Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. III. Immunostaining for laminin and its precursors

Reichert's membrane and the endodermal cells of the parietal yolk sac were examined for the presence of laminin antigenicity using anti-laminin antibodies and the peroxidase-antiperoxidase sequence. Immunostaining was observed through the full width of Reichert's membrane and within endodermal cells. In these cells immunostaining was observed in rough endoplasmic reticulum (rER) cisternae and Golgi apparatus. The Golgi staining could occur in any saccule, but predominated in components interpreted as the last saccule of the stack, the GERL element, and associated prosecretory granules. The secretory granules found in the ectoplasm were also immunostained. Finally, multivesicular bodies showed some staining. The immunostaining of Reichert's membrane indicates the presence of laminin itself, while that of rER cisternae and the Golgi apparatus is attributed to laminin precursors. Presumably the biosynthesis of laminin occurs along the usual protein pathway, that is, from rER through Golgi saccules and the GERL element to secretory granules, which release their content into Reichert's membrane. The laminin immunostaining of Reichert's membrane and endodermal cells is similar to that of type IV collagen. It is, therefore, likely that the two substances are processed and secreted simultaneously.     

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.     

Tridimensional architecture of the Golgi apparatus and its components in mucous cells of Brunner's glands of the mouse

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in thin and thick sections of mucous cells of mouse Brunner's glands by using low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of glands impregnated with osmium or treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase) or thiamine pyrophosphatase (TPPase) activity, the Golgi apparatus appeared, at low magnification, as a continuous network located in the supranuclear region. At higher magnifications and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following components were observed: on the cis-face of the Golgi stacks, an osmiophilic tubular network referred to as the cis-element; a cis-saccular-compartment composed of a distended porous saccule slightly reactive for NADPase and three or four underlying NADPase-positive, flattened, poorly fenestrated saccules; a trans-saccular-compartment consisting of four to six TPPase-positive saccules or sacculo-tubular elements, prosecretory granules, and "peeling off" trans-tubular networks. The saccules of the cis-compartment were often perforated by large pores in register. The cavities thus formed in the stacks were called wells and were pan-shaped with a mouth directed toward the cis-face of the stacks and a bottom closed by TPPase-positive saccules. The wells always contained 80-nm vesicles. The saccules of the trans-compartment were involved in the formation of secretory granules according to the following proposed sequence of transformation. The secretion product appeared initially as a granular material evenly distributed throughout a slightly distended, poorly fenestrated saccule. These saccules appeared to transform into fenestrated elements with irregular pores and with parts of them taking on the appearance of a tubular network; they were thus referred to as sacculotubular elements. The secretory material initially distributed throughout these elements accumulated in nodular dilatations randomly distributed along the tubular portions of the elements. The dilatations, considered as prosecretory granules, increased in size as they drained the secretory material from the rest of the sacculotubular elements. Such prosecretory granules, large and irregular in shape, "peeled off" from the stacks of saccules with residual saccular or tubular structures still attached to them, some of the latter forming trans-tubular networks. The prosecretory granules detached from such membranous residues, condensed, and finally transformed into spherical secretion granules.     

The cytomorphology of goblet cells of the fetal intestine. Studies of the large intestine of cattle (Bos primigenius taurus)

In the region of the base of the intestinal crypts undifferentiated goblet cells display a configuration and constellation of organelles and membrane structures that are indicative of their importance for function. These images at this stage of development deliver a scenario of the mechanism of secretory granule production: aggregates of protein vesicles from the "transitional elements" (PALADE) of the granular endoplasmic reticulum are, so to speak, rolled up on the trans side of the Golgi apparatus by inversion of peripheral membrane segments of the innermost Golgi lamellae, thereby forming corpuscles. The origin of the capsulated vacuoles, which contain vesicles as single elements or as conglomerates, is well established. Their capsule consists of a trilaminar external and external and internal membrane; between them lies condensed material of the Golgi apparatus. In the opinion of the present author, the development of the ensheathed vacuoles represents a basic, more general mechanism. In contrast, the further steps of synthesis, for the formation of secretory granules, are more heterogeneous. Condensation of the vesicles and the inner capsular membrane results in the formation of a prosecretory granule, which in the basic element in the process of secretory granule production. The prosecretory granules develop singly or by fusion with other granules to give primary secretory granules. The complexity of this mechanism of secretory granule formation, however, becomes evident when considering the apposition of capsulated vacuoles and prosecretory--primary--secondary secretory granules, of prosecretory and primary secretory granules as well as prosecretory granules and secondary secretory granules. Generally, primary granules show a tendency to become secondary secretory granules or to fuse with them. During maturation of the goblet cells the secretory granules fuse to form larger mucous bodies in the theca by fusion of the laminae of the membranes; a final product, there is a homogeneous mucous mass devoid of membranes.     

Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad

To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of "intermediate vesicles" including similarly structured tubules which may be over 300 nm long and are referred to as "intermediate tubules." The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled. The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.     

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.     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbonlike Golgi apparatus. 1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. 2) A first, poorly fenestrated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. 3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. 4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. 5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

Influence of antimicrotubular drugs on the Golgi apparatus of stomach secretory mucoid cells and small intestine absorptive cells

The effects of vinblastine and colchicine on the Golgi apparatus of stomach surface mucoid and absorptive intestinal cells were compared by cytochemical analysis. The two epithelial cells were chosen because of their different specific functions in the formation of secretory granules, the production of lysosomes and the intensity of membrane traffic in the cytoplasm. For the analysis, adult mice were injected with 1 mg/100 g b.w. of vinblastine and 1 mg/100 g b.w. of colchicine. For the demonstration of cis and trans cisternae of the Golgi apparatus, prolonged osmification, thiamine pyrophosphatase and acid phosphatase activity identification were applied. After treatment with vinblastine or colchicine, polarity of stacks in the Golgi apparatus of surface mucoid cells is preserved although the number of cisternae with thiamine pyrophosphatase or acid phosphatase activity decreases. However, the Golgi apparatus of intestinal absorptive cells completely disintegrates and only a few separated cis or trans cisternae can be identified. The main effect seems to be a reduction of vesicles which can be cytochemically identified as parts of the Golgi apparatus and an accumulation of vesicles which probably originate from budding ER. Communication between the ER and the Golgi apparatus seems to be interrupted.     

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.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

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.     

Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins.

We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.     

Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.     

Tridimensional structure of the Golgi apparatus in type A ganglion cells of the rat

The three-dimensional structure of the whole Golgi apparatus and of its components in type A ganglion cells was examined in thin and thick sections by low- and high-voltage electron microscopy. At low magnification, in 10-micron-thick sections of osmicated cells, the Golgi apparatus formed a broad, continuous perinuclear network. At higher magnification and in thinner sections of cells impregnated with uranyl acetate-lead-copper citrate or postfixed in K-ferrocyanide-reduced osmium, the Golgi apparatus appeared as a heterogeneous structure in which saccular regions characterized by stacks of saccules alternated with intersaccular regions made up of branching membranous tubules which bridged the saccules of adjacent stacks. The saccular regions consisted of the following superimposed elements: a cis-osmiophilic element made up of anastomosing tubules; two or three saccules negative for the phosphatases tested (i.e., nicotinamide adenine dinucleotide phosphatase = NADPase, thiamine pyrophosphatase = TPPase, and cytidine monophosphatase = CMPase); two saccules showing TPPase activity; and one to three trans-sacculotubular elements showing a "peeling-off" configuration, one of which showed CMPase activity. The saccules (phosphatase-negative) on the cis-side of the Golgi stacks showed, in addition to small circular pores, larger perforations in register. The cavities thus formed in the stacks of saccules, called "wells," always associated with small 80-nm vesicles, had a pan shape with the mouth directed toward the cis-face and the bottom closed by a TPPase-positive saccule. In face views of the saccules, the smallest of these perforations showed either a crescent shape, due to the presence of a bud on one side of the perforation, or a circular shape with a single small 80-nm vesicle in the center which was occasionally attached to the saccule by a filiform stalk. Such smaller cavities were considered as the precursors of the larger perforations and eventually of the wells. The small 80-nm vesicles seen in the small cavities or in the wells appeared to form in situ and possibly migrate toward the cisternae of endoplasmic reticulum seen proximal to the cis-face of the stack of saccules. Small 80-nm vesicles were also numerous in the intersaccular regions, along the lateral- and trans-aspects of the Golgi stacks, while larger, 150-to 300-nm vesicles, coated and uncoated, were seen only on the trans-face of the Golgi stacks in proximity to the trans-sacculotubular elements which appear to "peel off" from the Golgi stacks.     

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)