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.

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 The permeability barrier in mammalian epidermis.

1975 ◽  
Vol 65 (1) ◽  
pp. 180-191 ◽  
Author(s):  
P M Elias ◽  
D S Friend
Keyword(s):  
Plasma Membrane ◽  
Stratum Corneum ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Water Soluble ◽  
Structural Basis ◽  
Fracture Plane ◽  
Permeability Barrier ◽  
Stratum Granulosum ◽  
Lamellar Bodies

The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.

scholarly journals Induction of vacuolar apical compartments in the Caco-2 intestinal epithelial cell line

1991 ◽  
Vol 100 (3) ◽  
pp. 451-458 ◽  
Author(s):  
T. Gilbert ◽  
E. Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Inhibitors ◽  
Complete Disruption ◽  
Basolateral Plasma Membrane ◽  
Microtubular Network

Complete disruption of the microtubular network by colchicine or nocodazole in Caco-2 intestinal epithelial cells results in the appearance of basolateral microvilli and brush border-containing intracellular vacuoles (vacuolar apical compartment: VAC). These vacuoles are surrounded by a terminal web, express apical markers and exclude basolateral markers. The vacuoles do not originate from internalized apical or basolateral plasma membrane and their development is blocked by protein synthesis inhibitors, suggesting that they are newly synthesized. After removal of the microtubule inhibitors, VACs are usually degraded and/or released into the lateral intercellular space. Rarely was fusion with the apical membrane observed. These experiments support a role for microtubules in the biogenesis of the apical surface and indicate that, under some conditions, apical plasma membrane assembly may occur in the cytoplasm, as observed in some human pathological states.

Ultrastructural changes related to functional activity in gastric oxyntic cells

1981 ◽  
Vol 241 (5) ◽  
pp. G349-G358 ◽  
Author(s):  
J. G. Forte ◽  
J. A. Black ◽  
T. M. Forte ◽  
T. E. Machen ◽  
J. M. Wolosin
Keyword(s):  
Alternative Hypothesis ◽  
Membrane Surface ◽  
Apical Cell ◽  
Freeze Fracture ◽  
Weight Of Evidence ◽  
Ultrastructural Changes ◽  
Morphological Evidence ◽  
Apical Surface ◽  
Oxyntic Cell ◽  
Macromolecular Tracers

When stimulated to secrete HCl the gastric oxyntic cell undergoes profound morphological change. The identifiable apical cell surface is greatly expanded in the stimulated oxyntic cell as compared with nonsecreting ones. To account for this change, one hypothesis proposes that the expanded surface is derived from the fusion of cytoplasmic tubulovesicular membranes with the existing limited apical membrane surface. An alternative hypothesis suggests that the tubulovesicular compartment is actually confluent with the apical surface at all times and that the morphological appearance follows the expansion of this supercollapsed compartment as HCl secretion commences. A variety of morphological evidence is reviewed here including transmission electron microscopy during various stages of secretion and inhibition, analysis of freeze-fracture replicas, penetration of macromolecular tracers, and membrane surface-staining characteristics. It is concluded that the weight of evidence favors a membrane fusion process. Moreover, recent comparative studies of membrane fractions from resting and secreting stomachs show different morphological and functional properties that are also consistent with a fusion hypothesis as a fundamental event in the membrane transformation of the oxyntic cell.

Mosaic structure in the plasma membrane: spiral arrays of subunits in the cytoplasmic tubules of lamprey chloride cells

1982 ◽  
Vol 56 (1) ◽  
pp. 441-452
Author(s):  
T. Hatae ◽  
E.L. Benedetti
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
High Salinity ◽  
Petromyzon Marinus ◽  
Freeze Fracture ◽  
Chloride Cell ◽  
Chloride Cells ◽  
Ionic Homeostasis ◽  
Electron Dense Material ◽  
Basolateral Plasma Membrane

The membrane architecture of the cytoplasmic tubules in lamprey chloride cells has been studied by electron microscopy using thin-section and freeze-fracture techniques. The chloride cell of lampreys (Lampetra japonica, Petromyzon marinus) is largely occupied with a continuous network of cytoplasmic tubules, which are derived from the invagination of the basolateral plasma membrane. The lumenal surface of this tubular network is covered with spirally wound parallel rows of electron-dense material, which consist of linear aggregates of particles. Freeze-fracture of the membrane of the tubules also shows spiral arrangements of particles (approximately 9 nm in diameter) on the P-face and complementary shallow grooves on the E-face. These arrangements of particles are about 17 nm apart and wound at a pitch of about 45 degrees. These complex organizations of the membrane of the tubules are probably the sites of transport of ion and water, which is essential for the maintenance of ionic homeostasis in both low- and high-salinity environments.

Freeze fracture of the gill epithelium of euryhaline teleost fish

1980 ◽  
Vol 238 (3) ◽  
pp. R207-R212 ◽  
Author(s):  
C. Sardet
Keyword(s):  
Plasma Membrane ◽  
Tight Junctions ◽  
Teleost Fish ◽  
Freeze Fracture ◽  
Chloride Cells ◽  
Gill Epithelium ◽  
Functional Importance ◽  
Seawater Adaptation ◽  
Basolateral Plasma Membrane ◽  
Euryhaline Teleost

We briefly discuss the information one can obtain using freeze fracture. We used this technique to precisely identify the types of junctions that link the cells of the gill epithelium of euryhaline teleosts. In particular we demonstrate that seawater adaptation is characterized by the appearance of new apical tight junctions between chloride cells. Another particularity of these cells, the extensive network of tubules, extension of the basolateral plasma membrane, is shown to be made of repetitive units. We discuss the functional importance of the observations with respect to the adaptation of fish to different salinities.

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