Freeze-fracture characterization of the outermost Golgi cisterna (OGC) in rat pancreatic acinar cells

The outermost Golgi cisterna (OGC) frequently exhibits 55 nm diameter protuberances in P fracture faces and corresponding depressions, or pits, in E faces. When these protuberances (pits) appear regularly disposed, OGC faces are recognizable at relatively low electron microscopic magnifications. The mean particle width is less in OGC (6.4 nm) than in rough ER (7.9 nm) P faces, while particle number per unit area is respectively 70% and 100% greater in OGC P and E faces than in corresponding ER faces. The rather small OGC P face particles are better resolved at a relatively low shadowing angle. These differences in particle size (for P faces) and density between OGC and rough ER faces are detectable by inspection and may be used in the recognition of OGC faces devoid of protuberances (in P faces) and pits (in E faces). Fusion of presumably rough ER-derived microvesicles form short double-bossed tubular elements which constitute the OGC initial structure. These tubules enlarge by addition of microvesicle membrane and contents forming a characteristic sheet-like bossed structure.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.     

Fine structure of the palisade zone of the rat median eminence as revealed by freeze-fracturing

Summary Nerve terminals in the palisade zone of the rat median eminence were investigated with freeze-fracture electron microscopy. Fracture face P of the specific terminals showed two populations of intramembranous particles (IMP): a large and a small variety. The large IMP-s often formed small irregular clusters. In nerve terminals the total number of both populations of IMP-s was considerably less than that found on P membrane faces of ependymal feet. On fracture face E of the nerve terminals, the number of IMP-s was about a quarter of that seen on fracture face P.On both fracture faces of most terminals a few small round impressions (or elevations respectively) were found which may be interpreted as broken necks of either exo- or endocytotic vesicles. Neither gap nor tight junctions occurred at lateral membranes of the specific axon terminals. Similarly, no membrane specializations were observed with freeze-fracturing on membrane areas adjacent to the basement membrane. The findings are discussed in relation to a possible exocytosis mechanism of the hypothalamic releasing and inhibiting hormones.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.     

Freeze-fracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes

Membrane and cytoplasmic changes were observed after in vitro fertilization of hamster oocytes by examining freeze-fracture replicas. The density of intramembranous particles on areas of membrane between microvilli increased following fertilization. Although the intramembranous particle density of microvilli is higher than that on the intermicrovillar membrane of unfertilized eggs, it did not change significantly after fertilization. Cytoplasmic changes in the Golgi complex and mitochondria upon fertilization indicate a change in cellular activity. Lipid binding probes were applied to the oocyte membranes in order to study the distribution of specific lipids before and after fertilization. Probes included the B-hydroxy-steroid complexing molecules, filipin and tomatin, and an anionic lipid binding antibiotic, polymyxin B. Both tomatin and filipin complex with steroids in the P and E faces of the plasma membrane (including the polar bodies), cortical granules and vesicles deeper in the cytoplasm, and the Golgi complex, leaving mitochondria, pronuclei, endoplasmic reticulum, and the majority of vesicles unlabeled. Polymyxin B binding is dependent on its application before or after fixation or in association with EGTA. With its application we detected both minor membrane perturbations of wrinkles and particle redistributions and major perturbations of vesicle fusions, the formation of blebs, and the loss of membrane morphology. Neither the distribution nor apparent quantity of these probes changed overall following fertilization, but this impression does not include specific sites of sperm-egg fusion.     

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.     

Monocercomonas sp.: Cytochemistry and Fine Structure of Freeze-Fractured Membranes

Monocercomonas sp. from the wood-snake Tropidophis melanurus was studied using fast freezing of alive and fixed cells followed by freeze-fracture and deep-etching. Cytochemistry for enzymes (acid phosphatase, neutral phosphatase, and thiaminopyrophosphatase) and for carbohydrates and endocytosis of gold-labeled albumin were also performed. The Golgi complex is formed by 12-14 cisternae with typical cis and trans faces connected to a network of tubular and cisternal structures, and is positive for thiaminopyrophosphatase at the trans face. Intraluminal filamentous structures are seen connecting the two faces of the cisternae of the Golgi complex. Lysosomes appeared to contain acid and neutral phosphatases. Cytochemistry showed that lysosomes predominate among the unidentified vacuoles in the cytoplasm. Some vesicles are involved in the endocytic pathway, while others are derived from the Golgi complex. Hydrogenosomes have a rod-like or dumb-bell shape. Two of the anterior flagella present rosettes, when observed in replicas of freeze-fractured material, formed by circular arrangement of intramembranous particles on both P and E faces. The other anterior and the recurrent flagella do not show such rosettes but showed ribbon-like arrays of particles at the point where they emerge from the cell body.     

Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER

Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.     

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.     

Intramembranous particles and ripples in lipid-cytochrome c bilayers

Vesicles made from a mixture of phospholipids (PL) are unilamellar. When such lipid vesicles are incubated with reduced cytochrome c, multilamellar vesicles (PLC) are visualized by transmission electron microscopy. Both PL and PLC vesicles show a rippling in the freeze-fracture replicas. The rippling effect is likely to be due to the tilting of fatty acid chains. The freeze-fractured faces of PLC vesicles also show 3–4 nm intramembranous particles. Such particles are lacking from the comparable fractured faces of PL vesicles that are produced in the absence of cytochrome c. It is suggested that these intramembranous particles result from the presence of cytochrome c molecules; therefore, cytochrome c molecules may penetrate the lipid bilayer.     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

A correlated thin-section and freeze-fracture analysis of guinea pig adrenocortical cells

Comparison of the fine structural features of guinea pig adrenocortical cells as seen in thin sections with those revealed by freeze-fracture confirms the structural appearance of steroid-secreting cells as interpreted from thin sections and reveals significant new features of the membranous organelles. Smooth-surfaced endoplasmic reticulum appears as a network of tubules, interwoven or in parallel, and as cisternae, fenestrated and non-fenestrated. These elements are tightly packed in the deeper cortical cells, excluding other organelles from their domain. Tubules and fenestrated cisternae possess randomly distributed intramembranous particles on their PF faces, while closely packed non-fenestrated cisternae possess aggregates of particles interspersed with aparticulate regions on their PF faces. These differences in particle distribution suggest functional specialization among the various forms of reticulum. Mitochondria appear as elongated structures of varying shape. Freeze-fracture reveals that all their cristae have circular origins from the inner membrane. Sinuous tubules, which appear as tubules in section, and straight tubules, which appear as lamellae in section, arise from single sites. Flattened sac-like cristae may have multiple circular origins. Definite contact points seen between inner and outer membranes may facilitate passage of molecules, including steroids, into the mitochondrial compartments. Lysosomes and peroxisomes, which are easily identified in thin sections with the aid of cytochemistry, are difficult to identify with certainty by freeze-fracture. Single membrane-bound granules of slightly smaller diameter than mitochondria may represent lysosomes. Smaller granules interconnected with the tubular reticulum, as well as dilated regions of this organelle, may represent peroxisomes. Plasma membranes show no indication of tight junctions but do have abundant gap junctions which show a zonal differentiation: small gap junctions throughout the cortex, medium-sized regularly shaped gap junctions in zona fasciculata externa, and large irregular gap junctions in zona fasciculata interna and zona reticularis. The large junctions cover planar areas as well as surfaces of projections of one cell into another. Such junctions may allow passage of ions as well as of low-molecular-weight substances between the cells, facilitating or even amplifying the response to trophic hormone stimulation.     

Subcellular localization of B apoprotein of plasma lipoproteins in rat liver

Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.     

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.     

Immunocytochemical analysis of Uukuniemi virus budding compartments: role of the intermediate compartment and the Golgi stack in virus maturation.

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.     

Fine structure of Blastocrithidia culicis as seen in thin sections and freeze-fracture replicas

The fine structure of epimastigotes of Blastocrithidia culicis was studied by transmission electron microscopy of thin sections and freeze-fracture replicas. This parasite presents a well developed endoplasmic reticulum and Golgi complex systems. Differences in the density and organization of the intramembranous particles were observed between the membranes which enclose the cell body and the flagellum. Ridge-like elevations, visualized in freeze-fracture replicas, were observed in sites where the mitochondrial branches touched the plasma membrane. A special array of membrane particles was observed on both faces of the flagellar and the cell body membranes at the region where the flagellum adheres to the cell body. It appeared as strands made of two rows of membrane particles. Filipin-treated cells were used for the localization of membrane sterols in freeze-fracture replicas. The number of filipin-sterol complexes varied from cell to cell. In some cells, rows of filipin-sterol complexes were seen. No complexes were observed in the region of the attachment of the flagellum to the cell body.     

Membrane structure of caveolae and isolated caveolin-rich vesicles

 Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80–120 nm with an open porus of 30–50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30–50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles. Accepted: 16 September 1998     

Intramembranous particles are clustered on microvillus membrane vesicles

Many intramembranous particles in pig jejunal microvillus membranes cluster during cell disruption and membrane vesiculation with the MgCl2 aggregation technique (Hauser, H., Howell, K., Dawson, R.M.C. and Bowyer, D.E. (1980) Biochim. Biophys. Acta 602, 567-577). Isolated brush borders and purified microvillus membrane vesicles were jet-frozen and examined by freeze-fracture electron microscopy. From 30 to 60% of purified vesicles exhibited no intramembranous particles on their fracture face and 22-39% exhibited clustered or aggregated intramembranous particles. Only 6-15% of the vesicles exhibited the random distribution of intramembranous particles that is characteristic of intact enterocytes. Aggregation was not reversed after dialysis to remove divalent cations. Prior freezing of tissue or vesicles (-70 degrees C) gave the same results as fresh unfrozen material. Heterogeneity of microvillus vesicles may occur among the vesicles generated from a single microvillus.     

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.     

Quantitative analysis of intramembranous particles in the membranous system of rat liver cells at different stages of development and aging

The density of intramembranous protein particles was studied by freeze-fracture. Particle density on the fracture faces of the plasmalemma and the rough endoplasmic reticulum (RER), as well as the outer and inner membranes of the nucleus and the mitochondria in rat hepatocytes were quantified. Comparison among different age groups sampled days postcoitum (dpc), days postpartum (dpp), and months postpartum (mpp) shows age-related changes in particle density in each membrane system. With the exception of the RER, particle densities increased after the 16th dpc, reached a maximum at birth, and then decreased with increasing age. Simultaneously, the number nuclear pores shows a positive correlation with the particle density of the nuclear membranes. The particle density on the membranes of the RER shows a maximum on the 16th dpc, and on the 6th dpp. Thereafter, the density of the RER decreases slightly. In all membrane systems, the density of the particles on the external fracture faces is more variable than the density of the particles on the protoplasmic fracture faces.