Protoplasmic Face Research Articles

Intramembrane particles and responses of sensory axon terminals during reinnervation of the frog muscle spindle

Changes in the density of intramembrane particles (IMPs) of sensory nerve terminals in the bullfrog muscle spindle were correlated with recovery in the response of the spindle to stretch during postcrush reinnervation. A few IMPs on the protoplasmic (P) face in summer experiments (June to October) reappeared by the 3rd week after the nerve crush, then rapidly increased to 110% and 120% of control values 2 and 2.5 months after the crush. Afferent responses to stretch could be recorded after the mean IMP density on the P-face in terminal branches had recovered to more than 25% of the control value. The discharge rate showed a plateau pattern during the period of the excessive IMPs. This was supplanted by a normal pattern after a myelinated branch of the sensory axon was cut. The IMPs in winter experiments (November to April) reappeared by day 90 after nerve crush, and then slowly increased. The sustained responses to stretch reappeared after 5 months, when the mean IMP density on the P-face was restored to 25% or more of the control. Neither excessive density of the IMPs nor plateau pattern of the discharge rate were observed in winter experiments. The relation between the regenerated IMP densities and the functional recovery is discussed.

Effects of cigarette smoke condensate and 12-0-tetradecanoylphorbol-13-acetage on gap junction structure and function in cultured cells

The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.

Freeze-Fracture Morphometry on Normal and Cancer Breast Tissues

A stereological procedure to evaluate the number of particles per unit surface area (Np/micron2) and the particle density distribution in both protoplasmic (pf) and external face (ef) of the plasma membrane was applied on freeze-fracture preparations of human breast tissues with invasive carcinoma and with non-neoplastic lesions. A significant higher intramembrane particle density was recorded on the protoplasmic face of plasma membrane of epithelial cells from non-neoplastic breast tissues (2999 +/- 720 Np/micron2) compared to the particle density on the corresponding face of cancer cells (911 +/- 608 Np/micron2). Moreover, in the latter cells the external face had more than 50% of the surface area totally smooth while residual particles were often aggregated. The reduction of integral glycoproteins in the plasma membrane of malignant tissues fits apparently well with the increased activity of the methyltransferase-I, an enzyme methylating membrane phospholipids. These findings suggest a distribution of constituents and a lipid environment peculiar for the plasma membrane of breast cancer cells.

The haustorial host-parasite interface in rust-infected bean leaves after high-pressure freezing

The haustorial fine structure of the bean rust fungus, Uromyces appendiculatus var. appendiculatus, was studied within the cells of its host, Phaseolus vulgaris. Results were obtained after high-pressure freezing and subsequent freeze-substitution or freeze-fracturing. Good preservation of leaf tissue after freeze-substitution needed cryoprotection with 8% methanol. For freeze-fracturing, no chemical treatment was applied. In addition to the organelles which are generally found in fungi after cryo-fixation, tubular-vesicular complexes were found in the cytoplasm. Both techniques revealed an extrahaustorial matrix of even width, surrounding the haustorial body. The extrahaustorial membrane was not undulated, and the side facing the plant cytoplasm was lined with a delicate fringe of well-stained material. The extrahaustorial membrane was nearly devoid of intramembrane particles. The host plasma membrane in infected tissue, especially the protoplasmic face, had fewer intramembrane particles than those in uninfected tissue. The haustorial plasma membrane contained many intramembrane particles.

Momentary alteration of the postsynaptic membrane during transmission of a single nerve impulse.

Transmission of a nerve impulse at neuromuscular and other synapses is an extremely brief event. By using rapid-freezing and cryofracture techniques in the electric organ of Torpedo, synaptic transmission was found to be accompanied by significant changes affecting the postsynaptic membrane for a few milliseconds. In the replicas, the protoplasmic leaflet of this membrane was seen to contain intramembrane particles (IMPs) of two different forms, globular and elongated. Globular IMPs had a mean diameter of 8.8 nm; they were the most frequently found (80% in unstimulated specimens). Elongated IMPs had a major diameter of 17.9 nm, about twice that of globular IMPs. Transmission of a single nerve impulse was accompanied by a marked decrease in the number of globular IMPs and by an increase in the number of elongated IMPs, as if there were a coalescence of two adjacent round particles to form an elongated one. These changes started soon after the electrical stimulus and lasted for approximately 3 ms. IMPs in the postsynaptic protoplasmic face are thought to correspond to a certain proportion of nicotinic acetylcholine receptors that were extracted with this leaflet during the fracture process. The phenomenon described here reflects an abrupt change in the membrane, probably linked to activation of the acetylcholine nicotinic receptors.

Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells

Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly.

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of barriers to water flow in toad urinary bladder

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37°C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.

Freeze-fracture study of Blastocystis hominis.

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMP's). On all membrane replicas, the concentration of IMP's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMP's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.

Freeze‐Fracture Study of the Endosymbiont of Blastocrithidia culicis1

ABSTRACTThe two unit membranes which envelope the endosymbiont of the trypanosomatid protozoon, Blastocrithidia culicis, were studied using the freeze‐fracture technique. The distribution of the intramembranous particles on both fracture faces of the inner and outer membrane of the endosymbiont was analyzed in the replicas. The protoplasmic face of the inner membrane (PFi) had a higher density of membrane particles than that observed on the extracellular face (EFi), a pattern typical of plasma membranes. The extracellular face of the outer membrane (EFo) presented a density of membrane particles much higher than that observed on the P face of the outer membrane (PFo) a distribution significantly different from that found in the inner membrane of the endosymbiont and in the plasma membrane of the protozoon, but similar to that observed in Gram‐negative bacteria. The data obtained support the idea that the endosymbiont of trypanosomatids represents a Gram‐negative bacterium‐like microorganism enveloped by two unit membranes and lacking a peptidoglycan layer and which lives in direct contact with the cytoplasm of the protozoon.

Altered plasma membrane ultrastructure in multidrug-resistant cells

Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.

The ultrastructure of the cysts of Blastocrithidia triatomae Cerisola et al. 1971 (Trypanosomatidae): a freeze-fracture study.

Cysts of Blastocrithidia triatomae from hosts and from in vitro cultures were investigated by freeze-fracture and freeze-etch electron microscopy. No differences in structure were observed between cysts frozen immediately and those fixed and cryoprotected with glycerol. The ultrastructure of all cysts differed considerably from that known for mastigote stages of trypanosomatids: the nuclear pores were surrounded by circular fracture-plane transitions between the inner and outer nuclear membranes; the matrix of the kinetoplast appeared banded; the mitochondrion and the endomembrane system were poorly developed; no flagellar apparatus or basal body could be found; and the subpellicular microtubules characteristic of trypanosomatids were absent. Beneath the surface membrane, a special region, 80-90 nm thick and consisting of several rows of closely packed particles was observed. Intramembrane particle (IMP) distribution on the fracture faces of the surface membrane was highly asymmetric: the ectoplasmic face had very few IMPs, whereas the protoplasmic face contained numerous, closely packed IMPs. No external cyst wall was present.

The membrane component of the ribosomal bodies from the lizardLacerta sicula: a freeze-etching study

We have studied, using the freeze-etching technique, the cytomembranes of the flat cystenae, regularly interposed between the cristalline sheets of ribosomes, inside the ribosomal bodies of the hibernating lizardLacerta sicula. The results show the presence, on the protoplasmic face (PF), of numerous IMPs (intramembrane particles) which can be separated into two populations. The flatter ones appear to be arranged in a regular pattern, with the support of the optical diffraction analysis, can be correlated with the disposition of the ribosomes in the cristalline sheets.

Freeze fracture study of the schizonts and merozoites of Leucocytozoon caulleryi

The fine structure of the schizonts and merozoites of Leucocytozoon caulleryi was studied by the freeze fracture technique. Young schizonts were spherical, surrounded by a thick fibrous capsule and possessed numerous nuclei. The nucleus was surrounded by the outer and inner nuclear membrane. A number of nuclear pores were evident in the nuclear membranes. Advanced schizonts were characterized by the formation of cytomeres. Each cytomere was surrounded by a membrane. Several nuclei lay along the periphery of the cytomere. The protoplasmic face (P-face) of the membrane of the cytomere contained numerous intramembranous particles (IMP). The exoerythrocytic merozoites were surrounded by a two-layered pellicle consisting of the outer and inner membrane. During freeze fracturing, each of the two membranes could split along its hydrophobic interior to yield a total of four fracture faces. The P-faces of the outer and inner membranes were closely covered with evenly distributed IMP. The external faces (E-face) of these membranes had less IMP than the P-faces. The membrane architecture of the erythrocytic merozoites was similar to that of the exoerythrocytic merozoites. The erythrocytic merozoite was enveloped by an additional membrane probably derived from the plasma membrane of host cell.

Intramembrane particles and terminal responses following denervation of frog muscle spindles.

The sizes and population density of intramembrane particles in the non-myelinated sensory nerve terminals of frog muscle spindles were studied in comparison with changes in the responses to spindles to stretch 1-28 days after denervation. The sciatic nerve in the right leg was cut at the dorsal part of the hip joint. Spindle receptors in the left leg were used as controls. Muscle spindles were isolated from semitendinosus and iliofibularis muscles. The mean density of particles, which occurred mainly on the protoplasmic (P) face, declined rapidly to 60% of the control values 2 days after denervation, 30% after 4 days and to less than 10% after 7 days. The mean diameter of particles changed from 8.0 +/- 1.5 nm in the control to 12.8 +/- 2.5 nm 7 days after denervation. No significant difference between the results for the trunks (greater than 2 micron in diameter) and the branches (less than 0.5 micron) of the non-myelinated axons was observed. The peak rate of afferent discharges during a ramp-and-hold stretch survived up to 4 days after denervation, whereas the static component disappeared after 3 days. It is suggested that the decline to less than 20% of the control density of particles removes the mechanosensitivity of the sensory endings.

Quantitative analysis of freeze-fracture electron micrographs from red beetroot cells by microdensitometer-based technique

Profiles for the protoplasmic face of the freeze-fractured membrane from the root storage tissue of red beets are reconstructed by microdensitometry of electron micrographs of surface-shadowed platinum carbon replicas. Different distribution functions (heights, cross sections, volumes) related to the intramembrane particles are determined from the digitized profiles. Those distribution functions are used to obtain information about the size and shape of particles. Statistical functions such as autocovariance functions (ACFs) and roughness spectrum are also computed from quantized profiles. The initial portions of the ACFs have a Gaussian form whose parameters (root-mean-square surface roughness height δ and autocovariance length σ) are estimated. The parameter estimates deduced from ACFs are shown to be in good agreement with those retrieved from distribution functions. The method illustrated in this paper on plasma membrane particles from red beetroot cells is quite general and can be applied to any intramembrane particles.

Effect of Hyperthermia on the Plasma Membrane Structure of Chinese Hamster V79 Fibroblasts: A Quantitative Freeze-Fracture Study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.

Insulin modulates neuronal plasma membrane development in human fetal spinal cord neurons in culture.

The number of intramembrane protein particles (IMP) in the protoplasmic face of the perikaryal plasma membrane was evaluated in neurons from 9 week-old and 12 week-old human fetal spinal cord in culture. An increased number of IMP was observed in membranes from 12 week-old fetal neurons when compared to membranes from 9 week-old fetal neurons. The addition of insulin (100-2500 microU/ml) to the culture media resulted in a significantly increased number of IMP in neuronal membranes. Incubation with glucose (1.5-6 mg/ml) did not modify the number of IMP and glucose did not potentiate the effect of insulin when both glucose (3 mg/ml) and insulin (500 microU/ml) were added to the culture media. The results suggest that insulin may modulate the development of neuronal membranes and that this effect is not mediated by an increased glucose utilization.

The intramembrane structure of septate junctions based on direct freezing.

Smooth septate junctions from the midgut of the cricket, Acheta, and the horseshoe crab, Limulus, as well as Hydra-type septate junctions from the epidermis of Hydra have been studied by freeze-fracture after direct freezing using the liquid helium-cooled copper block/slam freezing method. The exoplasmic fracture face at both types of septate junction exhibits rows of closely packed but irregularly shaped intramembrane particles. Complementary to these particle rows, on the protoplasmic fracture face, are sharply defined grooves with a periodic variation in depth and width that was conspicuous in Hydra but less well defined in arthropods. The closely packed, irregular particles on the exoplasmic faces could represent plastically deformed portions of transmembrane proteins pulled through the bilayer during freeze-fracture. On the basis of this interpretation, the grooves on the protoplasmic faces represent a confluence of the bilayer disruptions occurring during fracturing. The structures observed here are different from those reported in replicas of glutaraldehyde-fixed and glycerol-cryoprotected tissue, in which the intramembrane junctional components partition with the protoplasmic face and often assume the appearance of continuous cylinders. This comparison illustrates some of the artifacts associated with freeze-fracturing and shadowing. On the basis of a comparison of freeze-fracture replicas and sections of lanthanum-infiltrated tissues, the relationship between intramembrane junctional components and intercellular septal elements is analysed.