Functional characterization of types of plasma membranes in cerebral cortex.

The cerebral cortex of normal oxygenated and of asphyxiated mice has been studied by freeze-fracturing technique with a twofold purpose. First, to investigate changes, if any, in the molecular organization of the plasma membrane of any specific cell type(s) that could be correlated with permeability changes thought to take place as a consequence of asphyxiation. Secondly, to attempt characterization of plasma membranes on the basis of the organization of their fractured faces. The decrease in the extracellular material in asphyxiated cerebral cortex seen in electron micrographs of thin sections could not be correlated with change(s), if any, in the molecular organization of the plasma membrane of any particular cell type. Plasma membranes of various types could be characterized on the basis of the arrangement of particles on the fractured faces. Some of these types correspond to identifiable cell processes, while others have not yet been identified with certainty. Fusion of synaptic vesicles with the presynaptic membrane is mediated through clustering of 100-150 A membrane-associated particles.     

Functional characterization of types of plasma membranes in cerebral cortex

The cerebral cortex of normal oxygenated and of asphyxiated mice has been studied by freeze-fracturing technique with a twofold purpose. First, to investigate changes, if any, in the molecular organization of the plasma membrane of any specific cell type(s) that could be correlated with permeability changes thought to take place as a consequence of asphyxiation. Secondly, to attempt characterization of plasma membranes on the basis of the organization of their fractured faces. The decrease in the extracellular material in asphyxiated cerebral cortex seen in electron micrographs of thin sections could not be correlated with change(s), if any, in the molecular organization of the plasma membrane of any particular cell type. Plasma membranes of various types could be characterized on the basis of the arrangement of particles on the fractured faces. Some of these types correspond to identifiable cell processes, while others have not yet been identified with certainty. Fusion of synaptic vesicles with the presynaptic membrane is mediated through clustering of 100–150 Å membrane-associated particles.     

The release of vaccinia virus from infected cells requires RhoA-mDia modulation of cortical actin

Prior to being released from the infected cell, intracellular enveloped vaccinia virus particles are transported from their perinuclear assembly site to the plasma membrane along microtubules by the motor kinesin-1. After fusion with the plasma membrane, stimulation of actin tails beneath extracellular virus particles acts to enhance cell-to-cell virus spread. However, we lack molecular understanding of events that occur at the cell periphery just before and during the liberation of virus particles. Using live cell imaging, we show that virus particles move in the cell cortex, independently of actin tail formation. These cortical movements and the subsequent release of virus particles, which are both actin dependent, require F11L-mediated inhibition of RhoA-mDia signaling. We suggest that the exit of vaccinia virus from infected cells has strong parallels to exocytosis, as it is dependent on the assembly and organization of actin in the cell cortex.     

Robust single-particle tracking in live-cell time-lapse sequences

Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.     

Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells

Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.     

Quick-freeze,deep-etch visualization of the ‘cytoskeletal spring’ of cochlear outer hair cells

Summary The lateral membrane system of the cochlear outer hair cell, consisting of the lateral plasma membrane, pillars, filamentous lattice and subsurface cisternae, is considered to be involved in the contractile movement of the isolated cochlear outer hair cell. The filamentous lattice, called the cytoskeletal spring, has been identified in the demembranated cochlear outer hair cell treated with the detergent Triton X-100. In this study, the quick-freeze, deep-etch method was applied to demonstrate the three-dimensional organization of both the filamentous and membranous structures of the lateral membrane system of cochlear outer hair cells. Treatment with saponin revealed that the inner leaflet of the lateral plasma membrane of the cochlear outer hair cell possesses more membrane particles than the outer leaflets, and that the pillars are closely associated with membrane particles in the inner leaflet of the lateral membrane. The presence of filamentous bridges between the filamentous lattice and the subsurface cisternae was also detected. We propose that the lateral membrane system in the cochlear outer hair cell may play an important role in the tuning mechanisms within the cochlea in normal hearing.     

Basic cell penetrating peptides induce plasma membrane positive curvature,lipid domain separation and protein redistribution

Basic cell penetrating peptides are tools for molecular cellular internalization of nonmembrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organization. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization.     

Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.     

Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web

Tetraspanins regulate cell migration, sperm–egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.     

Changes of the internal organization of the plasma membrane correlated to the regeneration potency of the cell

Internal organization of the plasma-membrane of rat thymocytes and pituitary tumor cells (GH₃) was experimentally altered by low temperature and either changing osmolarity or adding drugs destroying the cytoskeleton. These treatments induce reversible aggregation of intramembrane particles of the plasma membrane. Thin section electron microscopy of the hypotonically shocked cells show all the cell organelles to be extremely swollen and the cytoplasmic space to be rather empty. Returned to physiological conditions, the cells show normal morphological aspects, accompanied by ‘normal’ permeability properties of the plasma membrane; the aggregation of intramembrane particles is reversed. The proliferation behaviour of GH₃-cells is not affected by this treatment. This demonstrates a high regeneration potency of the mammalian cell and leads to the assumption that molecular components which are important for the survival of the cell must be structural (membrane) bound.     

Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells

Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.     

Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.     

Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance.

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (approximately 20- and 100-fold resistance, respectively), undergo a significant (approximately 2-fold) increase in the number of intramembrane particles (IMPs) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive cells. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable 'marker' for early diagnosis of drug resistance.     

Apoptosis and eryptosis: Striking differences on biomembrane level

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.     

Dynamics in the plasma membrane: how to combine fluidity and order

Cell membranes are fascinating supramolecular aggregates that not only form a barrier between compartments but also harbor many chemical reactions essential to the existence and functioning of a cell. Here, it is proposed to review the molecular dynamics and mosaic organization of the plasma membrane, which are thought to have important functional implications. We will first summarize the basic concepts of Brownian diffusion and lipid domain formation in model membranes and then track the development of ideas and tools in this field, outlining key results obtained on the dynamic processes at work in membrane structure and assembly. We will focus in particular on findings made using fluorescent labeling and imaging procedures to record these dynamic processes. We will also discuss a few examples showing the impact of lateral diffusion on cell signal transduction, and outline some future methodological challenges which must be met before we can answer some of the questions arising in this field of research.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

Freeze-fracture study of Trichomonas vaginalis

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.     

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins.