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 degrees 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.     

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes.

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.     

Rotary-Shadowed Freeze-Fracture Replicas: A Simple Method for the Analysis of Fracture Faces

In rotary-shadowed freeze-fracture replicas, intramembrane particles on the periphery of a membrane fracture face are not uniformly shadowed from all sides. Those eccentrically positioned intramembrane particles with a net centripetally directed shadowing are on a convex fracture face. In contrast, those eccentrically positioned intramembrane particles with a net centrifugally directed shadowing are on a concave fracture face. Centrally positioned intramembrane particles on convex faces are uniformly shadowed from all sides; however, central depressions of concave faces are often unshadowed.     

Rotary-shadowed freeze-fracture replicas: a simple method for the analysis of fracture faces

In rotary-shadowed freeze-fracture replicas, intramembrane particles on the periphery of a membrane fracture face are not uniformly shadowed from all sides. Those eccentrically positioned intramembrane particles with a net centripetally directed shadowing are on a convex fracture face. In contrast, those eccentrically positioned intramembrane particles with a net centrifugally directed shadowing are on a concave fracture face. Centrally positioned intramembrane particles on convex faces are uniformly shadowed from all sides; however, central depressions of concave faces are often unshadowed.     

Alterations in intramembrane particle distribution during interaction of erythrocyte-bound ligands with immunoprotein receptors

Freeze fracture studies have been performed on rabbit pulmonary alveolar macrophages and a nonphagocytic murine lymphoblastoid cell line, PU-5 Fc+, incubated with sheep erythrocytes, sheep erythrocyte-IgG Forssman antibody complex, sheep erythrocyte-IgG Forssman antibody-C complexes and aggregated IgG. Alveolar macrophages show redistribution of intramembrane particles after interaction with (EIgG) and E(IgM)C. The murine lymphoblastoid cell line shows intramembrane particle redistribution consequential to binding of E(IgG) and aggregated IgG. The results demonstrate that after specific immunoprotein receptor-ligand interaction, there is extensive plasma membrane reorganization which results in a redistribution and loss of intramembrane particles. Changes are observed in the protoplasmic face of the plasma membrane after the binding of ligand to the outer membrane surface. The findings suggest that interaction of erthrocyte-bound ligands with specific lymphoid and macrophage plasma membrane receptors leads to a generalized redistribution of integral membrane components in the membrane.     

Topographical relations of intramembrane particle distribution patterns in human sperm membranes

The distribution of intramembrane particles in human sperm membranes has been explored with particular reference to the topographical region of the sperm cell and the membranes' fracture face. Conspicuous differences in the size, arrangement, density, and lateral mobility of intramembrane particles between some topographically distinct membrane domains are demonstrated. The greatest regionality is exhibited by the plasma membrane. In sperm head regions, it shows a significant variability and changes its particle distribution during culture in capacitating medium. In contrast, little variability and no changes during the incubation are seen in the acrosomal and nuclear membranes. Striking is the difference in particle distribution on the E face of the outer acrosomal membrane between the acrosomal and equatorial regions. It is suggested that the invariable regional difference in the organization of the outer acrosomal membrane may bear on the different behavior of its two main domains during sperm capacitation and acrosome reaction.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

Membrane structure of caveolae and isolated caveolin-rich vesicles

 Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80–120 nm with an open porus of 30–50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30–50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles. Accepted: 16 September 1998     

Heterogeneity of the zymogen granule membranes in rat pancreas

Zymogen granules (ZG) of rat pancreas have been isolated by the procedure of Paquet et al. The granules lysed when exposed to alkaline pH (pH 8.2), and their membranes could be subfractionated by centrifugation on a sucrose gradient. Four discrete types of membranes corresponding to densities of 1.105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively and characterized both by morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands but in different proportions. Type A membranes are comprised of four major bands corresponding to molecular weights of 80, 69, 54, and 20 kDa, being in higher concentration than the others. Types B and C contain three major bands at 80, 54 and 20 kDa whereas type D is comprised of only two major bands at 69 and 54 kDa, the latter polypeptide corresponding to ATP-diphosphohydrolase activity which is present in all four membrane types. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM) showed that type A are large superimposed sheets of membranes with amorphous material between sheets. The surface area of these sheets corresponds grossly to the surface of an intact ZG with a few intramembrane particles (IMP) distributed at random or in small aggregates on large smooth fracture planes. Types B and C exhibit a totally different aspect, forming closed vesicles about the size of a small ZG with few IMP distributed at random or in small aggregates on smooth fracture planes. Type D membranes are very small vesicles with no detectable IMP on relatively smooth fracture planes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Freeze-fracture study of rat liver peroxisomes: evidence for an induction of intramembrane particles by agents stimulating peroxisomal proliferation

The membrane ultrastructure of isolated rat liver peroxisomes has been observed by rapid freezing and freeze-fracture techniques. Unidirectional and rotary shadowing allows a clear visualization of the intramembrane particles (IMPs) on both the protoplasmic fracture (PF) leaflet and the endoplasmic fracture (EF) leaflet and reveals an asymmetric distribution of IMPs. Both fracture faces were uniformly studded by IMPs, and the frequency was about seven times higher on the P face (2322 per 1.0 micron2) than on the E face (322 per 1.0 micron2). Administration of the peroxisomal proliferator clofibrate (ethyl-p-chlorophenoxyisobutyrate) induced a marked increase in the frequency of IMPs on both the P face (2.2-fold) and the E face (1.7-fold). The average size decreased (P less than 0.001) from 45.7 +/- 16.5 nm2 to 35.2 +/- 10.8 nm2 on the P face. A similar increase in the frequency of IMPs was observed on the P face (1.8-fold) and the E face (1.8-fold) of peroxisomes from rats fed a semisynthetic diet containing 20% (w/w) of partially hydrogenated fish oil. The average size increased (P less than 0.001) from 36.6 +/- 19.7 to 50.0 +/- 23.5 nm2 on the E face. This study demonstrates alterations both in frequency and size distribution of IMPs in liver peroxisomal membranes on exposure of rats to agents known to induce peroxisomal proliferation. The increase in frequency of IMPs was as expected from the observed increase in one of the major integral membrane polypeptides, with apparent molecular mass of 69 (or 70) kDa, in proliferating rat liver peroxisomes.     

Carotenoid-containing outer membrane of Synechocystis sp. strain PCC6714.

Outer membranes, free of cytoplasmic or thylakoid membranes and peptidoglycan components, were obtained from Synechocystis sp. strain PCC6714. Electron microscope studies revealed double-track outer membrane vesicles with a smooth-appearing exoplasmic surface, an exoplasmic fracture face covered by closely packed particles and a corresponding plasmic fracture face with regularly distributed holes. Lipopolysaccharide, proteins, lipids, and carotenoids were the constituents of the outer membrane of Synechocystis sp. PCC6714. Twelve polypeptides were found in outer membrane fractions, among them two dominant outer membrane proteins (Mrs, 67,000 and 61,000). Lipopolysaccharide-specific components were GlcN and an unidentified heptose. Outer membrane lipid extracts contained phosphatidylglycerol, sulfolipid, phosphatidylcholine, and unknown lipids. The carotenoids, myxoxanthophyll, related carotenoid-glycosides, zeaxanthin, echinenone, and beta-carotene were found to be true constituents of the outer membrane of Synechocystis sp. PCC6714.     

A freeze-fracture study of membrane events during neurohypophysial secretion

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.     

Correlation of structure and function of chloroplast membranes at the supramolecular level

Freeze-fracture electron microscopy has revealed that different size classes of intramembrane particles of chloroplast membranes are nonrandomly distributed between appressed grana and nonappressed stroma membrane regions. It is now generally assumed that thylakoid membranes contain five major functional complexes, each of which can give rise to an intramembrane particle of a defined size. These are the photosystem II complex, the photosystem I complex, the cytochrome f/b6 complex, the chlorophyll a/b light-harvesting complex, and the CF0 -CF1 ATP synthetase complex. By mapping the distribution of the different categories of intramembrane particles, information on the lateral organization of functional membrane units of thylakoid membranes can be determined. In this review, we present a brief summary of the evidence supporting the correlation of specific categories of intramembrane particles with known biochemical entities. In addition, we discuss studies showing that ions and phosphorylation of the membrane adhesion factor, the chlorophyll a/b light-harvesting complex, can affect the lateral organization of chloroplast membrane components and thereby regulate membrane function.     

Ultrastructural and chemical analysis of the outer membrane leaflet of the human red blood cell

Structural and biochemical analysis of the outer membrane leaflet of human erythrocytes freeze-fractured on positively charged supports showed that glycophorin A is its major constituent. Two classes of intramembrane particles can be discriminated on the external fracture face: those which are high but small in diameter and those which are low and large or elongated. The presence of small amount of band 3 protein in the outer membrane leaflet cannot be ruled out; it could be contained in the class of 'high' intramembrane particles on the external fracture face.     

Freeze-fracture study of the pavement cell in the lamprey gill epithelium. Analogy of membrane structure with the granular cell in the amphibian urinary bladder

Freeze-fracture studies of the lamprey gill epithelium reveal structural differences of the luminal and basolateral plasma membrane of the pavement cells. The luminal membrane is characterized by only a few intramembrane particles on the P face and numerous large (10-13 nm) particles on the E face, whereas the basolateral membrane shows the majority of intramembrane particles (6-8 nm) on the P face. The structural specialization of the luminal membrane and the differences between the luminal and basolateral membranes of the pavement cell are similar to those previously demonstrated for the unstimulated granular cell of the amphibian urinary bladder. Because of this similarity, it is suggested that the 2 cell types are analogous and that the luminal membrane of the pavement cell in the lamprey gill epithelium is functionally characterized by a low water permeability. A possible role of sodium uptake by the pavement cells from freshwater and putative differences of osmoregulatory mechanisms in the gills of lampreys and teleosts in freshwater environments are discussed.     

A Freeze-Fracture Study of the Microcyst Wall,Plasma Membrane,and Intramembrane Components of Physarum polycephalum1

ABSTRACT. Freeze-cleave replicas of encysted cells of the myxamoebae of Physarum polycephalum were examined to determine the intrawall substructure and to compare the intramembrane structural components. Cleaved areas of the cyst wall revealed a laminated substructure devoid of the macromolecular interruptions (intramembrane particles or IMP) visible in the cleaved cell membranes. The cyst wall adjacent to the cell membrane appears laminated, and a loose branching network of fibers and fibrils occurs at the wall periphery. The absence of intrawall particles is interpreted as a lack of protein or polypeptide components, thus suggesting additional support for the conclusion that polysaccharides are the major wall constituents. When cleaved cell membranes of encysted cells were examined, more intramembrane particles per unit area were observed on the extracellular membrane leaflet than on the protoplasmic membrane leaflet. In addition, homogeneous as well as aggregated particle distributions were visible on cleaved membrane leaflets. Moreover, the presence of aggregated and unaggregated particles on the same membrane leaflets similarly suggests asynchrony of the cell population. This paper examines and compares biological processes involving the cell membrane that may be related to different stages in the cell cycle or to periods of temporary stasis during the cell cycle.     

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.     

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.Abbreviations C carboxysome - CM cytoplasmic membrane - EF_s, EF_u exoplasmic fracture face of stacked and unstacked membrane area, respectively - ES exoplasmic surface - PF_s, PF_u plasmic fracture face of stacked and unstacked membrane area, respectively - PG peptidoglycan layer - TM thylakoid membrane Dedicated to Prof. Dr. D. Peters, Hamburg, on the occasion of his 75th birthday     

Comparison of the ultrastructure of adrenaline and noradrenaline storage granules of bovine adrenal medulla

The ultrastructure of the membranes of noradrenaline (NA) and adrenaline (A) granules of the bovine adrenal medulla (Terland, O., T. Flatmark, and H. Kryvi, Biochim, Biophys. Acta 553, 460--468 (1979)) was analyzed by transmission, negative staining and freeze-etch electron microscopy. The two types of storage granules can be distinguished mainly by two morphological criteria: (a) The NA-granules have a more electron dense matrix core than the A-granules, (b) the NA-granules revealed less asymmetry in the distribution of intramembrane particles (nPF:nEF = 4,5:1) than the A-granules (nPF:nEF = 9:1). Thus, the trilaminar structure, negative staining pattern and size distribution of the intramembrane particles of the two fracture faces on freeze-etch electron microscopy were very similar for the two types of granules. Freeze-etching revealed a wide range of the particle size distribution for both fracture faces in both types of granules, with an average diameter of 12.6 +/- 2.7 nm (A-granules) and 10.2 +/- 2.8 nm (NA-granules) for the E-fracture faces and 11.4 +/- 2.7 nm (A-granules) and 9.8 +/- 2.4 nm (NA-granules) for the P-fracture faces. Some of the particles on the P-fracture face (outer surface of the membrane) revealed a subunit structure, most clearly seen in the specimens of NA-granules. Morhpometric analyses of sectioned bovine adrenal medulla revealed that the chromaffin granules on an average account for approx. 13.5% of the cytoplasmic volume in the total population of chromaffin cells.     

Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.