Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Hemagglutinin-induced fusion of HAb2 and PLC cells: dynamics of fusion pore conductance

Infection of cells with influenza virus is mediated by the virus envelope protein hemagglutinin (HA) which induces fusion of viral and target membranes. Earlier we showed using fluorescent microscopy that HAb2 cells expressing HA on their plasma membranes fused with PLC cells when pH of the external medium was decreased to -5. In the present work we used double whole-cell recording to monitor the intercellular conductance in HAb2/PLC cell pairs during fusion. In approximately 40% of cell pairs the pH drop induced the intercellular conductance, which we interpret as the formation of a fusion pore. The following stages of the conductance growth were distinguished: initial fluctuations near zero (flicker), a subsequent slow increase up to 1-4 nS and a final rapid increase up to 10-100 nS (complete fusion). The first detectable intercellular conductance change (opening of a fusion pore) was accompanied by an increase in the conductances of both HAb2 and PLC cell membrane. This observation suggests that the early pore complex should be leaky. The dynamics of the intercellular conductance appeared to depend upon the voltage difference between the fusing HAb2 and PLC cells: voltages higher than 40 mV facilitated the conductance growth.     

Mammalian Wnt3a is Released on Lipoprotein Particles

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila . To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.     

Polarized distribution of Na+,K+-ATPase in giant cells elicited in vivo and in vitro

Giant cell formation was analyzed to determine whether it results in the high level of Na+,K+-ATPase expression that characterizes multinucleated cells such as osteoclasts. Giant cells and fusing alveolar macrophages were subjected to morphological, immunological, and biochemical studies. Both subunits of the Na+,K+-ATPase were found to be present on the plasma membrane of giant cells. Their localization was restricted to the non-adherent domain of the cell surface. Dynamic studies of giant cell differentiation demonstrated that on culture and/or multinucleation, an increase in sodium pump alpha-subunit synthesis occurred and led to a high level of expression of Na pumps. Conversely, the adherent plasma membrane of giant cells was enriched in a lysosomal membrane antigen. This study demonstrates that culture and/or multinucleation induces a significant increase in the expression of sodium pumps. The polarized distribution of these pumps and of a lysosomal component suggests that fusing macrophages undergo biochemical and morphological alterations which prepare them for a new and specialized function in chronic inflammatory reactions. Giant cells may offer a suitable model system to study the differentiation of other related multinucleated cells, such as osteoclasts.     

Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: formation of intercellular membrane bridges

Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainly comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.     

HIV-1进攻靶细胞的机制及相应环节抑制剂

HIV－1是导致获得性免疫缺陷综合症（AIDS）的流行最广、破坏力最强的病毒。HIV－1分两个步骤特异性地进攻CD4^ 细胞：一是利用表面糖蛋白gp120和靶细胞膜上的受体结合;二是通过跨膜糖蛋白gp41使病毒的包膜和靶细胞的质膜发生融合,经过上述步骤,病毒的核心蛋白和遗传物质得以进入人体,然其中进行复制,遇时,细胞膜的稳定性被破坏,细胞的内外环境失去平衡,最终导致细胞死亡。HIV－1进攻靶细胞的机制研究所取得的成就为研制安全有效的抗HIV／AIDS药物提供了新的思路和方向。     

Vesicle formation during electro-fusion of mesophyll protoplasts of Kalanchoë daigremontiana

Following electro-fusion of plant protoplasts the volume of the fused cell is the sum of the volumes of the parent cells. As shown for mesophyll protoplasts from leaves of Kalanchoë daigremontiana, the excess in membrane material arising from the reduction in membrane area is removed-at least to a larger extent — by the formation of vesicles which are visible in the light microscope. These vesicles, which may have been formed by the fusion of sub-microscopic vesicles, are observed in the contact zone of the fusing cells. The mechanism of the formation of vesicles during electro-fusion is discussed.     

Fine Structural Analysis of Animal Cell Surfaces: Membranes and Cell Surface Topography

Current research on the mechanism of transmembrane regulationof topographic modulation at the cell surface is described forthe sea urchin egg and Sarcoma 180 ascites tumor cells of SwissWebster white mice. The transmembrane system is characterizedin terms of three components: glycocalyx, membrane, and cytofibrillarstructures. The importance of membrane molecular architectureper se relative to the other surface components is assessedin terms of freeze fracture analysis of both cell types as wellas concanavalin A (ConA)-mediated long term agglutination, cytochalasinB effects, and other drug-induced changes at Sarcoma 180 cellsurfaces. A quantitative and qualitative assessment of intramembranousparticle (IMP) sizes and density distributions reveals intrinsicstructural changes of the fusing membranes at cortical reactionduring sea urchin egg fertilization and also with the post-fertilizedaccumulation of surface microvilli. Comparable changes in IMPare noted for microvillus retraction and membrane smoothingin Sarcoma 180 cells under a variety of experimental conditions.On the other hand, chemical perturbation of S-180 cell surfacesreveals a rather non-ubiquitous, though identifiable, involvementof microfilaments and no microtubule involvement in these topographicchanges. These observations suggest that the plasma membraneis a dynamic structure poised between "restrictive" and "lessrestrictive" states of fluidity or deformability and, hence,is a determinant component in topographic change.     

Cytophysiological Characteristics of the Vegetative and Dormant Cells of <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp. Strain FM3, a Bacterium Isolated from the Skin of a <Emphasis Type="Italic">Xenopus laevis</Emphasis> Frog

A bacterial strain (FM3) that is closely related to Stenotrophomonas acidaminiphila and S. maltophilia, was isolated from the skin surface of the frog Xenopus laevis. Cytophysiological studies on vegetative cells and cyst-like cells (CLCs) that were obtained in model experiments addressed the dynamics of transition of vegetative cells to the dormant state and their reversion to vegetative growth. The ultrastructural organization of the vegetative and dormant cells of strain FM3 possesses unique properties. Cultures that developed after inoculating vegetative cells were characterized by: (1) resistance to physical factors and sterilization procedures; (2) high antimicrobial activity with respect to some gram-positive and gram-negative bacteria; (3) resistance to polypeptide antibiotics; (4) the presence of an easily detaching S-layer on the cell surface; (5) the ability to secret outer membrane vesicles into the intercellular space; and (6) formation of S-layerderived tubular structures associated with outer membrane vesicles that are regularly arranged within the tubes. Dormant cells were characterized by: (1) resistance to dehydration; (2) resistance to high temperatures; and (3) the preservation of the S-layer on the surface of cystlike cells (CLCs). Depending on experimental conditions, strain FM3 formed three CLC morphotypes, which differed in their abundance and ultrastructural organization. The experimental conditions used for CLC formation approximated those under which bacteria survive in hospitals. A model of intermicrobial parasitism is suggested that applies to motile FM3 cells during the development of their populations (cultures).     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the basilar membrane, limbus spiralis and spiral ligament.These different parts continue one into the other and make up a single morphological and functional structure which may be called the supporting structure of Corti's organ (s.s.C.o.).It is formed by a tissue the components of which are the cells and an intercellular substance in which are arranged the capillary vessels.The cells can be classed in two groups, the first consisting of the cells proper (basilar membrane, limbus spiralis and spiral ligament cells) which present structural changes parallel with the growth mechanism of the intercellular substance; the second of the cochlear duct covering cells (Corti's organ cells, inner and outer spiral sulcus cells, interdental cells, stria vascularis cells).The intercellular substance is organised in laminae, fibrolaminae, bundles and microscopic fibers composed of filaments with an intervening ground substance.The filaments have a diameter ranging from 85 to 105 Å. Topochemical tests with polarised light microscope, enzymatic tests, diffractographic and chemical analyses suggest that these filaments unquestionably consist of protein material which have nothing to do with collagen or elastic fibers. Perhaps it may be classed in the K.E.M.F. group.The ground substance generally appear anhistous and transparent but in some parts of the basilar membrane it presents a cottony appearance.The possible different hypotheses about the classification of the s.s.C.o. tissue are discussed.The quantity and architecture of the cells and the intercellular substance vary appreciably in the basilar membrane, limbus spiralis and spiral ligament, which are examined in detail one by one.The demonstration that the s.s.C.o. is formed of a tissue possessing an intercellular substance containing filamentous scleroproteins clearly corroborates the theory that is performs supporting activity in respect of Corti's organ. The term supporting structure of Corti's organ is based on this interpretation.Research financed by C.N.R. grant.     

Organization and differentiation of the segmentary plate in common toad embryo Bufo bufo L., following heat shock (author's transl)

A heat shock greatly disturbs the shaping and the growth of embryos with 4 or 5 somites. One part of a segmentary plate with a size including four control somites stays inorganized, whereas posterior segmentation gets on normally. The differentiation of the unsegmented somitic block (fusing myoblasts, myogenesis) is the same as the one of the previous somites; the dermatome and the sclerotome issued from the unsegmented block are not cut up. Ultrastructural observations of treated segmentary plates show that the somitic block undergoes important changes. The non-segmented somitic mesoderm of the control animals obtains a loose aspect; its constitutive cells separated by lacunae are brought into contact by the means of filopods and junctions. 18 hours after the heat shock, the cells of the unsegmented block keep close to each other and are tightened together by many "pseudo-desmosomes", the intercellular spaces are reduced to small meatus. The nuclei of the somitic cells are clear and provided with a restricted amount of chromatine spots; dark granular nuclear like chromatin material is thrown out of the nucleus. In embryos fixed 40 hours after heat shock, several large plurinucleated cells, are formed in the non-segmented somitic block. Inside these plurinucleated cells, there is synthesis of myofilaments and differentiation of myofibrilles. Numerous intercellular lacunae are formed, some yet apparent "pseudo-desmosomes" form "pinchings" between the membranes of muscular cells and therefore confine series of intercellular lacunae.     

Human immunodeficiency virus envelope glycoprotein/CD4-mediated fusion of nonprimate cells with human cells.

Human immunodeficiency virus (HIV) infects human cells by binding to surface CD4 molecules and directly fusing with the cell membrane. Although mouse cells expressing human CD4 bind HIV, they do not become infected, apparently because of a block in membrane fusion. To study this problem, we constructed a recombinant vaccinia virus that can infect and promote transient expression of full-length CD4 in mammalian cells. This virus, together with another vaccinia recombinant encoding biologically active HIV envelope glycoprotein gp160, allowed us to study CD4/gp160-mediated cell-cell fusion in a wide variety of human and nonhuman cells in the absence of other HIV proteins. By using syncytium formation assays in which a single cell type expressed both CD4 and gp160, we demonstrated membrane fusion in lymphoid and nonlymphoid human cells but not in any of the 23 tested nonhuman cell types, derived from African green monkey, baboon, rabbit, hamster, rat, or mouse. However, in mixing experiments with one cell type expressing CD4 and the other cell type expressing gp160, all of these nonhuman cells could form CD4/gp160-mediated syncytia when mixed with human cells; in 20 of 23 cases, membrane fusion occurred only if the CD4 molecule was expressed on the human cells whereas in the other three cases, CD4 could be expressed on either one of the fusing partners. Interestingly, in one mouse cell line, CD4-dependent syncytia formed without a human partner, but only if a C-terminally truncated form of the HIV envelope glycoprotein was employed. Our results indicate that nonhuman cells are intrinsically capable of undergoing CD4/gp160-mediated membrane fusion, but this fusion is usually prevented by the lack of helper or the presence of inhibitory factors in the nonhuman cell membranes.     

Vacuolar symplast formation is due to highly permeable gap junctions between the tonoplast and endoplasmic reticulum membrane

An NMR method with a pulsed magnetic field gradient was applied to study changes in water permeability of the vacuolar symplast in maize (Zea mays L.) seedling roots treated with various inhibitors of cell metabolism. The results were qualitatively analogous to literature data on conductivity changes of intercellular gap junctions in animal cells exposed to similar treatments. Electron microscopy examination of root cells provided evidence for the existence of membrane contacts between the endoplasmic reticulum and the tonoplast. It is supposed that vacuoles of neighboring plant cells are interconnected through highly dynamical gap junctions between the tonoplast and the endoplasmic reticulum membrane.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Role of the hemagglutinin-neuraminidase protein in the mechanism of paramyxovirus-cell membrane fusion

Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth beta sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.     

Surface coat in steroid-secreting cells of the mouse ovary

Steroid-secreting cells (luteal, thecal and interstitial cells) of the mouse ovary have been studied with the ruthenium red method to stain the "cell coat". The results showed that a typical cell coat covers the entire surface of the plasma membrane except where the cells are connected by specific cell junctions. Further, particularly heavy concentration of ruthenium-red-reacting material was demonstrated in pericapillary and intercellular spaces. The differences in the thickness of the cell coat and its topographical modifications among different groups of steroidogenic cells may be related not only to intercellular adhesion and interactions but also concerned with a function of control in which the cell permeability is modified in relation with phenomena of cell recognition.