Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.     

Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1 s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.     

Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes

Summary The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.     

Morphological detection of filipin-sterol complexes in the cytoplasmic membrane of staphylococcal L-form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.     

Ionic-strength modulation of electrically induced permeabilization and associated fusion of mammalian cells

Application of a high electric field to cells in culture has been shown to make them both permeable and fusogenic. The molecular events involved in the phenomenon are still poorly understood. In this study we investigated the effects of the ionic strength of the pulsing buffer on the electropermeabilization and electrofusion of Chinese hamster ovary cells. Increasing the ionic strength of the pulsing medium results in an increase in sieving of transient permeant structures, but decreases the fusion index. Treatment of cells with trypsin or pronase before application of the pulses abolishes the ionic modulation of both electropermeabilization and electrofusion. A similar rate of expansion of permeabilization is obtained whatever the ionic content of the pulsing buffer, and cells fuse even at high ionic strength. This observation lends support to our hypothesis that membrane proteins play a role in electrofusion.     

Nucleus-anchoring cytoskeleton in chicken red blood cells

Cytoskeleton of chicken erythrocytes was studies studied after extraction of the cells with Triton X-100. In phase contrast microscopy the extracted cells were seen as ghost-like structures with preserved morphology, distinct nucleus and surrounding plasma membrane remnant. In electron microscopy, dense matrix-like nucleus, fibrillar plasma membrane residue and filaments, ca. 10 nm in diameter, traversing the cytoplasmic domain, were seen. Distinct bands of molecular weight 68000, 53000, 32000 and bands of both higher and lower molecular weight were obtained by polyacrylamide gel electrophoresis of the extracted cells. These results indicate that intermediate filaments, forming the nucleus-anchoring cytoskeleton, are present in nucleated chicken erythrocytes as part of cellular cytoskeleton.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Isolation and Characterization of the Fertility Factor P of Vibrio cholerae

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.     

Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.     

Mechanistic analysis of electroporation-induced cellular uptake of macromolecules

Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.     

Cellular localization of the bcl-2 protein and response to glucocorticoid stress

We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.     

Structure of mitochondrial DNA from Paramecium tetraurelia

Mitochondrial DNA (mtDNA) from endosymbiote-free stocks of Paramecium tetraurelia was isolated by 2 procedures. The buoyant density of the mtDNA in neutral CsCl was 1.702 gm/cm3, a value consistent with the melting temperature of the mtDNA. Only linear molecules were observed by electron microscopy. These molecules were homogeneous in size with a monomer molecular weight of 25.6 x 10(6) daltons. The size of the mtDNA determined after digestion with the restriction endonucleases EcoRI or Hind III agreed with the value obtained by electron microscopy. These studies also revealed that the digestion pattern of mtDNA from stock 172 differed from that of other 3 stocks (51, 127, 203) examined. Some mtDNA molecules exhibited snapback reassociation following denaturation.     

Electroporation in dense cell suspension--theoretical and experimental analysis of ion diffusion and cell permeabilization

Electroporation is a process where increased permeability of cells exposed to an electric field is observed. It is used in many biomedical applications including electrogene transfection and electrochemotherapy. Although the increased permeability of the membrane is believed to be the result of pores due to an induced transmembrane voltage U(m), the exact molecular mechanisms are not fully explained. In this study we analyze transient conductivity changes during the electric pulses and increased membrane permeability for ions and molecules after the pulses in order to determine which parameters affect stabilization of pores, and to analyze the relation between transient pores and long-lived transport pores. By quantifying ion diffusion, fraction of transport pores f(per) was obtained. A simple model, which assumes a quadratic dependence of f(per) on E in the area where U(m)>U(c) very accurately describes experimental values, suggesting that f(per) increases with higher electric field due to larger permeabilized area and due to higher energy available for pore formation. The fraction of transport pores increases also with the number of pulses N, which suggest that each pulse contributes to formation of more and/or larger stable transport pores, whereas the number of transient pores does not depend on N.     

Ultrastructural changes to the midgut of the black field cricket (Teleogryllus commodus) following ingestion of potato protease inhibitor II

Ultrastructural changes to the midgut epithelium of nymphs of the black field cricket (Teleogryllus commodus) after ingestion of potato protease inhibitor II (PPI-II) (0.6% (w/v) in artificial diet) were determined by light and electron microscopy. Crickets fed diet containing PPI-II grew more slowly than those fed control diet and changes observed to the PPI-II-fed nymphs included reduction of midgut wall depth, vacuolisation of the epithelial cells, swelling of the microvilli, cellular protrusions into the midgut and eventual rupture of individual or small groups of epithelial cells. These changes were first seen 2 days after PPI-II ingestion. Complete disintegration of the midgut to the basement membrane was not seen during the 27-day observation period and repair and regeneration of pockets of epithelial cells was observed. Immunocytochemistry revealed that PPI-II was localised within the ectoperitrophic matrix space of the gut. The location of the peritrophic matrix was determined by labelling with wheat germ agglutinin (WGA), but no rupture of this structure was observed in PPI-II-fed nymphs.     

Morphological changes during conoid extrusion in Toxoplasma gondii tachyzoites treated with calcium ionophore

Treatment of tachyzoites of Toxoplasma gondii with the calcium ionophore A23187 induced dramatic ultrastructural changes that were observed by light and electron microscopy. Light microscopy showed a higher percentage (22%) of tachyzoites with the conoid extruded when compared to control parasites. Electron microscopy confirmed the conoid extrusion by both transmission and scanning electron microscopy. Freeze-fracture replicas showed that the plasma membrane adjacent to cytoplasmic dense granules appeared devoid of intramembranous particles. Membrane-limited vesicles and filopodium-like structures at the cell surface were observed in treated cells. 3-D reconstruction from serial sections confirmed the data and showed a heterogeneity in dense granule shape not reported in control cells.     

Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy

Although exocytosis is widely believed to involve granule movement, membrane fusion and the emptying of granule content, direct study of these processes has been difficult in living cells because of the limited resolution of conventional light microscopy. Using video-enhanced microscopy and confocal laser microscopy, we have now studied these processes in living rat parotid and submandibular gland acinar cells. Under a differential interference contrast (DIC) microscope equipped with a CCD camera and a high speed image processor, secretory granules were in general stationary even after secretory stimulation with isoproterenol (IPR). Following IPR stimulation, however, there were abrupt changes in light intensity of secretory granules, and many granules disappeared. Confocal microscopy was then performed to confirm whether the observed changes in granules were related to membrane fusion and content release. For this, cells were perfused with the fluid-phase tracer Lucifer Yellow; confocal images thus obtained clearly demonstrated the appearance of fluorescence in omega-shaped invaginations of the apical plasma membrane which corresponded to the sites at which changes were observed in DIC images. The time sequence analyses of confocal images showed that there was a repetitive appearance and disappearance of omega-shaped fluorescent foci at the apical plasma membrane until most of the granules were depleted. During this time, there did not appear to be any significant expansion of the apical plasma membrane and if endocytic uptake of the tracer occurred, it was below the limit of detection. These observations provide new insights into the exocytotic process in salivary glands and are at variance in some respects with previous interpretations made from electron microscopy.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

Effect of trypsin on mouse mammary tumor virus.

Undisrupted mouse mammary tumor virus (MuMTV) derived from the milk of of RIII mice has been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy after treatment with insolubilized trypsin. No alterations were found in viral fine structure by either freeze-etch or negative-stain electron microscopy. No alterations were found in the ability of trypsinized virus to compete in a radioimmune assay for viral antigens. Infectivity experiments indicate no significant differences in the ability of treated virus to infect C57Bl mice. However, significant differences were observed in polypeptide composition. The intensely periodic acid-Schiff-positive band, gp140, was shown by galactose oxidase-borotritide labeling to be degraded into a fragment of 125,000 molecular weight. The major glycoprotein, gp55, was split into fragments of 36,000 and 23,000 molecular weight, both of which stained with periodic acid-Schiff stain. Gp68 was removed from the virus. Experiments with purified, iodinated gp55 showed that the trypsin-induced fragments of gp55 were immunologically active. We conclude that: (i) certain glycoproteins at the surface of MuMTV are accessible to an insoluble form of trypsin, (ii) the trypsin causes a nick in the polypeptide chain without affecting the configuration of the molecule; (iii) the nicked molecules remain bound to the virus; and (iv) the presence of these nicked molecules does not interfere with the biological or antigenic expression of virus function.     

Intracellular polycationic molecules cause reversible swelling of the rough endoplasmic reticulum

The microinjection of polycationic but not anionic molecules causes swelling of the rough endoplasmic reticulum (RER) in salivary gland cells of a fly larva. Ca-EGTA buffers, lanthanum chloride, lysozyme, bovine serum albumin, cationic and anionic ferritin were microinjected into salivary gland cells and their effects observed by light and electron microscopy. Immediately after the microinjection of polycationic molecules, the cytoplasm changed from transparent to opaque as the RER became swollen. Binding of polycationic molecules to the RER may cause the membrane to become permeable to some solute and swell due to osmotic forces.     

STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.