scholarly journals Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

1983 ◽  
Vol 97 (3) ◽  
pp. 749-755 ◽  
Author(s):  
L D Andrews ◽  
A I Cohen
Keyword(s):  
New York ◽  
Plasma Membrane ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Prolonged Exposure ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Apical Plasma Membrane ◽  
Frog Neuromuscular Junction ◽  
Vertebrate Photoreceptor

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

1981 ◽  
Vol 296 (1080) ◽  
pp. 47-54 ◽  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cholesterol Content ◽  
Intramembrane Particles ◽  
Exocrine Cells ◽  
Luminal Membrane

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.

scholarly journals Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.

1978 ◽  
Vol 78 (2) ◽  
pp. 577-596 ◽  
Author(s):  
P M Elias ◽  
J Goerke ◽  
D S Friend ◽  
B E Brown
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Lipid Vesicles ◽  
Cholesterol Content ◽  
Erythrocyte Membranes ◽  
Membrane Domains ◽  
Thin Sections ◽  
Multilamellar Liposomes ◽  
Fracture Identification

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol-digitonin structural complexes; (b) distinguishes cholesterol-rich and -poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.

Cholesterol in the plasma membrane of uterine epithelial cells: a freeze-fracture cytochemical study with digitonin

1985 ◽  
Vol 78 (1) ◽  
pp. 163-172
Author(s):  
C.R. Murphy ◽  
B. Martin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Tight Junctions ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Apical Plasma Membrane ◽  
Lesion Formation ◽  
Uterine Epithelial Cells ◽  
Cytochemical Study ◽  
Blastocyst Implantation

Freeze-fracture cytochemistry with digitonin has been used to examine the cholesterol content of the plasma membrane of uterine epithelial cells during the early stages of pregnancy in the rat. Lesions caused by digitonin complexing with cholesterol were seen on both lateral and apical portions of the membrane but tight junctions and desmosomes were lesion-free. Compared with day 1 of pregnancy, lesions on the apical plasma membrane were much more extensive and some were of different morphology on day 6 - the day of blastocyst implantation. We consider mechanisms of lesion formation and interpret the results to indicate a higher content and perhaps a different organization of cholesterol in the apical plasma membrane on day 6 of pregnancy. We also suggest how this increase may occur.

Evidence for microtubule nucleation at plasma membrane-associated sites in Drosophila

1987 ◽  
Vol 88 (1) ◽  
pp. 95-107 ◽  
Author(s):  
M.M. Mogensen ◽  
J.B. Tucker
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Cross Section ◽  
Plasma Membranes ◽  
Early Stage ◽  
Apical Cell ◽  
Apical Plasma Membrane ◽  
Cell Surfaces ◽  
Microtubule Organizing Centres ◽  
Oriented Parallel

This report is concerned with the nucleation and organization of microtubule bundles that assemble after ‘conventional’ centrosomal microtubule-organizing centres have been lost. The microtubule bundles in question span the lengths of wing epidermal cells. Bundles extend between hemidesmosomes at the apical cuticle-secreting surfaces of cells and basal attachment desmosomes that unite the dorsal and ventral epidermal layers of developing wing blades. Furthermore, each bundle includes up to 1500 microtubules and most of the microtubules are composed of 15 protofilaments. Individual cells were serially cross-sectioned at an early stage of bundle assembly. The number of microtubule profiles/cell cross-section decreased progressively by up to 59% of the most apical values in section sequences cut from fairly apical to more basal levels in the cells. The apical ends of microtubules were associated with numerous small dense plaque-like sites (diameter 0.1-0.2 micron), which were specialized regions of plasma membranes at the apical surfaces of cells. Many of the microtubules near apical plaques were not well aligned with each other; they ‘radiated away’ from cell apices. This was in contrast to the situation at more basal levels where most microtubules were oriented parallel to the longitudinal axes of cells. These findings indicate that the relatively dispersed arrays of apical plasma membrane-associated plaques act as microtubule-nucleating sites to initiate basally directed elongation of bundle microtubules. Apical cell surfaces and their plaques seem to operate as microtubule-nucleating and -organizing regions that functionally replace the centrosomal microtubule-organizing centres lost earlier in cell differentiation.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

1984 ◽  
Vol 98 (2) ◽  
pp. 685-698 ◽  
Author(s):  
T M Miller ◽  
J E Heuser
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Thin Section ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Freeze Fracture ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Coated Pits ◽  
Active Zones

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

scholarly journals Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors.

1984 ◽  
Vol 98 (5) ◽  
pp. 1788-1795 ◽  
Author(s):  
I Nir ◽  
D Cohen ◽  
D S Papermaster
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Photoreceptor Cells ◽  
Rod Photoreceptor ◽  
Rod Photoreceptors ◽  
Retinal Photoreceptors ◽  
Retinal Rod ◽  
Ros Formation ◽  
Developing Rat

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin-ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

scholarly journals Cytochemical evidence for the presence of phospholipids in epithelial tight junction strands.

1993 ◽  
Vol 41 (5) ◽  
pp. 649-656 ◽  
Author(s):  
F W Kan
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Phospholipase A2 ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Freeze Fracture ◽  
Fracture Face ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Specific Association

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.

scholarly journals Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

1990 ◽  
Vol 38 (10) ◽  
pp. 1421-1426 ◽  
Author(s):  
M R Torrisi ◽  
A Pavan ◽  
L V Lotti ◽  
G Migliaccio ◽  
M C Pascale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Viral Proteins ◽  
Low Ph ◽  
Surface Distribution ◽  
Transfected Cells ◽  
Cytosolic Side ◽  
Infected Cells

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

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