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D S Friend

Publications and source records attributed to D S Friend.

At least 91 records · Page 5Linked to original sources
PubMed1982

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm.

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin-phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.

Animals↗
PubMed1981

Orderly particle arrays on the mitochondrial outer membrane in rapidly-frozen sperm.

By deep-etching and rotary replication of unfixed, non-cryoprotected tissue frozen on a helium-cooled copper block, previously undemonstrable organellar surfaces and intramembranous structures can be examined. Among the more remarkable features of mammalian spermatozoa thus prepared are the highly ordered particulate arrays on the surface of the mitochondrial outer membrane. In the midpiece of the sperm, mitochondria curl around dense fibers and the axoneme. The surface of the mitochondrion that faces the plasmalemma carries closely packed rods in haphazard dispersement, composed of two to four 70-to 80-A particles, less than 20 A apart, while the concave aspect of the organelle contains rods in stepladder pattern. These ladders are parallel, with their particles in neighboring rungs apparently in register at a 40--45 degree angle relative to the mitochondrial axis. This organizational disparity between the convex and concave surfaces of the organelle not only affords evidence of a new mitochondrial substructure, but represents a type of topographical heterogeneity rarely found except within specialized areas of the plasma membrane. Other novel findings in the sperm cell include the observation of "lipid" tracts flanking intramembranous particle-strands in the plasmalemma of the cytoplasmic droplet, and a gridiron design on the cytoplasmic faces of the droplet's microcisternae, as well as both within and atop its plasma membrane--a motif consistent with the presence of exocytotic or endocytotic activity in this portion of the cell. Additional recent observations are the differing internal and external periodicities of axonemal microtubules and the subunit structure of rectangles on the tail surface overlying the intramembranous particles of the zipper.

Animals↗
PubMed1981

beta-Hydroxysterol distribution as determined by freeze-fracture cytochemistry.

Filipin, a polyene antibiotic, fluoresces and forms 15-25 nm aggregates when combined with beta-hydroxysterols, rendering sterols detectable by fluorescence microscopy and by electron microscopy of thin sections and freeze-fracture replicas. We applied filipin in a glutaraldehyde fixative to tissue-cultured cells of Drosophila melanogaster larvae, in which sterol concentration can be regulated. Since the number of filipin-sterol aggregates observed in membranes was found to be proportional to the amount of sterol experimentally inserted, utilizing filipin is a valid method for quantifying, as well as for mapping, sterol distribution in biological membranes. Other antibiotics may be similarly used for localizing some species of negatively charged phospholipids. In addition to cytochemical identification of specific lipids, rapid freezing and deep etching of unfixed, non-cryoprotected cells may permit us to examine membrane lipids in different physical states: liquid-crystalline and gel. Combining these several techniques has resulted in new data concerning the disposition of lipids during the intimate juxtaposition of membranes preceding fusion. For example, in guinea-pig sperm, foci of closely apposed membranes are bereft of beta-hydroxysterols and intramembranous particles. Such regions of membrane sometimes exist in a crystalline state and may be rimmed by negatively charged phospholipids. As previously noted in other areas of cytochemistry, the in situ localization of specific substances provides information unobtainable by morphological or biochemical techniques alone.

Animals↗
PubMed1980

Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

Polymyxin B was used to explore distribution of anionic phospholipids in sperm plasma membranes by electron microscopy of freeze-fracture replicas. After exposure to Hepes/Tris-buffered polymyxin at 4 mM, phosphatidylcholine liposomes showed no perturbations nor did they fluoresce with dansylated incubation. When phosphatidylethanolamine was included in the liposomes, they became perturbed and fluoresced. Plasma membranes of Drosophila larval cells, containing or lacking cholesterol, were also disrupted by polymyxin. The cell membranes of guinea pig sperm were likewise disrupted but in specific functional areas. Fusional membrane domains showed protrusions; the stable membrane of the flagellum revealed diffuse bubbling. Regions of well-defined particle arrays and the postacrosomal segment maintained smooth contours. By fluorescence microscopy, we detected the same heterogeneous binding of the polymyxin dansyl derivative.

Acrosome↗
PubMed1980

Freeze-fracture alterations in guinea pig sperm membranes preceding gamete fusion.

Normal fertilization in mammals depends on several events involving membrane fusion. One, a secretory phenomenon, is the fusion of the spermatozoan acrosomal granule membrane with the plasmalemma, and another concerns the union of this nascent hybrid membrane with that of the egg. Using freeze-fracture and ancillary cytochemical procedures, we can identify three general membrane constituents: proteins, apparent as intramembranous particles; phospholipids, visible as smooth, particle-free areas; and sterols, observed as 25-nm ellipsoid protuberances when complexed in situ with the polyene antibiotic filipin. Freshly removed from the epididymis, guinea pig sperm displays distinctive patterns of intramembranous particles and a nonhomogeneous distribution of sterols. When incubated in a Ca2+-free medium that permits processing spermatozoan membranes for fusion, these membrane components become altered. In the circular foci where union occurs, the particle pattern is modified from an orderly, geometric quilted design to a random distribution; filipin-reactive sterols are deleted, followed by the loss of particles from the nonesterified sterol-deficient patches. With the addition of Ca2+, the residual smooth areas meld as the number of particles equilibrates in the plane of the hybrid acrosomal-plasma membrane. Sterols appearing in the postacrosomal segment remain reduced in quantity. After acrosomal-plasma-membrane fusion, a second line of particle-cleared circles emerges behind the margin of the new membrane suture. These clearings appear to be identical to the circles observed in the acrosomal and plasma membranes preceding the acrosome reaction. We interpret this second series of clearings as the preparation of the spermatozoan membrane for fusion with the egg. In sperm, the focal alterations antecedent to organelle (acrosome) plasma membrane union are evidently comparable to those preceding plasma membrane (sperm) plasma membrane (egg) fusion. It will be of great interest to us to discover whether this type of protein and sterol/phospholipid juncture proves to be a unifying feature of fusions in all the various biological systems discussed in this volume.

Animals↗
PubMed1978

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

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.

Adrenal Glands↗
PubMed1977

Membrane alterations during cornification of mammalian squamous epithelia: a freeze-fracture, tracer, and thin-section study.

Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large molecules in non-keratinizing mammalian epithelia. However, in cornifying epithelia such as the epidermis, the importance of tight-junctional elements versus secreted intercellular lipid for barrier function is uncertain. In an attempt to resolve this question, we compared membrane structure in the stratum granulosum and stratum corneum of epidermis, esophagus, and vagina of newborn and adult humans and mice under both normal and various experimental conditions. We incubated pieces of epidermis in organ culture and infused tissues with lanthanum or horseradish peroxidase in vivo and in vitro. All were processed for electron microscopy of freeze-fracture replicas or thin sections. Lanthanum seeped outward to the stratum granulosum in all tissues examined--further apical migration was halted by lamellar-body contents in skin. A similar pattern of intercellular lamellar lipid deposition and membrane structure occurred in all epithelia studied. Freeze-fracture replicas of these obstructive regions revealed occasional, incomplete junctional strands (particularly in moist epithelia) and abundant lamellar material, but complete zonulae occludentes were never encountered. A possible relationship between moisture and tight junction formation was further suggested by organ culture experiments during which brief incubations stimulated an increase in the number of junctional strands and diminished numbers of lamellar bodies. We conclude that, in the epithelia studied, the deposition of secreted lamellar body contents forms the barrier to water-soluble tracer loss: tight-junctional elements are either absent or too fragmentary to constitute an effective barrier.

Animals↗
PubMed1977

Intramembranous particle distribution in human erythrocytes: effects of lysis, glutaraldehyde, and poly-L-lysine.

Freeze-fracture combined with quantitative electron microscopy of the intact human erythrocyte (RBC) and ghost revealed significant differences in their intramembranous particle coefficients. External (E) fracture-faces of unfixed ghost membranes were found to contain 40% fewer particles than those of intact unfixed RBC. The particle distribution of the intact RBC membrane depended on the use of glutaraldehyde fixation and glycerol cryoprotection. Whereas glutaraldehyde- and glycerol-treated cells disclosed 70% fewer E-face particles than did intact unfixed cells, poly-L-lysine-treated, intact, unfixed RBC showed no such differences. Treatment with a combination of poly-L-lysine and glutaraldehyde, however, increased the amount of E-face particles while reducing those of the protoplasmic (P) face. The poly-L-lysine effect varied with its concentration and was unaffected by previous application of neuraminidase. Nor did the lectin phytohemagglutinin induce particle rearrangement in intact cells. Our data demonstrate that the processes of glutaraldehyde fixation and glycerol cryoprotection modify the RBC membrane by decreasing the number of E-face particles present. In addition, the combination of poly-L-lysine and glutaraldehyde alters the affinity of some particles for one half of the membrane, suggesting that in freeze-fractured RBC, chemical bonds formed at the extracellular surface of the membrane can influence particle partitioning.

Aldehydes↗
PubMed1977

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa.

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

Acrosome↗
PubMed1977

Freeze-fracture of membrane fusions during exocytosis in pancreatic B-cells.

To examine the freeze-fracture appearance of membrane alterations at sites of exocytosis in mammalian cells, we studied the secretory granule and plasma membrane of rat pancreatic B-cells during glucose-stimulated insulin secretion. Constant features observed were the scarcity of particles in secretory-granule P-fracture faces and the almost total clearance of intramembranous particles in P-and E fracture faces of the plasma membrane in areas of close apposition of these two membranes preceding fusion; also observed was the temporary persistence of particle-cleared regions after the fusion was completed. Our observations thus support the concept that membranes fuse at sites of closely apposed, particle-free regions and that the physiologically created clear areas found in freeze-fracture replicas of the plasma membrane are the hallmarks of incipient or recent membrane fusion.

Animals↗
PubMed1977

Target-cell membrane alterations induced by lymphotoxin. Ultrastructural observations.

Upon in vitro stimulation by antigens or mitogens, lymphocytes release a series of lymphokines. One such lymphocyte mediator is lymphotoxin, which appears to be responsible for in vitro lymphocyte-mediated cytolysis. Employing immuno-electron microscopy with ferritin- or peroxidase-labeled antibody, we observed patchy localization of the mediator on the plasma membranes of target L cells exposed to lymphotoxin, often in areas overlying a microfilament web. When studying lymphotoxin-affected cells by electron microscopy of freeze-fracture replicas, we observed aggrlasma membrane, with intervening areas which were frequently particle free. Sometimes the affected cells revealed plasma-membrane lesions suggesting intramembranous "blisters."

Cell Membrane↗
PubMed1976

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation.

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.

Animals↗