Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules

1978 ◽  
Vol 30 (1) ◽  
pp. 151-174
Author(s):  
J.G. Robertson ◽  
M.P. Warburton ◽  
P. Lyttleton ◽  
A.M. Fordyce ◽  
S. Bullivant
Keyword(s):  
Sucrose Gradient ◽  
Phosphotungstic Acid ◽  
Nadh Oxidase ◽  
Osmotic Shock ◽  
Root Nodules ◽  
Freeze Fracture ◽  
Electrophoretic Analysis ◽  
Thin Sections ◽  
Peribacteroid Space ◽  
Gradient Fractionation

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1–153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.

scholarly journals STRUCTURE OF ISOLATED PLANT GOLGI APPARATUS REVEALED BY NEGATIVE STAINING

1966 ◽  
Vol 28 (2) ◽  
pp. 169-179 ◽  
Author(s):  
William P. Cunningham ◽  
D. James Morré ◽  
H. H. Mollenhauer
Keyword(s):  
Heavy Metal ◽  
Golgi Apparatus ◽  
Sucrose Gradient ◽  
Phosphotungstic Acid ◽  
Morphological Characteristics ◽  
Negative Staining ◽  
The Other ◽  
Functional Specialization ◽  
Thin Sections ◽  
Texture Size

Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO4-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.

Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

1992 ◽  
Vol 50 (1) ◽  
pp. 706-707
Author(s):  
Ji-da Dai ◽  
M. Joseph Costello ◽  
Lawrence I. Gilbert
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Manduca Sexta ◽  
Freeze Fracture ◽  
Cell Communication ◽  
Cellular Level ◽  
Thin Sections ◽  
Prothoracic Glands ◽  
Polyhydroxylated Steroids ◽  
Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

Structural cross-bridges between microtubules and mitochondria in central axons of an insect (Periplaneta americana)

1977 ◽  
Vol 27 (1) ◽  
pp. 255-272
Author(s):  
D.S. Smith ◽  
U. Jarlfors ◽  
M.L. Cayer
Keyword(s):  
Electron Micrographs ◽  
Mitochondrial Membrane ◽  
Periplaneta Americana ◽  
Freeze Fracture ◽  
Outer Mitochondrial Membrane ◽  
Thin Sections ◽  
Cross Bridge ◽  
Multiple Association ◽  
Random Models ◽  
Cross Bridges

The distribution of microtubules and mitochondria in central axons of an insect (Periplaneta americana) is assessed by comparison between counts on micrographs and computed axon random ‘models’. These studies show that the observed multiple association of microtubules with individual mitochondria is statistically highly significant. Electron micrographs of thin sections show that linkage is effected by physical cross-bridge, possibly comprising components from the microtubule and mitochondrion. Linear particle arrays are described on the outer mitochondrial membrane in freeze-fracture replicas, and tentatively related to the bridges seen in thin sections. The results are discussed in terms of proposed roles of microtubules in neurons and other cells.

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

2002 ◽  
Vol 205 (9) ◽  
pp. 1209-1219 ◽  
Author(s):  
Natalie Perzov ◽  
Vered Padler-Karavani ◽  
Hannah Nelson ◽  
Nathan Nelson
Keyword(s):  
Sucrose Gradient ◽  
Ph Sensor ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Immunological Assays ◽  
Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

Stages in the assembly of pleated and smooth septate junctions in developing insect embryos

1982 ◽  
Vol 56 (1) ◽  
pp. 245-262 ◽  
Author(s):  
N.J. Lane ◽  
L.S. Swales
Keyword(s):  
Embryonic Development ◽  
Freeze Fracture ◽  
Organizational Capacity ◽  
Intramembranous Particle ◽  
Septate Junctions ◽  
Thin Sections ◽  
Intramembranous Particles ◽  
Initial Event ◽  
Random Arrays ◽  
Insect Embryos

The stages that occur during the assembly of both pleated and smooth septate junctions in developing insect tissues have been examined. The oesophagus and mid-gut of the embryonic moth, and the oesophagus and central nervous system (CNS) of the locust embryo, have been investigated in thin sections and by freeze-fracture during the course of membrane biogenesis. The smooth septate junctions developing between the lateral borders of the mid-gut exhibit, in the early stages, individual intramembranous particles becoming aligned into short ridges. These ultimately migrate over the membrane face and fuse into longer arrays, which become stacked in parallel with other ridges to form the characteristic mature form of the junction just before hatching. Pleated septate junctions occur between the cells both of the oesophagus and of the perineurium, which ensheathes the neurones and the neuroglial cells in the locust CNS; these are also fully formed by the end of embryonic development. The pleated junctions appear to be assembled during the later stages of CNS or gut differentiation, arising first in embryos about two-thirds of the way through development. During their maturation, the initial event seems to be a membrane depression in the P face, which occurs in patches over the presumptive junctional membrane. Into these depressed regions or ‘formation-plaque’ areas, 8–10 nm particles appear to be inserted intramembranously in apparently random arrays. These particles are the most common elements but larger particles are also present; the former ultimately become aligned in a row. With time, other intramembranous particles come to lie in rows parallel to the original one. By hatching, the typical undulating stacks of parallel intramembranous particle rows are fully formed. Gap junctions also form between the same perineurial or oesophageal cells, usually before, but in some cases at the same time, or just after, the septate junctions have been assembled. Tricellular associations between cells also appear around the same time in embryonic development. The simultaneous assembly of these different junctions reflects a high degree of organizational capacity at the membrane level.

scholarly journals Antibodies against Sulphoquinovosyl-diacyl glycerol and their Reactions with Chloroplasts

1970 ◽  
Vol 25 (4) ◽  
pp. 412-419 ◽  
Author(s):  
Alfons Radunz ◽  
Richard Berzborn
Keyword(s):  
Bovine Serum Albumin ◽  
Ultrasonic Treatment ◽  
Sucrose Gradient ◽  
Osmotic Shock ◽  
Antirrhinum Majus ◽  
Coombs Test ◽  
Phosphatidyl Glycerol ◽  
Lamellar System ◽  
Intravenous Injections ◽  
Fucus Serratus

Using immunological techniques we tried to determine the localization and orientation of the sulpholipid of diloroplasts in thylakoids. During immunization of rabbits with lamellar systems of chloroplasts of Antirrhinum majus antisera were obtained which precipitated sulpholipid emulsions. Antibodies against the sulpholipid were not detected until a second series of intravenous injections.By immunizing purified sulpholipid of Fucus serratus, adsorbed to methylated bovine serum albumin, sulpholipid antisera were acquired. These antisera and the antisera against lamellar systems of Antirrhinum majus reacted with preparations of sulpholipid from widely different plant species. The sulpholipid antisera did not react with emulsions of the mono- or digalactosyl-diacyl glycerol, of lecithin or the anionic phosphatide phosphatidyl glycerol. Lamellar systems isolated without osmotic shock on a sucrose gradient were not agglutinated by sulpholipid antisera directly, but an indirect agglutination could be achieved in a Coombs-test or in a mixed antigen agglutination after Uhlenbruck. Broken chloroplasts, however, were agglutinated directly by sulpholipid antisera, as well lamellar systems which had been disrupted in a French-press or by ultrasonic treatment.From agglutination and agglutination inhibition experiments with lamellar systems prepared by different methods it is concluded that the determinant groups of the sulpholipid are accessible for antibodies in the lamellar system of chloroplasts. But probably the sulpholipid molecules are located inside the thylakoids.

Some ultrastructural aspects of Crithidia guilhermei n.sp. isolated from Phaenicia cuprina (Diptera: Calliphoridae)

1986 ◽  
Vol 64 (12) ◽  
pp. 2837-2842 ◽  
Author(s):  
Maurilio J. Soares ◽  
Reginaldo P. Brazil ◽  
Amilcar Tanuri ◽  
Wanderley de Souza
Keyword(s):  
Freeze Fracture ◽  
Kinetoplast Dna ◽  
Microscopical Level ◽  
Thin Sections ◽  
Crithidia Luciliae ◽  
Transmission Electron ◽  
Light Microscopical Level ◽  
Attachment Region ◽  
Phaenicia Sericata ◽  
A New Species

A flagellate trypanosomatid was isolated from the fly Phaenicia cuprina captured in Rio de Janeiro, Brazil. It grows well in liver infusion – trypticase medium, in the form of choanomastigotes, typical of the genus Crithidia. Morphometrical data obtained at the light microscopical level indicated that the new isolated Crithidia is smaller than Crithidia luciliae, a parasite isolated from Phaenicia sericata. Transmission electron microscopy of thin sections revealed that this trypanosomatid has a flagellar pocket divided into two compartments, one basal and the other apical, separated by a region of attachment of the flagellum to the cell body. The attachment region was characterized in freeze-fracture replicas. The flagellate has a compact kinetoplast DNA network. As in endosymbiote-containing trypanosomatids previously described, no subpellicular microtubules were seen in the regions where the mitochondria touched the plasma membrane, although no endosymbiotes were found in this flagellate. Electrophoretic mobility of six enzymes showed that the parasite could not be grouped in any of the isoenzymic pattern groups of other Crithidia spp. These observations indicate that the trypanosomatid isolated from P. cuprina is a new species of Crithidia. The flagellate is described as Crithidia guilhermei n.sp.

scholarly journals Membrane interactions between secretion granules and plasmalemma in three exocrine glands

1980 ◽  
Vol 84 (2) ◽  
pp. 438-453 ◽  
Author(s):  
Y Tanaka ◽  
P De Camilli ◽  
J Meldolesi
Keyword(s):  
Plasma Membranes ◽  
Intact Cell ◽  
Freeze Fracture ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Membrane Interactions ◽  
Thin Sections ◽  
Secretion Granules

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

scholarly journals A thin-section and freeze-fracture study of microfilament-membrane attachments in choroid plexus and intestinal microvilli.

1978 ◽  
Vol 79 (3) ◽  
pp. 774-787 ◽  
Author(s):  
N S McNutt
Keyword(s):  
Choroid Plexus ◽  
Thin Section ◽  
Brush Border ◽  
Freeze Fracture ◽  
Similar Type ◽  
Heavy Meromyosin ◽  
Thin Sections ◽  
Intestinal Brush Border ◽  
Fracture Study ◽  
20 Nm

Choroid plexus and intestinal microvilli in thin sections have microfilaments in the cytoplasm adjacent to the membranes, and in replicas have broken strands of filaments in both cytoplasm and on E faces of plasm membranes. The microfilaments contain actin as indicated by their binding of heavy meromyosin (HMM). In sections of choroid plexus, the microfilaments are 7-8 nm in diameter and form a loose meshwork which lies parallel to the membrane and which is connected to the membranes both by short, connecting filaments (8 times 30 nm) and dense globules (approximately 15-20 nm). The filamentous strands seen in replicas are approximately 8 nm in diameter. Because they are similar in diameter and are connected to the membrane, these filamentous strands seen in replicas apparently represent the connecting structures, portions of the microfilaments, or both. The filamentous strands attached to the membrane are usually associated with the E face and appear to be pulled through the P half-membrane. In replicas of intestinal brush border microvilli, the connecting strands attaching core microfilaments to the membrane are readily visualized. In contrast, regions of attachment of core microfilaments to dense material at the tips of microvilli are associated with few particles on P faces and with few filamentous strands on the E faces of the membranes. Freeze-fracture replicas suggest a morphologically similar type of connecting strand attachment for microfilament-membrane binding in both choroid plexus and intestinal microvilli, despite the lack of a prominent core bundle of microfilaments in choroid plexus microvilli.

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