. Tissue plasminogen activator (tPA)-Venus is released 40-fold slower than the three times smaller neuropeptide Y [NPY-monomeric GFP (mGFP)]. An NPY bearing two mGFPs in tandem [NPY-(mGFP)₂] as an intermediate-sized fusion probe is released most slowly. Although, the time–course of release varies substantially for a given probe. Coexpression of β-actin, actin-related protein 3 or mAbp1 slowed the release of the two larger cargo molecules but did not affect release of NPY-mGFP or of the granule-membrane-bound probe Vamp-pHluorin. Additionally, high concentrations of cytochalasin D slowed release of the tPA-Venus. Together these results suggest that fusion pore dilation is not the only determinate of release time–course and that actin rearrangements similar to those mediating actin-mediated motility influences the time–course of release without directly interfering with the granule membrane to cell membrane connection.     

Conformational regulation of alpha 4 beta 1-integrin affinity by reducing agents. "Inside-out" signaling is independent of and additive to reduction-regulated integrin activation

The alpha(4)beta(1)-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670-48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn(2+) in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.     

Bves: prototype of a new class of cell adhesion molecules expressed during coronary artery development

Bves is a protein expressed in cells of the developing coronary vascular system, specifically in the proepicardium, migrating epithelial epicardium, delaminated vasculogenic mesenchyme and vascular smooth muscle cells. Here, we show that Bves protein undergoes a dynamic subcellular redistribution during coronary vessel development. Bves is a membrane protein with three predicted transmembrane helices, an extracellular C terminus and an intracellular N terminus, and is confined to the lateral membrane compartment of epithelial cells. When epicardial cells are dissociated into single cells in vitro, Bves accumulates in a perinuclear region until cells make contact, at which time Bves is trafficked to the cell membrane. Bves accumulates at points of cell/cell contact, such as filopodia and cell borders, before the appearance of E-cadherin, suggesting an early role in cell adhesion. While Bves shares no homology with any known adhesion molecule, transfection of Bves into L-cells readily confers adhesive behavior to these cells. Finally, Bves antibodies inhibit epithelial migration of vasculogenic cells from the proepicardium. This study provides direct evidence that Bves is a novel cell adhesion molecule and suggests a role for Bves in coronary vasculogenesis.     

Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).     

A DIE responsive NIR-fluorescent cell membrane probe

It is challenging to achieve selective off to on modulation of the emissive state of a fluorophore within a complex and heterogeneous cellular environment. Herein we show that the dis-assembly of a non-fluorescent aggregate to produce individual fluorescent molecules, termed disaggregation induced emission (DIE), can be utilised to achieve this goal with an amphiphilic BF₂-azadipyrromethene (NIR-AZA) probe. Optical near-infrared properties of the NIR-AZA probe used in this study include absorption and emission maxima at 700 and 726 nm respectively when in the emissive non-aggregated state. Key to the success of the probe is the bis-sulfonic acid substitution of the NIR-AZA fluorophore, which is atypical for membrane probes as it does not contain zwitterionic lipid substituents. The aggregation/disaggregation properties of the NIR-fluorophore have been investigated in model surfactant and synthetic liposomal systems and shown to be emissive responsive to both. Real-time live cell imaging experiments in HeLa Kyoto and MC3T3 cells showed a rapid switch on of emission specific to the plasma membrane of viable and apoptotic cells attributable to a disaggregation-induced emission of the probe. Image analysis software confirmed localisation of fluorescence to the plasma membrane. Cell membrane staining was also effective for formaldehyde fixed cells, with staining possible either before or after fixation. This study adds new and important findings to recent developments of DIE responsive probes and further applications of this controllable emission-switching event are anticipated.     

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.