Intracellular and extracellular localization of fibronectin in primary cultures of aortic smooth muscle cells using immunoperoxidase cytochemistry in electron microscopy

This report describes the intracellular and extracellular localization of fibronectin at the ultrastructural level in primary cultures of aortic smooth muscle cells. Fibronectin was present in all the cisternae of the rough endoplasmic reticulum except the perinuclear cisterna, in large vesicles associated with the trans side of the Golgi complex, and in single large vesicles in the cytoplasm often associated with microtubules. The extracellular microfibrils were heavily stained. In sections parallel to the plane of growth bundles of extracellular microfibrils in continuity with arrays of intracellular microfilaments were observed (fibronexus). The basement membrane around the aortic smooth muscle cells was discontinuous and diffusely stained. The results indicate that fibronectin is localized in the cytoplasmic membranous apparatus of protein synthesis, processing, and secretion. The lack of reaction product in the flat cisternae of the Golgi complex let suggest either that fibronection may not be present in significant amounts within the flattened cisternae or that the method is insufficient in detecting the glycoprotein in this subcompartment off the Golgi complex.     

Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.     

Ultrastructural localization of concanavalin A-binding sites in the Golgi apparatus of various types of neurons in rat dorsal root ganglia: functional implications

The localization of concanavalin A (con A) binding sites has been determined at the electron-microscopic level in the six types of neurons (A1, A2, A3, B1, B2, C) of rat dorsal root ganglia. In all ganglion cells, con A stained the plasma membrane, the nuclear envelope, the cisternae of the rough endoplasmic reticulum, and the matrix of some multivesicular bodies. In contrast, the con A reactivity of the Golgi apparatus varied according to cell type. In type B1 and B2 cells and possibly in type A3 cells, the lectin was exclusively located in three or four saccules on the cis side of the Golgi stacks, whereas the TPPase-positive saccules and the trans sacculotubular elements were unstained with con A. In type A1, A2, and C neurons, all Golgi saccules as well as the trans sacculotubular elements were stained with the lectin. These results suggest that different types of glycoproteins were produced in these two groups of neurons. In the type A1, A2, and C cells, the persistence of the lectin reactivity in the TTPase-positive saccules or sacculotubular elements on the trans side of the Golgi stacks suggests the presence of glycoproteins with oligosaccharide side chains rich in alpha-D-mannosyl residues in terminal positions. In contrast, the disappearance of the con A reactivity in equivalent elements of the Golgi stacks in type B1, B2, and A3 cells suggests the addition at this level of other sugar residues characteristic of complex oligosaccharide side chains. The majority of the vesicular elements associated with the Golgi apparatus, as well as lysosomes, were unstained with con A.     

Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained. These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated and treated acinar cells.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Summary Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained.These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated und treated acinar cells.     

Involvement of endoplasmic reticulum in the origins of dictyosome-like structures of guinea pig spermatocytes

Summary Dictyosome-like structures (DLS) are formed in early spermatocytes first as single saccules. These saccules occur in association with forms of endoplasmic reticulum (ER) characterized by a paucity of ribosomes and luminal content, by a constriction of the lumina, and by a tendency to fragment or form myelin figures during fixation. Nascent DLS and the unusual ER cisternae share many characteristics in common including a pattern of staining with fixatives containing tannic acid where the membranes appear thin due to the inner membrane leaflet being unstained or poorly stained. DLS also appear to form in the region conventional Golgi apparatus but always in association with ER forms that frequently occupy portions of the Golgi apparatus zone.An ability to stain with phosphotungstic acid at low pH exhibited by DLS is given also by the specialized ER forms. One possibility for DLS formation suggested by the present study is that DLS cisternae differentiate from ER membranes after which they ultimately associate into the stacked configurations that characterize mature DLS.     

Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. I. Ultrastructure and phosphatase activity of endodermal cells

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.     

Structure of Golgi apparatus

Summary Golgi apparatus (GA) of eukaryotic cells consist of one or more stacks of flattened saccules (cisternae) and an array of fenestrae and tubules continuous with the peripheral edges of the saccules. Golgi apparatus also are characterized by zones of exclusion that surround each stack and by an assortment of vesicles (or vesicle buds) associated with both the stacks and the peripheral tubules of the stack cisternae. Each stack (sometimes referred to as Golgi apparatus, Golgi complex, or dictyosome) is structurally and functionally polarized, reflecting its role as an intermediate between the endoplasmic reticulum, the cell surface, and the lysosomal system of the cell. There is probably only one GA per cell, and all stacks of the GA appear to function synchronously. All Golgi apparatus are involved in the generation and movement of product and membrane within the cell or to the cell exterior, and these functions are often reflected as structural changes across the stacks. For example, in plants, both product and membrane appear to maturate from the cis to the trans poles of the stacks in a sequential, or serial, manner. However, there is also strong ultrastructural evidence in plants for a parallel input to the stack saccules, probably through the peripheral tubules. The same modes of functioning probably also occur in animal GA; although here, the parallel mode of functioning almost surely predominates. In some cells at least, GA stacks give rise to tubular-vesicular structures that resemble the trans Golgi network. Rudimentary GA, consisting of tubular-vesicular networks, have been identified in fungi and may represent an early stage of GA evolution.     

Transformation of Golgi membrane into the envelope of herpes simplex virus in rat anterior pituitary cells

Envelopment of herpes simplex virus type-1 (HSV-1) was investigated in relation to membrane differentiation in dissociated anterior pituitary cells. The number of cells stained positively with anti-HSV-1 serum was increased from 16 h to 31 h post infection. During this period, electron microscopy revealed that a number of nucleocapsids (unenveloped particles) were accumulated in the Golgi area, where they frequently became surrounded by a double membrane of short Golgi cisternae or by one with a Golgi associated endoplasmic reticulum lysosome (GERL)-like structure. The inner membrane of the cisterna surrounding the nucleocapsids showed regional specialization which was characterized by increased thickness and electron opacity. Acid phosphatase activity, a marker for GERL or trans Golgi cisternae, appeared in the cytoplasmic short cisternae surrounding the nucleocapsids, whereas glucose-6-phosphatase activity, a marker for the nuclear envelope or for endoplasmic reticulum, was not demonstrated in such cisternae. Monoclonal antibody against glycoprotein gD revealed that gD was localized in the trans Golgi membrane as well as in the envelope of the virion. The antibody-binding sites were highly concentrated in the area where Golgi membranes showed increased opacity. Furthermore, nucleocapsids were surrounded exclusively by gD-positive cisternal (Golgi or Golgi-derived) membranes. Thus, our results indicate that the envelope of HSV is derived from trans Golgi cisterna (GERL), and that some viral components, including gD, destined for the envelope may be assembled initially in the Golgi membrane, which is thereby transformed into the envelope of the virus.     

Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.     

Cytological differentiation in the uropygial gland.

Ultrastructural changes were studied in the cells undergoing secretory differentiation in zone I of the tubules of the uropygial gland of White Plymouth Rock chickens. A layer of basal cells and four secretory stages are recognized as the cells migrate from the periphery to the lumen of tubules and progressively elaborate a secretion product. Basal cells, containing rough endoplasmic reticulum and free ribosomes, rest on the basement membrane and are the source from which secretory cells arise. Dilated perinuclear cisternae and the proliferation of smooth endoplasmic reticulum in the form of vesicles, invaginated sacs and cusp-shaped cisternae indicate the onset of lipgenesis in stage I cells. The perinuclear cisternae are more dilated and the endoplasmic reticulum is composed on saccules and cisternae in stage II cells. Stage III cells are characterized by concentric lamellae of endoplasmic reticulum surrounding secretory droplets. Dilated cisternae of endoplasmic reticulum and secretory droplets both contain a reticular substance. The perinuclear cisternae of stage III cells have returned to normal dimensions. Large mature lucent secretory droplets, lined with electron-dense material, fill the cytoplasm ostage IV cells which degenerate and release their secretory product into the tubule lumen. Spherical membrane-bound compartments containing a mottled substance of moderate electron density occur in basal cells and all subsequent secretory stages. These mottled bodies are surrounded by saccules of endoplasmic reticulum in stage II cells and are intimately associated with secretory droplets in stage III cells, but there is no evidence that they give rise to secretory droplets and their role in secretory differentiation is unknown.     

The mystery of the unstained Golgi complex cisternae

The Champy-Maillet OsKI reaction has been used upon Golgi complexes to show two kinds of staining. It stains material being processed as it passes along the secretory pathway of the rough endoplasmic reticulum (RER) and Golgi cisternae (GC) up to crystallization in secretory vesicles. It also stains separately the environment within parts of the GC. This GC staining may occur in all compartments (transition vesicles, saccules, condensing vacuoles), but it is characteristically missing from any one of them. The unstained cisternae may be explained if outer saccules are made from either stained or unstained transition vesicles, both of which occur. The presence of empty, unstained transition vesicles is dictated by the surface to volume ratios of microvesicles in relation to saccules. Most transition vesicles must return their membrane to the endoplasmic reticulum, but from time to time it is presumed that they fuse to make a saccule. Saccules, stained and unstained, then mature through the stack. OsKI reactions with tissues and test molecules suggest that in the RER and GC the stain detects labile--S . S--bridges before they lock the tertiary configuration of proteins.     

RCA I-binding patterns of the Golgi apparatus

The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Golgi apparatus in the acinar cells of the developing embryonic pancreas: II. Localization of lectin-binding sites

The reaction patterns of the Golgi apparatus following staining with the lectins concanavalin A (ConA), Ricinus communis I agglutinin (RCA I), and Helix pomatia lectin (HPA) were studied in the pancreas acinar cells of rat embryos in the course of cell differentiation from day 13 through day 20 of gestation. The binding reactions were localized by means of pre-embedment incubation of 10-microns-thick cryosections of pancreas tissue, prefixed in a mixture of 4% formaldehyde/0.5% glutaraldehyde, using horseradish peroxidase for electron microscope visualization. ConA, which preferentially binds to alpha-D-mannosyl residues, consistently stained the cisternae of the cis Golgi side. The majority of the stacks also showed ConA staining of medial cisternae. The reaction of the trans side was variable; in each stage of development, the cisternae of the trans Golgi side either were devoid of labeling or appeared intensely stained. The reactions obtained with RCA I, which recognizes terminal beta-D-galactosyl residues, changed in the course of cell differentiation; in the protodifferentiated and early differentiated states, the system of "rigid lamellae," located at the trans side of the Golgi stacks, was intensely labeled, but became unreactive after production of secretion granules had started, the reaction then being restricted to the stacked saccules. In regard to the Golgi stacks in each of the developmental stages, RCA I binding sites either were confined to the trans cisternae, or, in addition, were found distributed across elements of the medial and cis compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-binding pattern in normal human gastric mucosa. A light and electron microscopic study

Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

Macromolecular differentiation of Golgi stacks in root tips ofArabidopsis andNicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

Summary The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to young and old, polysaccharideslime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips ofArabidopsis andNicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types.Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (30 nm) and are the least stained. Medial cisternae are narrower (20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4–6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network.Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.Dedicated to the memory of Professor Oswald Kiermayer