Ultrastructure of hamster bronchiolar epithelium in freeze fracture replicas

The morphology of the plasma membrane of ciliated and nonciliated bronchiolar epithelial cells of the hamster lung was studied using freeze fracture electron microscopy and transmission electron microscopy of thin sections. Tight junction structure of bronchiolar epithelial cells was evaluated with respect to the total number of junctional strands, and the organization of tight junctional fibrils. Tight junctional complexes seen on the surfaces of nonciliated cells had from 4 to 9 junctional strands (mean: 6.0±0.1). Ciliated cells displayed 4 to 10 tight junctional strands (mean: 6.9±0.1). Nonciliated bronchiolar epithelial cell (NC) membranes showed aggregates of 6 nm particles organized into rectilinear arrays of 2 to 30 particles. These particle clusters were seen predominantly at the basal pole of cells in close association with areas of intercellular interdigitation. NC cells also displayed membrane areas of particle clearing and aggregation consistent with intermediate phases of membrane fusion and exocytosis.     

Fate of gap junctions in isolated adult mammalian cardiomyocytes

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.     

Filipin and digitonin studies of membrane cholesterol in frog atrial fibers with unusual gap junction configurations

The gap junctions of frog myocardium present, in freeze-fracture, an atypical organisation of their junctional particles. Freeze-fracture cytochemistry with the cholesterol probes filipin and digitonin has been used to investigate whether the particular arrangement of the particles involves a lipid segregation in the plane of the membrane. Both probes labeled uniformly the non-junctional membrane, but no deformations were ever found inside the smooth membrane area circumscribed by the circle of junctional particles. At the level of junction formation zones almost no sterol probe complexes were found in the intramembranous particle free area which surrounded small clusters of junctional particles. These results suggest a regional variation in cholesterol related to the necessity of membrane fluidity during junction morphogenesis.     

Development of Junctions During Differentiation of Lens Fibers

Throughout the differentiation of eye lens epithelium into fibers, an extensive system of intercellular junctions develops. The junctional assembly is initially characterized by the accumulation of 9.0-nm intramembranous particles, forming linear rows in the matching plasma membranes of adjoining fibers. At the final stage of the fiber differentiation, the junctional particles are assembled in geometrically packed arrays. The formation of linear rows and bidimensional lattices of intramembranous particles probably favors reciprocal recognition of cell surfaces and specific cell-to-cell interlocking. Moreover, the existence of a rather rigid lipid core of the plasma membrane of eye lens fiber may promote the clustered distribution of intramembranous particles and facilitate the junctional assembly.     

Acute and subacute effects of injury on the canine alveolar septum

The effect of papain on the prevalence and distribution of alveolar macrophages, alveolar septal interstitial tissue gaps and epithelial cells in normal canine pulmonary alveoli was studied by light and electron microscopy. Serial sections of whole alveoli from control animals and from animals sacrificed 4 h, two weeks and one month after the instillation into one lung of crude papain in saline solution containing India ink as a marker were compared. In dogs, as in humans, there is normally a zonal distribution of alveolar macrophages and type 2 cells at alveolar junctional sites. We hypothesize that early alveolar septal injury takes place at these junctional sites, judging from concentration of alveolar macrophages and interstitial septal gaps at these sites following papain exposure, and also that septal repair activities are greatest at these sites, in view of the preponderance and high prevalence of type 2 cells occupying interstitial septal gaps in junctional zones. Consequently, the type 2 cell may play a role beyond that of merely replacing type 1 epithelial cells following alveolar septal injury.     

Freeze-fracture study of mast cell secretion.

Within seconds after exposure of rat peritoneal mast cells to polymyxin B, bulges appear on the surface of the cells. Freeze-fracture electron microscopy reveals that each bulge overlies a mast cell granule. In contrast to the even distribution of intramembranous particles in the plasma membrane of unstimulated cells, the intramembranous particles in the stimulated cells are unevenly distributed in the membrane of the bulges with large patches of membrane lacking intramembranous particles. The membranes over the most prominent bulges are entirely free of intramembranous particles, and in some instances there is an increased concentration of intramembranous particles at the margins of the bulges. Perigranule membranes exhibit the same changes in distribution of intramembranous particles. Electron microscopy of thin sections of rapidly fixed, stimulated mast cells shows a peculiar structure of the membrane overlying some bulges; instead of the pentalaminar membranes previously demonstrated, the membrane at these sites of presumptive fusion of perigranule and plasma membrane assumes the form of a single dense lamina with a fine fuzzy coating on either side. It seems possible that membrane fusion and subsequent pore formation proceed in the stimulated mast cell through a stage of flight of intramembranous particles and molecular rearrangement of the other membrane components.     

Zonal distribution of alveolar macrophages, type II pneumonocytes, and alveolar septal connective tissue gaps in adult human lungs

The distribution of alveolar macrophages, Type II cells, and alveolar septal connective tissue discontinuities or gaps in two neighboring alveoli from a human lung obtained at surgery, and preserved by vascular perfusion-fixation, was studied by electron microscopy. Discontinuities or gaps are defined as complete interruptions of all the connective tissue elements of the alveolar septum, including the basement membranes. Serial-sectioning of the alveoli, and the creation of montages of the entire circumference of each alveolus at intervals of every twentieth section (approximately 1.6 micron) at a magnification of X 2,160 permitted precise identification of cells and connective tissue gaps and allowed the reconstruction, by computer techniques, of the alveolar walls in 3 dimensions. These studies showed that all of the 48 alveolar macrophages identified, and over two thirds of all Type II cells and alveolar septal gaps, were located or bordered on alveolar septal junctional zones (within 10 microns of septal junctions). The profiles of 739 alveoli examined by light microscopy from 6 lungs similarly preserved by vascular perfusion-fixation, in which the alveolar surface lining was well fixed, also showed alveolar macrophages preferentially distributed in alveolar junctional zones. These were compared with 242 alveolar profiles from 3 other vascularly-fixed lungs and 971 alveolar profiles from 9 lungs fixed by way of the airways, in which the alveolar surface lining was lost. In these lungs, most of the alveolar macrophages were in the alveolar air spaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further development of a model for electrical transmission between myocardial cells not connected by low-resistance pathways.

We previously described a model for the electrical transfer of excitation from one cell to the next which utilized the electric field generated in the cleft between the cells. This model was analyzed only for the steady-state condition. In the present paper, we calculate the effects of membrane capacitance on the transmission of an action potential between two adjacent cardiac cells; the junction between cells was composed of two high-resistance excitable membranes separated by a narrow cleft. The parameters varied for this study included the threshold potential and capacitance of the junctional membranes. The calculations indicate that it is somewhat easier to achieve transmission when capacitive effects are included. Thus, the electric field model provides an alternative means of cell-to-cell propagation between myocardial cells which is electrical in nature but does not require the presence of low-resistance connections between cells.     

Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells.

We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free systems, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.     

The Application of Freeze-Cleaving Technics to Studies on Red Blood Cell Fine Structure

A simplified freeze-cleave and replication method of tissue preparation forexamination in the electron microscope is applied to studies on red blood cellfine structure. With this technic, the cytoplasm of red blood cells appearsto have a uniform pattern of packed granularity, with individual particles approximating the dimensions anticipated for replicas of individual hemoglobinmolecules. The cell surface is smooth and partially covered with small particles which may represent antigens, enzymes, or some structural proteins.The possibility that particles seen in cells and on cell membranes may represent an artifact is discussed. Pretreatment of cells prior to freezing influencesthe plane of cleavage through packed cells so that the plane of cleavage canbe preferentially directed either through the cytoplasm or along red cellmembranes. The freeze-cleave technic may be of particular value in applications where extensive areas of membrane must be surveyed, such as searchingfor leukemogenic viruses budding through cell membranes.

Submitted on August 16, 1966 Accepted on November 24, 1966     

Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.

The gene family encoding gap junction proteins (connexins) consists of several known members, and multiple connexins are frequently coexpressed by coupled cells. To characterize the channel properties of the major rat liver gap junction protein (connexin 32) in isolation from other gap junction proteins, we have introduced the cDNA encoding it into a human hepatoma cell line (SKHep1) in which we have identified a gap junction deficiency. In this cell line, dye coupling was absent and junctional conductance was near zero. Connexins and connexin 32 mRNA were not detectable by immunocytochemistry and Northern blot analysis. After transfection and selection, cells were strongly coupled with regard to dye and electrical current, and connexin 32 mRNA and punctate connexin 32-immunoreactive membrane contacts were abundant. Functional gap junction channels were still expressed after 19 passages of the cells, indicating stable transfection. When junctional conductance was rendered reversibly low by exposing the cells to agents that uncouple other cell types, currents through single gap junction channels could be observed. The unitary conductance of these expressed channels was about 120-150 pS, a value that is distinctly larger than in heart cells, which express a different gap junction protein.     

In vitro assembly of the nonglycosylated membrane protein (M) of Sendai virus.

The nonglycosylated membrane protein (M) of Sendai virus was purified from virions and conditions were found under which the protein assembled in vitro into three types of ordered structures: narrow tubes, wide tubes, and sheets. These structures were examined by high resolution electron microscopy by using negative staining and metal shadowing techniques. The tubes and sheets are formed from strands 7.2 nm wide that are composed of annular subunits. The wide tubes appear to be formed by the rolling of a sheet into a cylinder in which the 7.2-nm strands are inclined with a pitch of 26-33 degrees and have a left-handed orientation. In addition to the strong reflections corresponding to the 7.2-nm spacings generated by the strands, optical diffraction patterns also showed weak reflections that could be indexed on a lattice corresponding to real-space lattice constants of 7.6 nm and 5.3 nm, with an included angle of 71 degrees. The dimensions and arrangements of these structures formed in vitro are strikingly similar to those of ordered arrays of particles found by others to be associated with the inner surface of the plasma membrane of infected cells. The results support the concept that ordered arrays of M protein, similar to those assembled in vitro, are involved in the assembly of the virus particle by budding from the cell membrane and that they provide specific recognition sites for the viral nucleocapsid at the cytoplasmic surface of the plasma membrane.     

Lipid membranes in poxvirus replication

Poxviruses replicate in the cytoplasm, where they acquire multiple lipoprotein membranes. Although a proposal that the initial membrane arises de novo has not been substantiated, there is no accepted explanation for its formation from cellular membranes. A subsequent membrane-wrapping step involving modified trans-Golgi or endosomal cisternae results in a particle with three membranes. These wrapped virions traverse the cytoplasm on microtubules; the outermost membrane is lost during exocytosis, the middle one is lost just prior to cell entry, and the remaining membrane fuses with the cell to allow the virus core to enter the cytoplasm and initiate a new infection.     

Junctional Structures in Haemopoiesis: a Study of Bone Marrow using Freeze-Fracture and Lanthanum Impregnation Techniques

Intercellular regions of contact in the haemopoietic compartment of normal rat bone marrow were studied using freeze-fracture and lanthanum tracer techniques. Small adhering junctions (like desmosomes and their variants) were found between haemopoietic and stromal cells but tight, gap or septate junctions could not be identified. These findings are in agreement with the concept that extensive junctional structures may be inconsistent with orderly development of this transient cell system, preventing the delivery of mature cells into the circulation and resulting in ineffective haemopoiesis. Occasionally 'pinching off' of a portion of the cytoplasm of erythroid cells by stromal cells was seen, providing a means for intercellular communication. Structures similar to intercellular bridges responsible for direct intercellular communication were also seen.     

Gap Junctions between Photoreceptor Cells in the Vertebrate Retina

In the outer plexiform layer of the retina the synaptic endings of cone cells make specialized junctions with each other and with the endings of rod cells. The ultrastructure of these interreceptor junctions is described in retinas of monkeys, rabbits, and turtles, in thin sections of embedded specimens and by the freeze-fracturing technique. Cone-to-rod junctions are ribbon-like areas of close membrane approximation. On either side of the narrowing of the intercellular space, the junctional membranes contain a row of particles located on the fracture face A (cytoplasmic leaflet), while the complementary element, a row of single depressions, is located on fracture face B. The particle rows are surrounded by a membrane region that is devoid of particulate inclusions and bears an adherent layer of dense cytoplasmic material. Cone-to-cone junctions in some places are identical to cone-to-rod junctions, while in other places they closely resemble typical gap junctions (nexus). Interreceptor junctions, therefore, represent a morphological variant of the gap junction, and probably mediate electrotonic coupling between neighboring photoreceptor cells.     

Hydrophobic ion transfer between membranes of adjacent hepatocytes: a possible probe of tight junction structure.

The topology of the tight junction is probed by introducing dipicrylamine (dpa-), a lipid-soluble anion, into the membranes of hepatocyte pairs in culture. Once partitioned into the membrane, dpa- ions are free to move in the hydrophobic core of the membrane, where their mobile charges greatly increase membrane capacitance. If tight junctions are lines of membrane fusion, dpa- will cross the tight junction without traversing a polar headgroup layer. Furthermore, the electric potential across the tight junction will be equal to the difference in membrane potentials of the two cells. dpa- can therefore be expected to move electrophoretically from cell membrane to cell membrane across the junction in response to an intercellular voltage difference. Experiments performed under double whole-cell clamp show that this transfer occurs as follows: First, dpa- causes an intercellular current unrelated to gap junctions to flow in response to an intercellular voltage difference. Second, this electrophoretic removal or addition of dpa- from a cell's membrane through the tight junction must reduce or increase its dpa- content and thus its capacitance. Experiments confirm this prediction: We detect rapid, symmetric, and reversible changes in membrane capacitance in response to changes in the membrane potential of the neighboring cell. Finally, we find that hepatocyte membranes contain a negatively charged endogenous molecule that contain a negatively charged endogenous molecule that can move from cell to cell like dpa- under the influence of an intercellular potential difference. We conclude that membrane fusion occurs at tight junctions and that this hydrophobic intercellular pathway can play a role in intercellular communication.     

Localization of high concentrations of phosphotyrosine-modified proteins in mouse megakaryocytes

Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P- Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.     

Immunoelectron microscopic studies on the cell surface location of the thyroid microsomal antigen

The cell surface location of the thyroid microsomal antigen was studied by immunoelectron microscopy. Isolated, open human thyroid follicles were incubated with patient sera containing high titers of microsomal autoantibodies. Cell surface-bound antibodies were detected by the immunogold technique using IgG-coated colloidal gold particles (10 or 15 nm). Immunocytochemical incubations were performed at 4 degrees C. Gold particles were concentrated at the apical cell surface of the follicle cells, while the basolateral cell surface was almost completely unlabelled. Quantitative evaluation of four experiments with follicle cells prepared from different patients showed that about 90% of the gold particles at the apical cell surface was associated with microvilli and that the concentration of gold particles at the microvillus membrane was, although with great intercellular variation, several times higher than that at smooth portions of the apical plasma membrane. This suggests that the microsomal antigen is organized in microdomains in the apical plasma membrane. In follicles labelled immunocytochemically at 4 degrees C and then incubated at 37 degrees C, gold particles were slowly internalized. The particles appeared in smooth and coated pits of the apical plasma membrane as well as in vesicles, vacuoles and lysosomes in the apical part of the cytoplasm. Membranes of TSH-induced pseudopods were always unlabelled. Our observations indicate that thyroid microsomal antigen immunoreactivity is present in the apical but not in the basolateral plasma membrane. The antigen with bound antibodies is internalized by micropinocytosis but not by macropinocytosis. The selective location of bound microsomal antibodies at the apical plasma membrane and their absence from the membrane of TSH-induced pseudopods are compatible with the idea that the microsomal antigen and thyroperoxidase are identical.     

Visualization of the protein associations in the erythrocyte membrane skeleton.

We have obtained clear images of the erythrocyte membrane skeleton from negatively stained preparations that originate directly from the intact cell but in which the spectrin meshwork is artificially spread to allow close inspection. Our procedure requires less than 2 min at 5 degrees C in phosphate buffers. We find 200-nm-long spectrin tetramers crosslinked by junctional complexes. Each junction contains a regular 37-nm rod, probably an actin oligomer of approximately 13 monomers. Densities appear at variable places in the meshwork but distinct globules occur with great frequency 78 nm from the spectrin tetramer's junctional insertion end, very close to the known binding site for ankyrin. Most frequently, five or six spectrin tetramers insert into each junction, producing a meshwork that displays remarkably regular long range order.