Intracellular transport of proteins in active and resting secretory cells of the venom gland of Vipera palaestinae

The intracellular transport of venom proteins has been studied in active and resting venom glands of the snake Vipera palaestinae by electron microscope radioautography after an intra-arterial injection of [3H]leucine. In the active gland, most of the label is initially (10 min) found over the RER. By 30 min, the relative grain density of the Golgi complex reaches its maximum, with concomitant increase in the labeling of the condensing vacuoles. Later on, a steep increase in radioactivity of the secretory granules is observed. At 3 h, these granules, which comprise about 2% of the cell volume, contain 22% of the total grains. At the following hour, their labeling declines and at the same time the radioactivity of the secreted venom is increased. It is concluded that, in the active cell, venom proteins are transported via the Golgi apparatus into membrane-bounded granules which are the immediate source of the secreted venom. An alternative pathway, which involves the RER cisternae as a storage compartment, seems unlikely, since incorporated label does not accumulate in this compartment after prolonged postpulse intervals. The route of intracellular transport of proteins in the resting glands is similar to that of the active ones, but the rate of synthesis and transport is much slower. The present results and earlier data, thus, show that the increase in the rate of secretion after initiation of a new venom regeneration cycle is the result of accelerated rates of both synthesis and transport.     

A morphometrical study of the exocrine pancreatic cell in fasted and fed frogs

The influence of feeding on the ultrastruct of the frog exocrine pancreatic cell was studied by morphometrical procedures. Volume and surface of various cell structures were measured and expressed per unit cell volume. The average cellular size was not influenced by feeding. Though protein synthesis changes 5-to 10-fold (van Venrooij, W. J., and C. Poort. 1971. Biochim. Biophys. Acta. 247:468-470), no significant differences were observed in the amount of membrane that constitutes the rough endoplasmic reticulum (RER) and that represented the major part of total cellular membranes. The appearance of the RER changed. When fasted, most of its membrane was arranged in stacks of tightly packed, narrow cisternae. Within 4 h after feeding, these cisternae were separated and irregularly dilated, and ribosomes became ordered in typical rosettes on their surface. The total volume of the Golgi system increased twofold after feeding. The vesicular and tubular elements at the Golgi periphery did not change, but the volumes of the Golgi cisternae and the condensing vacuoles increased 2.5- and 6-fold, respectively. The increased in the amount of membrane present in these structures was only 1.6- and 3.5-fold, which reflects the more distended appearance of the cisternae and the rounded shape of the condensing vacuoles after feeding. Feeding halved the number of secretory granules per cell, and signs of exocytosis were more common than in fasted animals. These findings suggest that, in the frog pancreatic cell, fluctuations in the production of secretory proteins are not accompanied by an important breakdown and renewal of cellular membranes. This may favor a rapid and strong response of the cell to feeding.     

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta)

Summary Frog pancreatic tissue was pulse-labelled in vitro with ³H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions.Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae.Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues.The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30° C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.     

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well- developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post- Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.     

Temperature and energy dependence of secretory protein transport in the exocrine pancreas.

Pancreatic lobules pulse-labeled with [3H]leucine have been incubated at temperatures between 0 and 37 degrees C in the presence or absence of ongoing oxidative phosphorylation. Subcellular fractionation methods and electron microscopic autoradiography have been used to monitor the progress of intracellular transport of newly synthesized secretory proteins. Over the period studied, exit from the rough endoplasmic reticulum (RER) occurs only at greater than 10 degrees C while traversal of the Golgi complex and entry into condensing vacuoles requires greater than 22 degrees C. Both steps of transport require ongoing ATP production. Incubation at 10 or 20 degrees C does not diminish ATP levels, relative to 37 degrees C controls. Remarkable and unprecedented alterations of the ultrastructure of transitional elements of the RER accompany the arrest of exit from the RER: at 10 degrees C transitional elements are much more numerous and longer than in controls; in the absence of ATP production they are essentially absent. These observations are interpreted in terms of a cyclic model of RER-to-Golgi vesicular traffic. Inhibition of ATP production also causes an increase in the rigid cisternae and coated elements in the distal Golgi area.     

Glycoprotein transport in the surface mucous cells of the rat stomach

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.     

Evidence for a satellite secretory pathway in neuronal dendritic spines

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.     

RADIOAUTOGRAPHIC ANALYSIS OF THE SECRETORY PROCESS IN THE PAROTID ACINAR CELL OF THE RABBIT

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-³H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.     

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla

Summary The synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-³H and L-3,4-dihydroxy [ring 2,5,6-³H] phenylalanine (L-dopa³H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine ³H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine ³H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa ³H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine ³H or L-dopa ³H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals. Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group of Hypertension of the Clinical Research Institute of Montreal and by the Canadian Heart Foundation     

Radioautographic analysis of the secretory pathway for glycoproteins in principal cells of the mouse epididymis exposed to [3H] fucose

The secretory process for glycoproteins in principal cells of the mouse caput epididymis was studied by electron microscope radioautography at intervals after exposure to [3H] fucose in vitro. The large Golgi apparatus showed very heavy labeling at the initial interval, followed by a steady decline in percent of grains and relative grain concentrations. Conversely, the epididymal lumen and the apical cell surface began low and increased in radioactivity at the 30-min interval. The extensive sparsely granulated endoplasmic reticulum showed modest increases in percent of grains and relative grain concentrations 30 min after administration of the percursor. Subdivision of the sparsely granulated reticulum into "intermediate" profiles (some ribosomes attached to the membranes) and "smooth" profiles (lacking ribosomes) showed that this increase was due to silver grains assigned to the smooth portions. After the initial interval, high relative grain concentrations were calculated for vesicles. The results indicate that glycosylation of epididymal secretory glycoproteins occurs in the Golgi apparatus, which is, therefore, not bypassed as its morphological features had suggested. The kinetics of the secretory process in the principal cells includes 15 to 30 min for synthesis of the polypeptide parts of secretory products and addition of sugars in the Golgi apparatus, and a similar time for subsequent release from the Golgi apparatus, transport to the apical end of the cell and discharge to the lumen. Ribosome-studded (intermediate) portions of the sparsely granulated endoplasmic reticulum are probably involved in synthesis of polypeptide parts of secretory products, while vesicles or smooth portions of the sparsely granulated reticulum may play a role in intracellular transport of glycoproteins.     

A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex

We have identified a vesicle fraction that contains alpha 1-antitrypsin and other human HepG2 hepatoma secretory proteins en route from the rough endoplasmic reticulum (RER) to the cis face of the Golgi complex. [35S]Methionine pulse-labeled cells were chased for various periods of time, and then a postnuclear supernatant fraction was resolved on a shallow sucrose-D2O gradient. This intermediate fraction has a density lighter than RER or Golgi vesicles. Most alpha 1-antitrypsin in this fraction (P1) bears N-linked oligosaccharides of composition similar to that of alpha 1-antitrypsin within the RER; mainly Man8GlcNac2 with lesser amounts of Man7GlcNac2 and Man9GlcNac2; this suggests that the protein has not yet reacted with alpha-mannosidase-I on the cis face of the Golgi complex. This light vesicle species is the first post-ER fraction to be filled by labeled alpha 1-antitrypsin after a short chase, and newly made secretory proteins enter this compartment in proportion to their rate of exit from the RER and their rate of secretion from the cells: alpha 1-antitrypsin and albumin faster than preC3 and alpha 1-antichymotrypsin, faster, in turn, then transferrin. Deoxynojirimycin, a drug that blocks removal of glucose residues from alpha 1-antitrypsin in the RER and blocks its intracellular maturation, also blocks its appearance in this intermediate compartment. Upon further chase of the cells, we detect sequential maturation of alpha 1- antitrypsin to two other intracellular forms: first, P2, a form that has the same gel mobility as P1 but that bears an endoglycosidase H- resistant oligosaccharide and is found in a compartment--probably the medial Golgi complex--of density higher than that of the intermediate that contains P1; and second, the mature sialylated form of alpha 1- antitrypsin.     

Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1

To study the in vivo processing and secretion of Apolipoprotein A-I (Apo A-I), young chickens were administered individual L-[3H]amino acids intravenously and the time of intracellular transport of nascent Apo A-I from rough endoplasmic reticulum (RER) to the Golgi apparatus was measured. Within 3 to 9 min there was maximal incorporation of radioactivity into Apo A-I in both the RER and the Golgi cell fractions. By contrast, the majority of radioactive albumin was also present in the RER by 3 to 9 min, but did not reach peak amounts in the Golgi fraction until 9 to 25 min. Both radioactive Apo A-I and albumin appeared in the blood at about the same time (between 20 and 30 min). NH2-terminal amino acid sequence analysis of nascent intracellular Apo A-I showed that it contains a pro-hexapeptide extension identical to that of human Apo A-I. After 30 min of administration of radioactive amino acids radioactive Apo A-I was isolated by immunoprecipitation from the liver and serum. NH2-terminal sequence analysis of 20 amino acids indicated that chicken liver contained an equal mixture of nascent pro-Apo A-I and fully processed Apo A-I, whereas the serum only contained processed Apo A-I. Further studies showed that the RER only contained pro-Apo A-I, whereas a mixture of pro-Apo A-I and processed Apo A-I was found in the Golgi complex. These results indicate that, in chicken hepatocytes, there is a more rapid transport of Apo A-I than of albumin from the RER to the Golgi cell fractions, and that Apo A-I remains in the Golgi apparatus for a longer period of time before it is secreted into the blood. In addition these studies show that the in vivo proteolytic processing of chicken pro-Apo A-I to Apo A-I occurs in the Golgi cell fractions.     

Glycosylation process in gonadotrophs: a quantitative electron microscope autoradiographic study with labeled glucosamine

Incorporation of [3H]glucosamine into dispersed anterior pituitary cells was studied by electron microscope autoradiography. Gonadotrophs were examined to determine the intracellular route and kinetic patterns of glycosylation. Studies were performed with cells from; (a) normal adult male rats; (b) rats orchidectomized 3 wk earlier; and (c) orchidectomized rats treated with tunicamycin. Our results show that incorporation of [3H]glucosamine first occurs in the rough endoplasmic reticulum (RER), then proceeds in the Golgi elements (where peripheral carbohydrates are attached). Treatment with tunicamycin results in a decrease in labeling of these 2 organelles. Comparison of the kinetic patterns in normal and castrated male rats shows that the accumulation of labeled glycosylated proteins in granules reaches a plateau within 2 hr post-pulse in normal rats, and rises during a 6-hr chase in castrated rats. However, because of the necessity for a rather long 15 min pulse, we cannot exclude the possibility that incorporation of glucosamine during the pulse may occur concomitantly in the RER and the Golgi saccules, to be followed by rapid transfer to the secretory granules.     

Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway

The transport of the apolipoprotein (apo) constituents of hepatic very low density lipoprotein (VLDL) through the secretory pathway was investigated with estrogen-induced chick hepatocytes in primary culture. Cell monolayers were pulse-labeled with [3H]leucine and, after differing periods of chase with unlabeled leucine, were subjected to subcellular fractionation for 3H-apoprotein analysis. The first-order rate constants for transit of apoB, apoA-I, and apoII through the endoplasmic reticulum (ER) and Golgi were estimated using a three-compartment (ER, Golgi, and extracellular medium) kinetic analysis. The results indicate that apoB resides in the ER (t1/2 = 26 min) for a shorter period of time than in the Golgi (t1/2 = 43 min). For apoII, the t1/2 for transport through the ER and Golgi are 43 and 49 min, respectively. ApoA-I transits the ER at a rate (t1/2 = 6 min) much faster than apoB, apoII, and virtually all other secretory proteins. Upon reaching the Golgi, the rate of movement of apoA-I is markedly reduced (t1/2 = 28 min). Thus, in contrast to current models of protein secretion, the rate-limiting step in the secretion of VLDL apoproteins from the cell is transport through the Golgi, not the ER. Examination of the steady-state distribution of the apoproteins in the ER and Golgi support this conclusion. To characterize the intracellular transport process further, the distribution of apoproteins between the lumenal contents of the ER and Golgi and the membranes which delineate these compartments was determined after steady-state labeling with [3H]leucine. Approximately 50% of the apoB in the ER and in a dense, early Golgi fraction was membrane-associated, whereas in a less dense or late Golgi compartment, only 20% was bound to membranes. ApoII was also associated with the membranes of the ER and Golgi to a significant extent. In contrast, apoA-I was primarily localized lumenally throughout the secretory pathway. The occurrence of membrane-associated apoproteins in the Golgi, coupled with their slow rate of transit through this compartment suggests a major role for the Golgi in the assembly of the constituents of VLDL, and suggests that interaction of apoproteins (apoB) with the membranes of the Golgi is required for the maturation of VLDL.     

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.     

Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.     

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0- 10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Multi‐step down‐regulation of the secretory pathway in mitosis: A fresh perspective on protein trafficking

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non‐dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down‐regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin‐dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.