Analysis of desmosomal intramembrane particle populations and cytoskeletal elements: Detergent extraction and freeze-fracture

1985 ◽  
Vol 241 (2) ◽  
pp. 341-351 ◽  
Author(s):  
Mike Pirbazari ◽  
DouglasE. Kelly
Keyword(s):  
Freeze Fracture ◽  
Intramembrane Particle ◽  
Detergent Extraction ◽  
Cytoskeletal Elements

SEM of non-coated hepatocellular cytoskeleton of perfused livers

1984 ◽  
Vol 42 ◽  
pp. 306-307
Author(s):  
S.W. French ◽  
N.C. Benson ◽  
C. Davis-Scibienski
Keyword(s):  
Ambient Temperature ◽  
Critical Point ◽  
Protease Inhibitors ◽  
Metal Deposition ◽  
Heat Damage ◽  
Detergent Extraction ◽  
Cytoskeletal Elements ◽  
Triton X ◽  
Excised Tissue

Previous SEM studies of liver cytoskeletal elements have encountered technical difficulties such as variable metal coating and heat damage which occurs during metal deposition. The majority of studies involving evaluation of the cell cytoskeleton have been limited to cells which could be isolated, maintained in culture as a monolayer and thus easily extracted. Detergent extraction of excised tissue by immersion has often been unsatisfactory beyond the depth of several cells. These disadvantages have been avoided in the present study. Whole C3H mouse livers were perfused in situ with 0.5% Triton X-100 in a modified Jahn's buffer including protease inhibitors. Perfusion was continued for 1 to 2 hours at ambient temperature. The liver was then perfused with a 2% buffered gluteraldehyde solution. Liver samples including spontaneous tumors were then maintained in buffered gluteraldehyde for 2 hours. Samples were processed for SEM and TEM using the modified thicarbohydrazide procedure of Malich and Wilson, cryofractured, and critical point dried (CPD). Some samples were mechanically fractured after CPD.

Fine structure of the kidneys of osmotically stressed Mytilus, Mercenari, and Anodonta

1986 ◽  
Vol 64 (12) ◽  
pp. 2779-2787 ◽  
Author(s):  
Hamidur R. Khan ◽  
Mary Lou Ashton ◽  
A. S. M. Saleuddin
Keyword(s):  
Basal Membrane ◽  
Ultrastructural Changes ◽  
Freshwater Bivalve ◽  
Septate Junctions ◽  
Intramembrane Particle ◽  
Marine Bivalves ◽  
Fluorescent Stain ◽  
Cytoskeletal Elements ◽  
Lateral Cell ◽  
Membrane Infoldings

Osmotically induced ultrastructural changes in the kidneys of the freshwater bivalve Anodonta and the marine bivalves Mytilus and Mercenaria were studied. Osmotic stresses were given to Anodonta by keeping them in distilled water or in 6% seawater, and to Mytilus and Mercenaria by keeping them in 50% seawater for various periods. In all of these bivalves, the convoluted, single cell layered kidney epithelia displayed wide lateral intercellular spaces as well as extracellular spaces in the basal membrane infoldings during hyposmotic stress. These spaces were greatly reduced when the animals were kept in isosmotic media (i.e., isosmotic to their respective hemolymphs). The kidney cells contained abundant cytoskeletal elements and microfilaments were often observed in bundles in the basal membrane infoldings. Actin was observed in the basal membrane infoldings using the specific fluorescent stain nitrobenzoxadiazole-phallacidin. The cell contacts of the kidney epthelia were studied in platinum replicas of freeze-fractured tissues. The lateral cell membrane and basal membrane infoldings contained many gap junctions. Many rows of dense intramembrane particles of septate junctions were observed in the kidneys of animals from isosmotic media. The septate junctions in the kidneys of aminals from hyposmotic media contained either fewer intramembrane particle rows or many sinuous intramembrane particle rows. The site of prourine formation in mollusks are discussed.

Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281

1985 ◽  
Vol 78 (1) ◽  
pp. 49-65 ◽  
Author(s):  
N.J. Maihle ◽  
B.H. Satir
Keyword(s):  
Plasma Membrane ◽  
Mutant Strain ◽  
Protein Secretion ◽  
Tetrahymena Thermophila ◽  
Freeze Fracture ◽  
Size Class ◽  
Secretory Product ◽  
Wild Type ◽  
Intramembrane Particle ◽  
Mutant Cells

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

scholarly journals Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections.

1988 ◽  
Vol 107 (6) ◽  
pp. 2511-2521 ◽  
Author(s):  
G Knoll ◽  
K N Burger ◽  
R Bron ◽  
G van Meer ◽  
A J Verkleij
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Morphological Evidence ◽  
Epithelial Cell Line ◽  
Rapid Freezing ◽  
Intramembrane Particle ◽  
Local Point ◽  
Basolateral Plasma Membrane

The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

The extrarhabdomeral cytoskeleton in photoreceptors of Diptera. I. Labile components in the cytoplasm

1984 ◽  
Vol 220 (1220) ◽  
pp. 339-352 ◽  
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Compound Eye ◽  
Freeze Fracture ◽  
Dynamic Processes ◽  
Ionophore A23187 ◽  
The Status ◽  
Thiol Proteases ◽  
Cytoskeletal Elements ◽  
Nm Range

Labile cytoskeletal structures in the cytoplasm of photoreceptors of the blowfly Lucilia and of Drosophila were stabilized before primary fixation for electron microscopy by retinal infiltration with two inhibitors of thiol proteases, Ep-459 or Ep-475. Alternatively, pretreatments employed EGTA in combination with the Ca 2+ ionophore A23187. The following cytoskeletal structures were revealed, (i) Radial, robust filaments run roughly parallel to the axes of the rhabdomeral microvilli and may be continuous with the axial microvillar filaments. They have diameters of 8 nm upwards, and are occasionally seen to be in association with radial microtubules and with pigment granules. (ii) Slender radial filaments with diameters in the 6-8 nm range extend for shorter distances from the bases of microvilli, and are also associated with endocytotic structures. (iii) The receptor cytoplasm is densely occupied by an ill-defined, filamentous network. (iv) Bundles of slender filaments run longitudinally on each side of rhabdoms of R 1-6 in Lucilia , close to the plasma membrane. Dimensions cited for all categories of filament must be treated with caution because of problems of resolution. Photoreceptors do not bind the fluorescent F-actin probe NBD-phallacidin either without or after treatment with thiol protease inhibitors, and slender filaments are of greater diameter than the 4-5 nm obtained for identified actin filaments in the basement membrane of the compound eye of Lucilia . Infiltration of retinae with Ep-459 or Ep-475 neither prejudices phototransduction, nor impairs the radial migrations of granules of screening pigment in response to light or dark adaption. The status of these cytoskeletal elements is discussed in terms of the dynamic processes of the photoreceptors, and of various labile filaments described from recent studies of vertebrate material using the deep-etch freeze-fracture technique.

A freeze-fracture analysis of intramembrane particle densities on dystrophic hamster heart sarcolemma

1984 ◽  
Vol 7 (7) ◽  
pp. 513-523 ◽  
Author(s):  
Kathryn A. Graham ◽  
Richard R. Shivers ◽  
Burr G. Atkinson
Keyword(s):  
Freeze Fracture ◽  
Fracture Analysis ◽  
Intramembrane Particle ◽  
Heart Sarcolemma ◽  
Hamster Heart

Studies on Trifluoperazine Inhibition of Trichocyst Expansion and Secretion in Paramecium

1981 ◽  
Vol 39 ◽  
pp. 576-579
Author(s):  
Robert S. Garofalo ◽  
Birgit H. Satir.
Keyword(s):  
Picric Acid ◽  
Freeze Fracture ◽  
Secretory Vesicle ◽  
Paramecium Tetraurelia ◽  
Intramembrane Particle ◽  
Cellular Processes ◽  
Vesicle Membranes ◽  
Stimulus Secretion Coupling ◽  
Secretory Products ◽  
Secretory System

The importance of Ca2+ ion in regulation of many cellular processes is well established, as is its role as the mediator of “stimulus-secretion coupling” in exocytic release of secretory products. However, while the Ca2+ requirement in many secretory systems is clear, its mode of action is not.To probe the site of Ca2+ action, we have employed the protozoan, Paramecium tetraurelia as a model secretory system. Secretion in Paramecium requires Ca2+ as well as specific components in both the plasma and secretory vesicle membranes that can be visualized in freeze fracture as organized intramembrane particle arrays. The fusion of plasma and secretory organelle membranes, and subsequent release of vesicle contents, is routinely elicited by exposure to picric acid, and stimulation results in synchronous release of hundreds of secretory products called trichocysts. A rise in cytoplasmic Ca2+ triggers release and expansion of the trichocysts. This is accompanied by a reordering of the trichocysts' paracrystalline matrix, transforming the 5 μm long organelle to a 20-40 μm long needle-like structure.

scholarly journals Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells.

1980 ◽  
Vol 85 (1) ◽  
pp. 83-95 ◽  
Author(s):  
J Muller ◽  
W A Kachadorian ◽  
V A DiScala
Keyword(s):  
Urinary Bladder ◽  
Freeze Fracture ◽  
Toad Urinary Bladder ◽  
Granular Cells ◽  
Thin Sections ◽  
Intramembrane Particle ◽  
Luminal Membrane ◽  
Particle Aggregates ◽  
Cytoplasmic Structures ◽  
Induced Changes

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.

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