Endocytosis in absorptive cells of cultured human small-intestinal tissue: Horseradish peroxidase,lactoperoxidase, and ferritin as markers

Summary The occurrence of endocytotic mechanisms in human small intestinal absorptive cells was investigated by culturing biopsy specimens in the presence of horseradish peroxidase (HRP), lactoperoxidase (LPO), and ferritin. The results indicate that both HRP and LPO entered the cells by apical endocytosis, after which they were transported via apical vesicles and tubules to the lysosome-like bodies. Ferritin, which showed a distinct affinity for the cell-coat glycoproteins, was not interiorized by the absorptive cells.These findings suggest that although human absorptive cells have an endocytotic mechanism, possibly fluid-phase endocytosis, cell-coat glycoproteins are not taken up by the cells, as indicated by the absence of ferritin in the apical vesicles and tubules, as well as the lysosome-like bodies. These findings provide indirect support for our hypothesis that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport via a crinophagic mechanism (fusion of apical vesicles and tubules with lysosome-like bodies) rather than via an exocytotic-endocytotic mechanism.     

Morphological studies of the goldfish hindgut mucosa in organ culture

Summary The morphology of the absorptive cells of the goldfish hindgut mucosa, and their capability for horseradish peroxidase (HRP) uptake, were investigated by electron microscopy after a 24-h organ culture. The columnar appearance and the fine structure of the absorptive cells were well preserved for 24 h at room temperature and 37° C with 5% CO₂ in air, in all the media used in this study. Mitoses were frequently observed in the epithelium at the bottom of cultured mucosal folds, and re-epithelization was also observed in many explants.Some structural changes were, however, noted in the cultured absorptive cells, as compared with the non-cultured absorptive cells; the deep invaginations of the surface membrane between the microvilli decreased in number; supranuclear giant vacuoles were reduced in size or almost disappeared; the distributional pattern of mitochondria in the absorptive cells was altered.The HRP uptake experiments showed that the absorptive cells cultured for 24 h could still take up HRP by endocytosis and transport it, indicating that the absorptive cells maintained their capability of macromolecule uptake and transport after 24 h of culture. In addition, HRP experiments, in which reaction product was detected within numerous cytoplasmic tubules (CT), various vacuoles and CT-vacuole complexes, suggested a close relationship between CT and vacuolar system in the apical cytoplasm during endocytotic events in the absorptive cells.     

The effect of chloroquine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue

Summary The effect of chloroquine, an inhibitor of intralysosomal catabolism, on the synthesis, transport, and degradation of cell-coat glycoproteins in absorptive cells of cultured human small-intestinal tissue was investigated by morphometrical, autoradiographical, and biochemical methods. Neither synthesis nor transport of cell-coat material was affected by the drug, but culturing of the absorptive cells in the presence of chloroquine led to a dose- and time-dependent enlargement of the dense bodies; other cell structures showed no alterations. ³H-fucose-labelled material accumulated in the dense bodies of the absorptive cells of these cultures. Since no increase of -glucuronidase and acid phosphatase activity (both lysosomal enzymes of glycoprotein nature) was found, this accumulation of radiolabelled material can be explained as a chloroquine-mediated inhibition of the degradation of cell-coat glycoproteins. These macromolecules probably enter the lysosome-like bodies by a crinophagic mechanism, i.e., fusion of these organelles with the apical vesicles and tubules involved in intracellular transport. These findings suggest that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport in human intestinal absorptive cells, i.e., the degradation of excess cell-coat material.     

Colchicine-induced tubular, vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. II.--Endocytosis studies

Administration of the antimicrotubular agent colchicine to adult rats (0.5 mg/100 g of body weight for 6 hr) induces formation of extended aggregates of tubular, vesicular, and cisternal organelles in the absorptive cells of the small intestine. The phosphatase reaction pattern (thiamine pyrophosphatase, acid phosphatase, acid trimetaphosphatase) suggests that the majority of them belongs to the lysosomal system (Ellinger and Pavelka, 1984). The present study extends these findings and examines the uptake and fate of intravenously injected horseradish peroxidase (HRP) at the basal and lateral cell surfaces and of intraluminally applied HRP at the apical cell surface. HRP, applied to control animals and animals pretreated with colchicine, was internalized at both apical and basolateral cell surfaces of the absorptive cells, and delivered into endosome-like vesicles, multivesiculated bodies (mvbs), dense bodies (dbs), and in several instances into Golgi cisternae. Following intraluminal application, evidence was obtained for the transport of HRP across the cell; in contrast, intravenously applied HRP was never detected at the apical cell surface. Colchicine pretreatment did not stop the uptake of HRP, which was rapidly sequestered to the clustered tubules, vesicles, and cisternae, as well as to the mvbs and dbs. After longer intervals, the portion of HRP-reactive tubules, vesicles, and cisternae within the clusters increased: 60 min after HRP-administration all of them contained HRP-activity. These results indicate that the colchicine-induced clustered organelles are recipients of endocytic materials internalized at the apical as well as at the basolateral cell surface.     

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D

Summary The effect of cytochalasin B (CB) and cytochalasin D (CD) on the endocytotic uptake of horseradish peroxidase (HRP) by intestinal absorptive cells was investigated by morphometric methods. The results showed that CD inhibited endocytosis considerably, and without any detrimental side-effects. CB had hardly any effect on the endocytosis of HRP, but caused a significant decrease in the number of apical vesicles and tubules involved in the transport of cell-coat glycoproteins from the Golgi apparatus to the brush border.Electron-microscopic autoradiographic analysis of the effect of CD showed that although endocytosis is inhibited significantly by the drug, the amount of radiolabelled cell-coat material entering the lysosome-like bodies was unaltered compared with control cultures. These observations support our hypothesis that the cell-coat glycoproteins of the absorptive cells enter the lysosome-like bodies by a crinophagic rather than by an exocytotic-endocytotic mechanism.     

Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer

Summary The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled cancanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4° C, incubated for different times in Con-A free medium at 37° C, and prepared for electron microscopy. After 1 min incubation at 37° C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37° C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Uptake and transepithelial transport of nerve growth factor in suckling rat ileum

Nerve growth factor (NGF) is necessary for the development of sympathetic and some sensory neurons. Milk may be a source of NGF for suckling young, but sites of intestinal absorption of the protein have not been identified. To determine whether NGF is transported across the absorptive epithelium of suckling rat ileum, we assessed binding, uptake, and transport of 125I-NGF by light microscopy and EM autoradiography. Blood and tissue extracts were analyzed by biochemical and immunological methods to determine whether NGF was taken up structurally intact. NGF binding sites were identified on microvilli and apical invaginations of ileal absorptive cells in vitro. Injected into ileal loops in vivo, NGF radioactivity retained by fixation was evident after 20 min in apical regions of absorptive cells, in endocytic tubules (which mediate the uptake of membrane-bound ligands), in vesicles (which mediate nonspecific endocytosis), and in the supranuclear lysosomal vacuole. At 1 and 2 h, radiolabel in these compartments increased and silver grains were evident at the basal cell surface, and in cells, matrix, and vessels of the lamina propria. In blood and liver, radiolabeled molecules that were immunologically and electrophoretically indistinguishable from NGF and that co-eluted with NGF on gel filtration columns were detected, confirming that some NGF was transported across the epithelium structurally intact. Thus, absorptive cells of suckling rat ileum can take up NGF by both receptor-mediated and nonspecific endocytosis, and direct NGF either to the lysosome for degradation, or into a transepithelial transport pathway.     

Intracellular transport of horseradish peroxidase in the absorptive cells of goldfish hindgut in vitro,with special reference to the cytoplasmic tubules

Summary This study was undertaken to determine whether the numerous cytoplasmic tubules (CT) in the apical cytoplasm of goldfish hindgut absorptive cells are directly involved in the endocytotic transport of macromolecules into the cells, or whether they are derived from the intracellular membrane components. The absorptive cells were exposed to horseradish peroxidase (HRP)-containing medium in organ culture and subsequently fixed and prepared for electron microscopy. Analysis revealed that 5 sec after exposure, many vesicular structures, including coated vesicles, were labelled with reaction product whereas almost all CT were negative. After a 1-min exposure, reaction product was detected in about 11 % of the CT, and thereafter, the percentage increased to about 95% after 15 min exposure. As labelled CT increased in number, the number of densely labelled vacuoles with attached CT also increased. CT connected to vacuoles with a peripheral margin of dense reaction product were always HRP-positive, whereas those connected to vacuoles which were not distinctly labelled were themselves also devoid of HRP reaction product. This indicated that the labelling of CT was closely associated with the labelling of the inner surface of the vacuolar membrane. These results indicate that CT are probably formed by a budding off from these vacuoles, rather than being directly involved in endocytosis.     

Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in clutured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease.

The transport of 3H-fucose- and 3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a smiliar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies.     

Fine structure of the gastric epithelium of the ascidian botryllus schlosseri vacuolated and zymogenic cells

Summary Vacuolated and zymogenic cells, which are two of five cell types identified by electron microscopy in gastric epithelium of B. schlosseri, are described in detail. The vacuolated cells are characterized by one, or a few, supranuclear vacuoles containing myelin figures. A peculiar Golgi apparatus is consistently found at the base of the vacuoles; it consists of cisternae frequently containing small vesicles and tubules of constant diameter and/or a strong electron-opaque material. A variety of vesicles and multivesicular bodies are visible in the apical cytoplasm below long ribbon-like microvilli. The se findings suggest that the vacuolated cells are involved in absorptive and perhaps secretory activity. The zymogenic cells are characterized by a highly developed RER, numerous apical secretory granules and a well developed supranuclear Golgi apparatus. At the apical end, autophagosomes are frequently encountered, some of which contain also zymogen granules. Both cell types contain numerous lipid droplets, which are interpreted as an energy reserve available for the cells and for the entire colony during the change of generation. Correlation between structure and function in both cell types is discussed by taking into account the peculiar life cycle of B. schlosseri, as well as previously reported data on similar cells in other ascidians.We would like to dedicate this work to Prof. Giuseppe Reverberi on the occasion of his 70th birthday.The authors are indebted to Profs. A. Sabbadin and G. Mazzocchi for their most helpful suggestions and advice. We would also like to thank Mr. G. Tognon for technical assistance and the staff of the Stazione Idrobiologica di Chioggia for their assistance in collecting material. — This research was supported by a grant of C.N.R., contract from the Istituto di Biologia del Mare, Venezia, No. 7100396/04115542 and with the E.M. facilities of C. N. R. contract No. 70.01798.04.115.876.     

Regional specialization in the malpighian tubules of the new zealand glow-worm Arachnocampa luminosa (diptera: Mycetophilidae). The structure and function of type I and II cells

The Malpighian tubules of the glow-worm Arachnocampa luminosa are divided into four morphologically distinct regions (Parts 1--4) each comprised of a different cell type (Types I--IV). The ultrastructure of Type II cells is indicative of a transport function. The basal cell surface is highly invaginated and at the apical surface the lumen is lined with microvilli about 80% of which contain mitochondria. Spherites contained in these cells are formed from small vesicles produced by the Golgi apparatus. They have a central uric acid core enclosed by laminations of phosphates of calcium and magnesium. Cells of Part 2 of the tubule secrete a fluid high in potassium (173 mM) and low in sodium (18 mM). The cell is 30 mV negative and the lumen 44 mV positive to the bathing solution. This is consistent with the proposal of an apical cation pump. The secretion produced by Part 2 of the tubules is modified by the Type I cells by the reabsorption of potassium (162 mM) and the addition of sodium (24 mM) to the primary excretory fluid. Type I cells are 20 mV negative and the lumen 22 mV positive with respect to the bathing medium. From ultrastructural observations, Type I cells exhibit features characteristic of transporting cells thought to have an absorptive function. The basal and apical cell surfaces are extensively folded, and mitochondria are found in bands above the basal infoldings and below the microvilli. Mitochondria do not penetrate the microvilli. On comparative grounds, the fine structure of Type I cells suggest that they reabsorb ions from the tubule lumen. Energy for these processes may come from the breakdown of lipids by microperoxisomes contained within these cells. Alternatively, the fluid produced by Part 2 of the tubule may be modified passively by diffusional processes across Type I cells.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.     

Excurrent duct system of the male turtle Chrysemys picta

The epididymis and efferent duct system of the turtle Chrysemys picta were examined. Seminiferous tubules are drained by a series of ducts that form a rete exterior to the tunica albuginea. The rete is located lateral to the testis and consists of anastamosing tubules of varying diameters, lined by a simple epithelium consisting of squamous to cuboidal cells. The rete is highly vascularized. A series of tubules (efferent ductules) connect the rete to the epididymis proper. The efferent ductules are highly convoluted, running between the epididymal tubules and are of varying diameters. The simple columnar epithelium lining these tubules possesses tight junctions, with every third or fourth cell possessing long cilia that protrude into the lumen. The cytoplasm of these epithelial cells contains abundant mitochondria. In the central portion of the efferent ductule, epithelial cells possess granules that appear to be secreted into the lumen by an apocrine process. The epididymis proper is a single, long, highly convoluted tubule that receives efferent ductules along its entire length. It is lined by a pseudostratified epithelium containing several cell types. The most abundant cell (vesicular cell) lacks cilia, but has a darkly staining apical border due to numerous small vesicles immediately beneath the luminal membrane. The small vesicles appear to fuse with each other basally to form larger vesicles. These cells appear to have an absorptive function, and occasionally sperm are embedded in their cytoplasm. The second-most abundant cell is a basal cell found along the basement membrane. The number of these cells fluctuates throughout the year, being most abundant in late summer and early fall. A small narrow cell with an oval nucleus and darkly staining cytoplasm, extending from the basement membrane to the apical surface, is present in small numbers, particularly in the caudal regions of the epididymis. This cell is frequently found in association with another narrow cell having a rounded nucleus and abundant mitochondria in its cytoplasm.     

The effect of thioglycolate on intermediate filaments and membrane translocation in rat urothelium during the expansion-contraction cycle

Summary The functional role of cytokeratin intermediate filaments in the translocation of asymmetric membrane plaques between cytoplasm and surface of apical urothelial cells was investigated during contraction and expansion of rat urinary bladders. A stereological investigation of electron micrographs provided estimations of surface area, volume, and number of discoidal vesicles and infoldings per unit volume of urothelial apical cell cytoplasm. Contracted and distended bladders incubated in 0.01 M sodium bicarbonate were compared to identical preparations experimentally incubated in 5 mM thioglycolic acid. The latter reagent disrupts the intermediate filament network by reducing sulfhydryl bridges. Densities of discoidal vesicles in cells contracted after incubation in thioglycolate were similar to density estimations in cells expanded under control conditions. Similarly, densities of vesicles in cells expanded after exposure to thioglycolate were comparable in number to those in normally contracted cells. Thus, membrane translocation to and from the luminal surface was blocked by thioglycolate treatment. The lack of normal membrane transfer at the luminal surface induces apical cells exposed to experimental conditions to undergo extraordinary adjustments in response to external pressures of bladder contraction and distension. During contraction, the apical-intermediate cell interface unfolded while the luminal surface ballooned out into the lumen. In distended bladders, large intercellular spaces formed between apical cells along their lateral margins. The results support a model published earlier implicating the filament network as a critical mediator of membrane translocation.     

Translocation of dimeric IgA through neoplastic colon cells in vitro.

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.     

INTESTINAL TRANSPORT OF ANTIBODIES IN THE NEWBORN RAT

Evidence has been reported that the proximal small intestine of the neonatal rat selectively transports antibodies into the circulation. This study describes the morphology of the absorptive epithelial cells in this region of the intestine and their transport of several immunoglobulin tracers: ferritin-conjugated immunoglobulins (IgG-Ft) and antiperoxidase antibodies. Cells exposed to rat IgG-Ft bound the tracer on the membrane of tubular invaginations of the apical cell surface. Tubular and coated vesicles within the cell also contained the tracer, as did the intercellular spaces. Uptake of tracer was highly selective and occurred only with rat or cow IgG-Ft; when cells were exposed to chicken IgG-Ft, ferritin-conjugated bovine serum albumin, or free ferritin, tracer did not enter the cell or appear in the intercellular spaces. Experiments with rat and chicken antiperoxidase showed a similar selective uptake and transport of only the homologous antibody. When cells from the distal small intestine were exposed to the tracers, all tracers were absorbed nonselectively but none were released from the cells. Cells from the proximal small intestine of the 22-day-old rat failed to absorb even rat IgG-Ft. A model is presented for selective antibody transport in proximal cells of the neonatal rat in which antibodies are selectively absorbed at the apical cell surface by pinocytosis within tubular vesicles. The antibodies are then transferred to the intercellular space within coated vesicles. Distal cells function only to digest proteins nonselectively.     

Mitochondria-rich (chloride) cells in the gill epithelia from four species of stenohaline fresh water teleosts

Summary The mitochondria-rich (chloride) cells have been found to be present in the gill epithelia of four species of stenohaline fresh water teleosts. The cytoplasm of these chloride cells contains an extensive network of cytoplasmic tubules which communicate with intercellular spaces bordering the lateral and basal cell surfaces. Numerous vesicles with fairly electron-dense interiors are also present in the apical cytoplasm of chloride cells. The apical surface of a chloride cell forms an apical pit, but the lumen of the pit does not appear to be in continuity with the interior of the apical vesicles and tubules inside the cell.When Carassius auratus were kept in 100, 200, 300, and 400 mOsm-diluted sea water for a month, no appreciable changes occurred in the number and fine structure of the chloride cells, except for a dilation of the apical vesicles and a slight decrease in diameter of the cytoplasmic tubules in these cells in the fishes kept in 300 and 400 mOsm.These results suggest that chloride cells may be a rather common occurrence in the gill epithelia of stenohaline fresh water teleosts, and may function in ion-transport in these fishes in fresh water environments.     

Membrane differentiations of an absorptive epithelium covering embryonic trophotaeniae in a goodeid teleost

Summary The trophotaenial absorptive cells (TACs) in goodeid embryos facilitate nutrient absorption during prolonged periods of intraovarian gestation. In a study of membrane differentiations associated with solute and ligand transfer in the trophotaeniae of Xenotoca eiseni, embryos were incubated in vivo with cationized ferritin (CF) prior to freeze-cleaving. This exposure to high concentrations of an adsorptive ligand was meant to induce swelling of the endosomal compartment. Macromolecular trafficking in TACs occurs via an apical endocytic complex consisting of plasma membrane invaginations, a large population of small vesicles, uniformly thick apical tubules, and endosomes. Freeze-fracture replicas showed that the microvillar plasma membrane P-face of TACs was studded with intramembrane particles (IMPs) at a fairly high density, whereas that of the cell surface proper contained a distinctly lower density and the tubulovesicular endocytic pits contained almost no IMPs. The majority of small vesicles and apical tubules in a near surface position displayed P-fracture faces with only a few odd IMPs, indicating that membrane, shuttling between the apical plasma membrane and intracellular sorting organelles, obviously does not carry along many large-sized integral membrane proteins. The distended endosomal compartment had many P-face-associated particles primarily clustered into patches. Specializations of the lateral plasma membrane included 4–8 tight junctional strands, relatively large complements of gap junction proteins, and numerous plaques of desmosomal membrane particles. A system of lamellar cisternae underlay the lateral cell surface that was in continuity with the intraepithelial space by numerous tubular canals, giving rise to an intracellular amplification of the basolateral plasma membrane. Their outward openings appeared as tiny pits on the cytoplasmic faces of freeze-cleaved cell membrane. The density of IMPs on the P-faces of the surface plasma membrane was apparently lower than that on its invaginated lamellar complex. Hence, it is concluded that the mobility of integral membrane proteins in the plane of the membrane may be hampered in movement across the surface pores.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Ultrastructural changes in the coronet cells of the saccus vasculosus from rainbow trout,Salmo gairdneri (Richardson), kept in sea water

Summary The coronet cells of saccus vasculosus of fresh-water living and sea-water adapted rainbow trout were studied with the electron microscope, with special regard to changes in the latter group. Only quantitative differences were observed, namely a raised number of mitochondria in the apical region and the head and also a concentration of the agranular endoplasmic reticulum with a higher amount of electron-dense material and vesicles around the Golgi saccules. Together, these findings suggest a secretory function for the coronet cell. A supposed transport of vesicles from the head region of the coronet cell out into the globules is suggested. Interrelation between primary and secondary vesicles is discussed.This work was supported by grants from the Royal Physiographical Society of Lund and the Faculty of Natural Sciences at the University of Lund.—I am greatly indebted to Mrs. Lena Svenre and Miss Inger Norling for excellent technical assistance.