Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.     

Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells

We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. The results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.     

Ultrastructure of Pseudomonas saccharophila at Early and Late Log Phase of Growth

The fine structure of Pseudomonas saccharophila, a soil bacterium, is similar to that of the marine Pseudomonas reported by Wiebe and Chapman. The unit membrane of the plasma membrane is clearly seen in some areas of thin sections. The ribonucleoprotein granules are distributed in the cytoplasm of the cell. Cells of P. saccharophila during early exponential phase are large, and most of them contain a large number of poly-beta-hydroxybutyrate granules. Some of the granules are quite large and occupy up to three-fourths of the cross section of the cell. Thin sections of the cells in the late log phase, however, show fewer and smaller poly-beta-hydroxybutyrate granules located in the central region of the cell. Negative-stained and freeze-fracture preparations show that the outer surface of the cell wall of P. saccharophila is covered with a large number of tiny granules and long, slender flagella. The outer surface of the plasma membrane appears to be smoother than the outer surface of the cell wall, and it also contains numerous granules. Since the outer surface of the cell wall is quite smooth in freeze-fracture preparations, the wrinkled appearance in thin sections is probably an artifact of fixation and dehydration. The poly-beta-hydroxybutyrate did not solidify at the freezing temperature used (approximately -150 C), and it was consequently pulled out in a spikelike structure during the fracturing process. P. saccharophila, under the conditions in our study, appears to multiply by the constrictive type of cell division.     

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.     

Fine structure of Blastocrithidia culicis as seen in thin sections and freeze-fracture replicas

The fine structure of epimastigotes of Blastocrithidia culicis was studied by transmission electron microscopy of thin sections and freeze-fracture replicas. This parasite presents a well developed endoplasmic reticulum and Golgi complex systems. Differences in the density and organization of the intramembranous particles were observed between the membranes which enclose the cell body and the flagellum. Ridge-like elevations, visualized in freeze-fracture replicas, were observed in sites where the mitochondrial branches touched the plasma membrane. A special array of membrane particles was observed on both faces of the flagellar and the cell body membranes at the region where the flagellum adheres to the cell body. It appeared as strands made of two rows of membrane particles. Filipin-treated cells were used for the localization of membrane sterols in freeze-fracture replicas. The number of filipin-sterol complexes varied from cell to cell. In some cells, rows of filipin-sterol complexes were seen. No complexes were observed in the region of the attachment of the flagellum to the cell body.     

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotin-avidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy.When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.     

Effect of the local anesthetic dibucaine on the surface membrane in sterol-manipulated Tetrahymena pyriformis studied by freeze-fracture electron microscopy

The effect of the local anesthetic dibucaine on the membrane ultrastructure of sterol-manipulated Tetrahymena pyriformis (NT-1 strain) was studied by freeze-fracture electron microscopy. Dibucaine-treated, ergosterol-replaced Tetrahymena cells had marked alterations in their plasma membranes. IMP-free small depressions (exoplasmic fracture face) and protrusions (protoplasmic fracture face) were formed on the plasma membranes which was in contact with the outer alveolar membrane. In addition, large IMP-free surface "blebs" covered with hexagonally-arranged depressions and protrusions appeared on both the plasma and outer alveolar membranes. These "blebs" were pinched off when the membranes were severely affected. Our previous study (28) demonstrated that the plasma membrane of dibucaine-treated native Tetrahymena cells that contain tetrahymanol showed vertical displacement of its intramembranous particles and that subsequently a smooth, flat surface appeared. Therefore, the structural changes in ergosterol-replaced membranes produced by dibucaine differ strikingly from changes in the native membranes. The remarkable difference in the ultrastructural deformation of the plasma membrane probably is due to a difference in the membrane lipid composition induced by sterol-manipulation.     

Morphological changes of the adipose cell plasma membrane during lipolysis

Morphological changes of the plasma membrane in the white adipose cell associated with lipid mobilization were assessed qualitatively and quantitatively on freeze-fracture replicas of epididymal adipose tissue from fasted and from streptozotocin-diabetic rats. The number of plasma membrane invaginations and intramembranous particles were evaluated per square micrometer of membrane and per entire adipocyte. These two determinations show that the number per square micrometer (local concentration) of both structural features progressively increases with the duration of diabetes and fasting, while that at the same time their number per entire cell (total content) remains unchanged. These data thus show: (a) a reorganization of the adipose cell plasma membrane during lipolysis; and (b) that this reorganization can be detected only by determining the concentration and the total content of the structural features of the membrane involved.     

Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions

Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4- 6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7- 6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7- 6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques.     

Distribution of intramembranous particles and filipin-sterol complexes in the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Membrane specializations in flagellar ribbons of elasmobranch fish

Specialized epithelial cells lining the elasmobranch nephron bear numerous flagella which are organized into closely-packed, parallel rows forming ribbons (Lacy et al., 1989a). The compact arrangement of the adjacent flagella comprising each ribbon suggests they are structurally bound together, forming a single unit which functions to force urine along the nephric tubule. In the present study, the structural basis of the interflagellar connections was investigated by scanning electron microscopy (SEM) and by transmission electron microscopy (TEM) of thin sections and freeze fracture replicas. Various fixatives and histochemical stains were used to elucidate the structure and composition of the interflagellar adhesive material. SEM of the luminal cell surface showed the organization of the flagella in ribbons. In TEM, fixation in a solution containing glutaraldehyde and tannic acid, Ruthenium red or Alcian blue, or postfixation in reduced OsO(4) revealed that the plasma membrane of each flagellum of a ribbon was surrounded by a thin layer of surface coat composed of very short filaments more prominent at sites where adjacent flagella were in close apposition. In comparable locations, freeze-fracture replicas disclosed small aggregates or plaques of particles arranged in an irregular, discontinuous line on both faces P and E of the flagellar membrane. In areas where the flagella were not arranged into ribbons (most frequently after immersion fixation), the surface coat was thick and expanded and, in replicas, the intramembranous particles were randomly scattered. All of these plasma membrane specializations appear to function in binding adjacent flagella and thus facilitate a coordinated flagellar ribbon beat.     

Freeze-Fracture Study of the Site of Attachment of Cryptosporidium muris in Gastric Glands

ABSTRACT The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Addition of lipid to the photosynthetic membrane: effects on membrane structure and energy transfer

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the addition of lipid from a defined source under experimental conditions. Liposomes prepared by sonication are mixed with purified photosynthetic membranes obtained from spinach chloroplasts and are taken through cycles of freezing and thawing. Several lines of evidence, including gel electrophoresis and freeze-fracture electron microscopy, indicate that an actual addition of lipid has taken place. Structural analysis by freeze-fracture shows that intramembrane particles are widely separated after the addition of large amounts of lipid, with one exception: large hexagonal lattices of particles appear in some regions of the membrane. These lattices are identical in appearance with lattices formed from a single purified component of the membrane known as chlorophyll-protein complex II. The suggestion that the presence of such lattices in lipid-enriched membranes reflects a profound rearrangement of photosynthetic structures has been confirmed by analysis of the fluorescence emission spectra of natural and lipid- enriched membranes. Specifically, lipid addition in each of the cases we have studied results in the apparent detachment of chlorophyll- protein complex II from photosynthetic reaction centers. It is concluded that specific arrangements of components in the photosynthetic membrane, necessary for the normal functioning of the membrane in the light reaction of photosynthesis, can be regulated to a large extent by the lipid content of the membrane.     

Organization of calcium pump protein dimers in the isolated sarcoplasmic reticulum membrane.

The arrangement of the calcium pump protein in the isolated sarcoplasmic reticulum (SR) membrane was examined by optical diffraction of freeze-fracture electron micrographs. Several states of protein particle organization were observed: random, hexagonal and tetragonal packing, and a mixture of hexagonal and tetragonal packing. This suggests that the time-averaged positions of protein particles in the plane of the SR membrane are weakly defined. In addition, there appears to be a greater degree of local or short-range order compared to long-range order within the field of freeze-fracture particles. We utilized measurements from tetragonally or hexagonally packed arrays to determine a unit cell area occupied by each freeze-fracture particle and its associated lipid matrix. When these unit cell areas and the stereologically determined area per freeze-fracture particle were compared to the cross-sectional area occupied by a single calcium pump protein and its associated lipid, obtained by x-ray and neutron diffraction methods, we concluded that each freeze-fracture particle probably represents a dimer of pump protein molecules in the plane of the SR membrane.     

Particle Assemblies in the Plasma Membrane of Tetrahymena: Relationship to Cell Surface Topography and Cellular Morphogenesis1

During a freeze-fracture electron microscopical study of the plasma membrane of Tetrahymena, several different types of organized particle assemblies were observed. Three of these were found only on the protoplasmic face and were localized in the anterior-ventral region of the cell. These consisted of plate-like arrays composed of 4–25 triplet rows of small 3–4 nm particles; long, paired linear arrays localized at the tops of cortical ridges and composed of 7–8 nm particles; and elongated tetragonal arrays located in the grooves between ridges and composed of approximately 10 nm particles. The distribution of these arrays is consistent with roles in cellular morphogenesis, chemoreception, or cell-cell pairing during conjugation. In addition, a unique particle track associated with the cytoproct (anal pore) was observed in the external face of the plasma membrane. Furthermore, the protoplasmic face of the plasma membrane is characterized by a high density of particles organized into localized microarrays, consisting of small paracrystals or strings, which exhibit a loose higher-order patterning most evident toward the anterior end of the cell. Particle distributions on the protoplasmic face do not appear to be significantly altered by conditions that cause clumping of alveolar membrane particles. Taken together, these observations are consistent with the idea that the proteins of the plasma membrane are highly ordered and relatively immobile and that the structure of the plasma membrane is regionally differentiated.     

Freeze-fracture images of exocytosis and endocytosis in anterior pituitary cells of rabbits and mice

Summary Freeze-fracture images of exocytosis and endocytosis were studied in various kinds of secretory cells of the anterior pituitary of mice and rabbits. Exocytotic figures are frequently observed in thin section of the anterior pituitary cells. In freeze-fracture images, small elevated membrane areas without membrane particles are often seen on the PF of the plasma membrane of the secretory cells. There is a secretory granule in the cytoplasm just beneath the particle-free membrane area, and limiting membrane of the granule is also devoid of the membrane particles at the part facing the plasma membrane. The fusion of membranes for exocytosis may occur at this particle-free area.The limiting membrane of the granule which is continuous with the plasma membrane is almost always coated after release of the granule core. This invagination of coated membrane may be an initiation site for the membrane retrieval after exocytosis. In freeze-fracture images, this depressed region with an accumulation of the membrane particles is observed on the PF of the plasma membrane. This particle-rich depressed region is thought to correspond to the coated area of the plasma membrane observed in thin section. It is thought that the membrane retrieval by pinocytosis initiates at the particle-rich depressed region of the plasma membrane.This study was supported by a grant from the Japan Ministry of Education     

Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography]

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.     

Freeze-fracture replica immunolabelling reveals urothelial plaques in cultured urothelial cells

The primary function of the urothelium is to provide the tightest and most impermeable barrier in the body, i.e. the blood-urine barrier. Urothelial plaques are formed and inserted into the apical plasma membrane during advanced stages of urothelial cell differentiation. Currently, it is supposed that differentiation with the final formation of urothelial plaques is hindered in cultured urothelial cells. With the aid of the high-resolution imaging technique of freeze-fracture replica immunolabelling, we here provide evidence that urothelial cells in vitro form uroplakin-positive urothelial plaques, localized in fusiform-shaped vesicles and apical plasma membranes. With the establishment of such an in vitro model of urothelial cells with fully developed urothelial plaques and functional properties equivalent to normal bladder urothelium, new perspectives have emerged which challenge prevailing concepts of apical plasma membrane biogenesis and blood-urine barrier development. This may hopefully provide a timely impulse for many ongoing studies and open up new questions for future research.     

Phenotypic Characterization of Paranoiac and Related Mutants in Paramecium Tetraurelia

Paranoiac and related mutants of Paramecium tetraurelia display altered membrane excitability. We describe an extension of behavioral characterizations of the paranoiac, fast-2, and tetraethylammonium-insensitive mutants, comparing in detail their reactions to sodium stimulation under standard culture conditions, when grown at various temperatures and when starved. We also use freeze-fracture electron microscopic techniques to analyze in these stocks the morphology of organized arrays of membrane particles, the ciliary plaques. This group of mutants is diverse, showing differences in behavior under standard culture conditions and different reactions to temperature and starvation stresses. Ciliary plaque morphology is altered in some, but not all, of the mutants. The possibility is discussed that these plaques may be sites of potassium or sodium transport.