Endocytosis and vesicular traffic in fetal and adult colonic goblet cells

Structural and functional differences between adult and fetal colonic goblet cells have not been clearly defined. To compare the binding, uptake, and intracellular pathway of internalized apical membrane in fetal and adult goblet cells, cationic ferritin (CF) was used as a nonspecific probe. The initial distribution of membrane anionic sites was determined in segments of proximal and distal colon from fetal (18-22 days) and adult rats that were fixed prior to a 10-minute exposure to CF at 4 degrees C. Uniform binding along the apical membrane and microvilli was noted at all ages. To assess uptake and intracellular transport, segments of proximal and distal colon from fetal and adult rats were exposed to CF for 10 minutes at 4 degrees C prior to a saline wash and incubation in saline or Liebovitz L-15 medium for 3, 6, 15, 30, or 60 minutes at 37 degrees C. In addition, fetal rats between 18 and 22 days gestation (birth) were exposed to CF continuously for 10-30 minutes via an intracaecal injection. The results showed extensive uptake of CF in the fetal goblet cells and a more variable intracellular pathway than in with the adult. Within 3 minutes, numerous CF positive vesicles and tubules were present within the apical cytoplasm as well as interspersed among the secretory granules of the fetal goblet cell. Most of these vesicles were smooth surfaced, although some were coated. By 15 minutes, CF was frequently seen in multivesicular bodies, and occasionally in vacuoles in the vicinity of the Golgi. No CF was detected in Golgi cisternae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocytic appartus and transcytosis in epithelial cells of the vas deferens in the rat

The apex of the principal epithelial cells lining the vas deferens of the rat contains coated pits in continuity with the apical plasma membrane and large subsurface-coated vesicles (100–125 um). In the apical cytoplasm, large, pale, uncoated vesicles (150–300 nm), small coated and uncoated vesicles (50–60 nm), uncoated vesicles about 75–90 nm, and membranous apical tubules are present, in addition to large, vacuolar, pale, multivesicular bodies, dense multivesicular bodies, and secondary lysosomes seen deeper in the cytoplasm amongst numerous ER cisternae, saccules of the Golgi apparatus, and mitochondria. The endocytic activity of these cells was investigated by using cationic ferritin (CF) as a marker of adsorptive endocytosis and native ferritin (NF) for demonstrating fluid-phase endocytosis. These tracers were injected separately into the lumen of the vas deferens, and the animals were killed at various time intervals thereafter from 2 to 90 minutes. At 2 minutes CF was seen bound predominantly to microvilli and to areas of the apical plasma membrane delimiting coated pits as well as in large, coated vesicles. At 5 and 15 minutes the tracers were seen in apical tubules and pale multivesicular bodies; at 30 minutes moderately dense multivesicular bodies were labeled. At 1 hour and longer time intervals dense multivesicular bodies and secondary lysosomes were labeled. NF followed the same pathway as CF; however, no binding to microvilli or areas delimiting coated pits was observed. The numerous other vesicular structures, i.e., the large uncoated vesicles (150–300 nm) and the small coated and uncoated vesicles (50–60 nm), never became labeled with the tracers and therefore were not involved in the endocytic process. There was, however, and exception in the case of several small (75–90 nm) uncoated vesicles seen deeper in the apical cytoplasm of these cells which were labeled exclusively with CF. With time such vesicles appeared along the lateral and basal surfaces of these cells and discharged their content of CF into the lateral intercellular space or the connective tissue space at the base of these cells. Thus the principal epithelial cells in addition to sequestering the endocytosed tracers within secondary lysosomes where they are presumably degraded also appear to be involved in the transcytosis of material from the lumen of the vas deferens to the underlying lamina propria.     

Endocytic apparatus and transcytosis in epithelial cells of the vas deferens in the rat

The apex of the principal epithelial cells lining the vas deferens of the rat contains coated pits in continuity with the apical plasma membrane and large subsurface-coated vesicles (100-125 nm). In the apical cytoplasm, large, pale, uncoated vesicles (150-300 nm), small coated and uncoated vesicles (50-60 nm), uncoated vesicles about 75-90 nm, and membranous apical tubules are present, in addition to large, vacuolar, pale, multivesicular bodies, dense multivesicular bodies, and secondary lysosomes seen deeper in the cytoplasm amongst numerous ER cisternae, saccules of the Golgi apparatus, and mitochondria. The endocytic activity of these cells was investigated by using cationic ferritin (CF) as a marker of adsorptive endocytosis and native ferritin (NF) for demonstrating fluid-phase endocytosis. These tracers were injected separately into the lumen of the vas deferens, and the animals were killed at various time intervals thereafter from 2 to 90 minutes. At 2 minutes CF was seen bound predominantly to microvilli and to areas of the apical plasma membrane delimiting coated pits as well as in large, coated vesicles. At 5 and 15 minutes the tracers were seen in apical tubules and pale multivesicular bodies; at 30 minutes moderately dense multivesicular bodies were labeled. At 1 hour and longer time intervals dense multivesicular bodies and secondary lysosomes were labeled. NF followed the same pathway as CF; however, no binding to microvilli or areas delimiting coated pits was observed. The numerous other vesicular structures, i.e., the large uncoated vesicles (150-300 nm) and the small coated and uncoated vesicles (50-60 nm), never became labeled with the tracers and therefore were not involved in the endocytic process. There was, however, an exception in the case of several small (75-90 nm) uncoated vesicles seen deeper in the apical cytoplasm of these cells which were labeled exclusively with CF. With time such vesicles appeared along the lateral and basal surfaces of these cells and discharged their content of CF into the lateral intercellular space or the connective tissue space at the base of these cells. Thus the principal epithelial cells in addition to sequestering the endocytosed tracers within secondary lysosomes where they are presumably degraded also appear to be involved in the transcytosis of material from the lumen of the vas deferens to the underlying lamina propria.     

Endocytic activity of Sertoli cells grown in bicameral culture chambers

Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface charge distribution in normal and transformed rat bladder epithelial cells in vitro

Cationized ferritin (CF) was used as an ultrastructural marker to study differences in the distribution and density of surface anionic charges between normal and neoplastic cells. In anchorage-independent cell systems, CF induces a redistribution of cell surface receptor-ligand complexes into clusters, patches, and caps on the surfaces of transformed cells, but not on the surfaces of normal cells. In the present report, the authors have, for the first time, extended the CF labeling studies to a system of anchorage-dependent, rat bladder epithelial cell lines: normal RBTC cells and RBTCC-8 carcinoma cells. Cells were grown to confluency and labeled with 500 micrograms/ml CF for 3 minutes. After completion of CF labeling, cells were incubated for 0, 15, 60, 120 minutes, or overnight and were fixed then with buffered glutaraldehyde for electron microscopy. The results show that (a) CF induces grouping of surface anionic charge sites into patches and clusters on RBTCC-8 carcinoma cells (CF covers 53.86 +/- 2.15% of the plasma membrane), but not on normal RBTC cells (CF covers the entire plasma membrane); (b) CF densities are well correlated to the cell surface sialic acid contents of RBTC (2.42 +/- 0.30 microM/10(9) cells) and RBTCC-8 cells (1.26 +/- 0.25 microM/10(9) cells); and (c) CF-labeled membrane is internalized at equal rates by RBTC and RBTCC-8 cells (30% in 15 minutes; 60% in 60 minutes; 70% in 120 minutes; 100% overnight). These data indicate that CF-induced patching of anionic sites is a surface characteristic that is common to anchorage-dependent and independent tumor cells. Mechanisms of CF patching in neoplastic cells are discussed.     

Endocytosis in epithelial cells lining the rete testis of the rat

The endocytic activity of the low cuboidal cells lining the rete testis was analyzed by electron microscopy following injection of various tracers into the lumen of these anastomotic channels. At 1 and 5 minutes after injection, cationic ferritin (CF) and concanavalin A-ferritin (Con A) were seen bound to the apical plasma membrane and to the membrane of subjacent vesicles or invaginations connected to this apical membrane. At 30 and 60 minutes, these tracers were found in intracytoplasmic vesicles and in vesicles connected to the lateral or basal plasma membrane as well as in the lateral intercellular space and in the lamina lucida of basal lamina. At 30 minutes, CF and Con A also appeared in the matrix of pale multivesicular bodies while at 1 hour dense multivesicular bodies were labeled. At 2 hours and later time intervals, the tracers accumulated in dense granules identified as lysosomes. Native ferritin (NF), concanavalin A-ferritin in presence of α-methyl-D-mannoside, and horseradish peroxidase or albumin bound to colloidal gold were all to be incorporated by the lysosomal system of these epithelial cells, as just described for CF and Con A, but these various tracers were not bound to the apical plasma membrane or to the membrane of cytoplasmic vesicles, nor were they found in the intercellular spaces or the lamina lucida at the base of the cells. Thus, the epithelial cells of the rete testis do not appear to be only involved in the uptake of substances from the lumen and their disposal by the lysosomal system, but also appear to contribute to the transport of certain macromolecules from the lumen to the laterobasal surfaces of the cells. These cells may thus play a role in determining the composition of the rete testis fluid.     

Three-dimensional demonstration of the intracellular structures of mouse mesothelial cells by scanning electron microscopy

Intracellular structures of mouse mesothelial cells lining the small intestine were studied by scanning electron microscopy. Specimens were prepared by the freeze-polishing method which permitted demonstration of the attenuated thin layered organization of the intracellular structures. When the surface cell membrane had been partially turned over during the specimen preparation procedure, many pinocytotic vesicles were observed attached to the cytoplasmic side of the cell membrane. We also demonstrated the external side of the basal cell membrane on which many openings of the pinocytotic vesicles were clearly shown as those recognized on the free cell surface. Two adjacent pinocytotic vesicles often appeared fused together. The multivesicular vacuole formed by the congregation of the pinocytotic vesicles were sometimes recognized on the cytoplasmic side of the surface and basal cell membrane. On the nuclear surface, nuclear pores and polysomes were visible. From our SEM findings, we classified the rough endoplasmic reticulum into three types: type I, II and III. The type I rough endoplasmic reticulum was composed of flat cisternae with fenestration, the type II of polygonal flat cisternae without fenestration, and the type III of vesicular cisternae. Polysomal formation is evident in the type III, but less in the type I and II. All of the endoplasmic reticulum appeared connected each other forming a continuous system in the cell. Two kinds of the Golgi apparatus were recognized: a stretched type with highly extended Golgi cisternae and a shrunken type with less developed Golgi cisternae. In the latter, lots of Golgi vesicles were attached on the periphery of the cisternae, and the outermost cisterna was commonly fenestrated.     

The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen

Biosynthesis of type IV collagen in the parietal endodermal cells of 12 day gestant Sherman rats was examined following intraconceptal injection of 3H-proline. The concepti were removed at times varying from 2 minutes to 24 hours after the injection. The parietal wall of the yolk sac, including endodermal cells and the associated basement membrane known as Reichert's membrane were processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Radioactivity was high in endodermal cells during the first 2 hr after 3H-proline injection and later dropped to some extent, while radioactivity rose in Reichert's membrane. Examination of endodermal cell organelles showed some early labeling over rER and Golgi apparatus without a clear-cut trend, except for a drop in Golgi label at late times after 3H-proline injection. The density of silver grains over secretory granules rose significantly by 40 min, reached a high peak by 4 hr and then declined at the time when radioactivity increased over Reichert's membrane. Furthermore, the radioactively-labeled secretory granules were localized mainly at the trans Golgi face soon after injection and near the cell surface adjacent to Reichert's membrane at later times. Biochemical reports indicate that a substantial amount of the proline taken up by the 12-14.5 day rat embryo endodermal cells is incorporated into type IV collagen. Since there is high labeling of the secretory granules from 40 min to 4 hr and the labeled granules are associated with the Golgi apparatus at early times, it is proposed that collagen precursors are processed through rER and Golgi apparatus, packaged into secretory granules and then transported to the cell surface where type IV collagen or its precursors are released and subsequently deposited into Reichert's membrane.     

Subpopulations of microtubules with differential sensitivity to nocodazole: role in the structural organization of the Golgi complex and the lysosomal system

The role of cytoplasmic microtubules (MTs) in the structural organization of the Golgi complex and the lysosomal system was studied in L929 mouse fibroblasts using a combination of cytochemical and electron microscopic methods. Immunocytochemical staining demonstrated a radiating pattern of MTs, originating in the juxtanuclear region containing the centrioles. The stacks of Golgi cisternae with associated vesicles and tubules (the trans Golgi network), as well as most of the endosomes and lysosomes, were also located in and immediately around this region. Wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) bound to the plasma membrane at 4 degrees C. After warming to 37 degrees C, the conjugate was rapidly internalized by endocytosis and via small vesicles transferred to endosomes and lysosomes in the juxtanuclear region. Slightly later it was also found in the trans Golgi network. A weaker but otherwise similar staining was obtained if the conjugate was applied to fixed and permeabilized cells. Treatment with 3.0 microM nocodazole induced depolymerization of MTs and rounding up of the cells. At the same time, the Golgi complex was disorganized with dispersion of its stacks of cisternae throughout the cytoplasm and atrophy of the trans Golgi network. The lysosomes gathered around the dispersed cisternal stacks and the endosomes became fewer in number. Moreover, the uptake of WGA-HRP was markedly inhibited. Following withdrawal of the drug, MTs reformed and the structural organization of the cell was again normalized. If the recovery was allowed to take place in the presence of low concentrations of nocodazole (0.03 or 0.15 microM), MTs of short and medium length were formed, the latter showing a curly path. In parallel, a partial or nearly complete normalization of cell morphology occurred. Internalization and transport of WGA-HRP labeled membrane to the lysosomes and the trans Golgi network was also resumed. In contrast, the cells were not able to pass through mitosis in a normal manner and multimicronucleated cells accumulated. Taken together, the findings indicate that L929 cells contain a subset of nocodazole-resistant MTs which play an important role in the structural organization of the cell, especially with regard to the Golgi complex and the lysosomal system. They support recent notions that there exists subpopulations of MTs with different physicochemical properties and functions.     

Proposed role for small cytoplasmic vesicles in cytokine secretion by mouse macrophages

This report represents an extension of a prior report hypothesizing that cytokines in mouse macrophages are secreted by a morphologic array of small vesicles which fuse with the cell membrane and which originate in the Golgi complex [Med Hypoth 53 (1999) 107]. The Golgi complex in macrophages is distinguished by a characteristically multicentric configuration and shows budding of vesicles from the closely approximated tips of the lamellae. The location of small vesicles which extend from the Golgi complex to the cell membrane supports the hypothesis that there is one type of vesicle which fuses with the cell membrane and secretes its content of cytokines. The other type of vesicle has been shown to fuse with pinocytic vacuoles to form hydrolase positive cytoplasmic granules. Consideration of cytokines produced by macrophages will help to clarify the immunologic functions of these cells.     

Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons

SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5–10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20–30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25–positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.     

Budding of small vesicles from the rough-surfaced endoplasmic reticulum in secretory ameloblasts of rat molar tooth germs

The budding of small vesicles from the rough-surfaced endoplasmic reticulum (rER) was examined in the secretory ameloblast of rat molar tooth germs by ordinary fixation or prolonged osmium fixation. The budding of small vesicles from the rER was observed not only at the special region (transitional region) of the rER system, which abutted on the cis-face of the Golgi apparatus, but also at other regions of the rER in the secretory ameloblast. Small vesicles (presumed to be transitional vesicles) were adjacent to the rER, which also showed budding of vesicles. After prolonged osmium treatment, osmium deposits appeared in small vesicles, as well as in the cisternae of the cis saccule of the Golgi apparatus. Small vesicles containing osmium deposits were located at various regions of the cell, including the cis-face of the Golgi apparatus. These findings indicate that the budding of small vesicles from the rER is not restricted to the transitional region of the rER system of the secretory ameloblast, but is found at various regions of the cell. This indicates that newly synthesized proteins may be transferred from the rER cisternae to the transitional vesicles not only in the transitional region of the rER system adjacent to the Golgi apparatus, but also in other regions of the secretory ameloblast.     

Quantitative changes of membranes in rat parathyroid cells related to variations of serum calcium

Parathyroid glands of rats were exposed to low, normal, or high serum calcium concentration by infusion of either ethylenglycol-bis-(2-aminoethylether)-tetraacetic acid (EGTA), NaCl, or CaCl2 for 90 minutes. Electron microscopic morphometry revealed that the cell volume remained constant, whereas the cell surface increased significantly after EGTA infusion and decreased significantly after CaCl2 infusion compared with parathyroid cells of NaCl-infused rats. The surface of the Golgi complex including the surrounding vesicles increased significantly after CaCl2 infusion. Significant changes were not obvious in the volume and surface of the nucleus or in the surface of the rough endoplasmic reticulum. These results suggest, first, that parathyroid hormone is released by exocytosis leading to increased cell surface within 90 minutes. Second, it is considered likely that plasma membrane constituents are retrieved by endocytosis and incorporated into the Golgi complex supporting the idea of membrane recycling.     

Morphological aspects of type II alveolar pneumocytes following treatment with puromycin in vivo

Ultrastructural modifications of type II pneumocytes (PNM-II) in mice were analysed 125 and 155 minutes after puromycin treatment (12 mg/100 gm at 0, 30, 60 and 90 minutes). A quantitative evaluation of the cell compartments was carried out and the inhibition of protein synthesis in PNM-II was monitored by light microscopic radioautography, following ³H-leucine injection. In electron micrographs, following a 125-minute puromycin treatment, the number and size of lamellar bodies, the precursors of lung surfactant material, appeared markedly reduced. The multivesicular bodies (MVB), which are normally very frequent in PNM-II, had almost completely disappeared, as had composite bodies. Golgi saccules were dilated, while the area occupied by Golgi vesicles was enlarged. Observations following the 155-minute puromycin treatment showed a strong enhancement of these modifications. Smooth and coated vesicles of the Golgi area, as well as peroxisomes, did not appear modified by puromycin. Elongated zones of autophagy were more prevalent after 125-minute treatment than after the 155-minute one. Small bodies were frequently observed in the cytoplasm, near the Golgi zone. They were bounded by a smooth membrane and contained tiny vesicles and/or electron-dense lamellae similar to those present within the lamellar bodies. Parallel membranes formed folds, some of them in continuity with lamellar bodies, thus encircling portions of cytoplasm. These structures, which were few in number in controls, were very frequently observed in treated cells, mainly after the 125-minute treatment. These extensive alterations of PNM-II morphology appeared to be related to a disturbed production of pulmonary surfactant.     

Characterization of the interstitial cells associated with the submuscular plexus of the guinea-pig colon

Interstitial cells associated with the submuscular plexus of the guinea pig colon were studied by electron microscopy and by light microscopic wholemount stretch preparations. Their cytoplasmic features are similar to those of fibroblasts and they contain a well-developed Golgi apparatus, granular endoplasmic reticulum and many mitochondria. Intermediate filaments are abundantly distributed throughout the perinuclear region and processes. Numerous caveolae, a basal lamina and subsurface cisterns are observed on the cell membrane as in smooth muscle cells. The most characteristic feature of this cell type is the existence of many large gap junctions that interconnect these cells to each other and with the smooth muscle cells. Nerve varicosities containing synaptic vesicles are observed in close apposition with cells of this type. Whole-mount preparations stained by the zinc iodide-osmic acid method and by vimentin immunohistochemistry clearly demonstrated the stellate form of these gap junction-rich cells and suggested that they correspond to the interstitial cells of Cajal.     

Glycoprotein containing vesicles in the surface epithelial cells of the ascending colon of the rat

Recently, radioautographic studies have shown that cell coat glycoproteins are transported to the cell surface by vesicles both in the amoeba (Flickinger, '75) and in the epithelial cells of the ascending colon of the mouse (Michaels and Leblond, 1976). In the current morphological and cytochemical study of the surface epithelial cells of the rat ascending colon, it is shown that filamentous material, resembling the cell coat, is contained in saccules toward the mature face of the Golgi apparatus and in vesicles close to the apparatus and near the terminal web. The vesicles are limited by a unit membrane composed of asymmetric osmiophilic leaflets and similar to the plasma membrane. When stained by the periodic acid-chromic acid-silver methenamine technique, silver was precipitated on the cell components containing the filamentous material indicating the presence of glycoproteins. Narrow invaginations from the cell surface that may correspond to vesicles undergoing exocytosis were also positive for glycoproteins. The distribution of the filamentous material that was glycoprotein positive parallels the pathway followed by material that had been found to be labeled with a tritiated glycoprotein precursor (³H-fucose) in the epithelial cells of the ascending colon of the mouse. It is suggested that the system of vesicles in the rat colon cells is acting in a manner similar to the vesicles in the mouse cells to transport cell coat glycoproteins from the Golgi apparatus to the cell surface.     

Sorting and transepithelial transport of adsorbed protein tracers: effects of temperature

The epithelium of the guinea pig yolk sac is involved in the selective transport of macromolecules to the fetus. We studied the compartments involved in sorting and transepithelial transport of protein tracers and the effect of lowered temperature (18 degrees C) on these events. Explants of yolk sac were incubated with a mixture of cationized ferritin (CF) and horseradish peroxidase (HRP, Sigma type VI). At 4 degrees C, both tracers were bound to the cell surface and binding of an HRP-gold complex was shown to be inhibited by mannan. At 37 degrees C and 18 degrees C, both tracers were taken up into tubules and vesicles in the apical cytoplasm. Usually the tubules contained a mixture of tracers, but they often showed a polarized distribution with CF and HRP at opposite ends. The vesicles also contained mixtures of the tracers, but some contained only one. In addition, there were some irregularly shaped vacuoles composed of saccules that contained either a mixture, HRP alone, or CF alone. These results suggest that these adsorbed ligands are binding to unique microdomains of the endocytic complex. After 20 min at 37 degrees C coated vesicles 100 nm in diameter were located in the apical cytoplasm and coated vesicles of the same size were located at the lateral cell membrane. Usually they contained only HRP or CF, although occasional mixtures were seen. At 18 degrees C, HRP was transported across the cells in 100 nm vesicles. However, transport of CF was completely inhibited at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface membrane traffic in guinea pig basophils exposed to cationic ferritin

Surface membrane traffic patterns can be influenced by a number of factors, including the functional state of the cell. We used transmission electron microscopy to investigate the fate of surface membrane in guinea pig basophils exposed to cationized ferritin (CF) in vitro. CF bound to the plasma membrane and was internalized on the membranes of vesicles and vacuoles, a process that was particularly prominent at the uropod of basophils exhibiting a polarized ('motile') configuration. The vesicles/vacuoles moved to the Golgi area, or, in the case of degranulating basophils, were observed in continuity with the degranulation sac, a structure formed largely by the fusion of individual cytoplasmic granule membranes. However, CF-positive vesicles were never observed to fuse directly with the membranes of intact cytoplasmic granules.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with ³H-fucose. Control rats received ³H-fucose only. All rats were sacrificed 90 min after ³H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

Ultrastructural organization and histochemical profile of adult fowl choroid plexus epithelium

The choroid plexus epithelial cell of adult fowl is columnar with an irregular apical border of smooth-surfaced microvilli and cilia. Bi-nucleate cells occur frequently. Coated plasmalemma pits and vesicles (～ 1000 Å) are present at all margins of the cell. Smaller coated vesicles (600–700 Å), found in variable numbers throughout the apical zone, tend to be concentrated in the Golgi region. Sparse to profuse amounts of granular endoplasmic reticulum (RER) are found throughout the cell with tubular whorled configurations occasionally noted in the subnuclear cytoplasm. Parallel arrays of RER extend from the cell base, around the nucleus, to the Golgi zone. Oval, fine granular masses occur in the subnuclear area. Free ribosomes are distributed throughout the cell. An array of diverse dense bodies and a few multi-vesicular bodies lie near the Golgi complex. Variable amounts of smooth ER are present in the supra-nuclear region. Toluidine blue, pH 4.0, demonstrates a heavy subnuclear condensation of cytoplasmic RNA. After sulfation, the apical margin and basement membrane are made metachromatic by Toluidine blue. A diastase-resistant, strong PAS positive reaction occurs in the apical border and basement membrane. Lateral cell margins are lightly stained with PAS. Alcian blue and colloidal iron intensely color the microvillous border. Bromphenol blue and bromsulfalein heavily stain the apical and basal zones of all cells.