The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

Secretion of collagen by insects: Autoradiographic study of l-proline 3H-5 incorporation by the firebrat Thermobia domestica

The biosynthesis of collagenous endoskeletal endosterna by fibroblasts was studied by electron microscope autoradiography after tritium-labelled proline administration in the firebrat Thermobia domestica at various time intervals. Autoradiographs were quantitatively analyzed and the relative concentration of label was determined for various cellular compartments. The labelling in the rough endoplasmic reticulum, ground cytoplasm, Golgi apparatus and endosterna was compared during the secretion of radioactive products. The label, initially located in the rough endoplasmic reticulum was found in the Golgi apparatus before it accumulated in the endosternum. The involvement of the rough endoplasmic reticulum and Golgi apparatus in the transport of secreted collagen is discussed, comparing our results with current knowledge of collagenous secretion.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the ciliary epithelium of the eye: effects of microtubule-disrupting drugs

3H-fucose was injected intravenously or intravitreously into albino rats. After time intervals of 10, 40, and 50 min, 1, 1.5, and 4 hr, 1, 3, and 7 days, and 1, 2, and 4 weeks after injection, the animals were sacrificed by intracardiac perfusion with glutaraldehyde. Samples of the ciliary body were prepared for light and electron microscope radioautography. Light microscope autoradiographs showed that the cells of both the inner and outer layers of ciliary epithelium actively incorporated 3H-fucose label in a reaction that peaked in intensity at 4 hr after injection, and then progressively declined. Electron microscope radioautographs revealed that, at early time intervals, most of the label was localized to the Golgi apparatus. With time, the plasma membrane of both cell types became increasingly labeled, and accounted for 60-70% of the total silver grains at 4 hr after injection. Adjacent to the basal cell surface of the inner layer cells, the fibers of the zonula became increasingly labeled from 1.5 hr onwards, providing strong evidence that these cells secrete glycoproteins to the zonula. When vinblastine was administered 30 min before 3H-fucose injection, followed by sacrifice 1.5 hr later, a much larger proportion of label remained localized to the Golgi apparatus than in controls, and the plasma membrane and zonula were much less labeled. These results suggest that, as documented in other cell types, microtubules may play a role in the intracellular transport of membrane and secretory glycoproteins in these cells.     

ETUDE QUANTITATIVE PAR RADIOAUTOGRAPHIE AU MICROSCOPE ELECTRONIQUE DE L'UTILISATION DE LA DL-LEUCINE-3H PAR LES CELLULES DE L'HYPOPHYSE DU CANARD EN CULTURE ORGANOTYPIQE

The synthesis, intracellular transport, storing, and excretion of proteins by duck hypophyseal cells in organ culture were studied with tritiated DL-leucine and high resolution radioautography (pulse-labeling experiments). Quantitative study of the radioautographs allowed a determination of the relative proportions of cytoplasmic radioactivity located in each cellular compartment (ergastoplasm, Golgi apparatus, and protein granules) as well as the variations in these proportions as a function of time. The number of labeled protein granules as opposed to the total number of granules in the cell was also determined (RSg). These data were separately analyzed for the two types of cells present in the explants: prolactin cells and "MSH" cells. The synthetic process follows a course common to both cell types, each of which is distinguished by its particular modalities. The labeled proteins, synthesized within several minutes in the ergastoplasm, are concentrated in the Golgi zone within 30 min. They then migrate out of this area, the emptying of which is accomplished in about 4 hr. These proteins become equally distributed between the protein granules, on the one hand, and the cytoplasm ("sedentary" proteins), on the other. The RSg reaches its maximum when the Golgi zone is emptied, but this figure remains very low (3%). The RSg then decreases slowly (1% in 40 hr). It is concluded that hypophyseal cells are able to store protein in their granules and that their processes of synthesis and excretion are not continuous. The prolactin cells differ from the "MSH" cells in that they have a slower migration of newly synthesized proteins, and these proteins pass via the dilated ergastoplasmic cisterns in which they may possibly be stored.     

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)     

SYNTHESIS OF THE CARBOHYDRATE OF MUCUS IN THE GOLGI COMPLEX AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY OF GOBLET CELLS FROM RATS INJECTED WITH GLUCOSE-H3

It is known that colonic goblet cells utilize glucose to synthesize the carbohydrate portion of mucus glycoprotein. To determine the intracellular site of this synthesis, glucose-H³ was injected into 10-g rats. At 5, 20, 40 min, 1, 1½, and 4 hr after injection, segments of colon were fixed and prepared for electron microscope radioautography. By 5 min after injection, label had been incorporated into substances present in the flattened saccules of the Golgi complex. At 20 min, both Golgi saccules and nearby mucigen granules were labeled. By 40 min, mucigen granules carried almost all detectable radioactivity. Between 1 and 4 hr, these labeled granules migrated from the supranuclear region to the apical membrane; here, they were extruded singly, retaining their limiting membrane. The evidence indicates that the Golgi saccule is the site where complex carbohydrate is synthesized and is added to immigrant protein to form the complete glycoprotein of mucus. The Golgi saccule, distended by this material, becomes mucigen granules. It is roughly estimated that one saccule is released by each Golgi stack every 2 to 4 min: a conclusion implying continuous renewal of Golgi stacks. It appears that the Golgi synthesis, intracellular migration, and release of mucus glycoprotein occur continually throughout the life of the goblet cell.     

BETA GRANULE FORMATION IN ISOLATED ISLETS OF LANGERHANS : A Study By Electron Microscopic Radioautography

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.     

Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments

Recently we demonstrated that low-pH compartments can be visualized with the electron microscope using a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), which concentrates in acidic compartments and can be detected by immunocytochemistry with a monoclonal anti-dinitrophenol antibody. We now report that DAMP also accumulates in cisternae and vesicles associated with the trans face of the Golgi apparatus. DAMP rapidly leaves this compartment when cells are incubated with the ionophore monensin, which indicates that accumulation is due to the acidic pH in this compartment. Using indirect protein A-gold immunocytochemistry, we localized fibronectin, a major secretory protein in fibroblasts, to the trans Golgi vesicles that took up DAMP. Therefore, the trans cisternae of the Golgi apparatus and forming secretory vesicles have an acidic pH.     

Anti-horseradish peroxidase associated with Golgi complex of antibody-forming cells.

Observations are reported on the localization of anti-horseradish peroxidase in antibody-forming cells in popliteal lymph nodes following a single injection of antigen. Reaction product, marking the site of antibody, was observed not only in the ergastoplasm and cisternae of the Golgi complex, but also in globules associated with the Golgi complex.     

LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, STORAGE, AND SECRETION : I Electron Microscopic Radioautographic Study of Liver after Injection of Tritiated Palmitate or Glycerol in Fasted and Ethanol-Treated Rats

A time sequence study of intracellular movement of labeled lipid in the liver was carried out on fasted and ethanol-treated rats injected with either palmitate-³H or glycerol-³H by electron microscopic radioautography. The elimination of water-soluble lipid precursors during specimen preparation was checked and found to be complete. The labeled lipid product in the tissue was identified as mostly triglyceride. A dehydration procedure was adapted to minimize the loss of lipid during specimen preparation. At 2 min after injection, the earliest time interval studied, both precursors were found to have penetrated the liver cells, and the label was found over both rough and smooth elements of the endoplasmic reticulum, which is the site of glyceride esterification. From 5 min on, in fasted and especially in ethanol-treated rats, the label was seen also over lipid droplets 0.5–2.0 µ in diameter, which represent "storage lipid" (slowly turning over compartment). Mitochondria became labeled mostly at later time intervals after injection. From 10 min on, concentration of label was seen over the Golgi apparatus, containing small osmiophilic particles. Association of label with groups of particles in smooth-surfaced vesicles and vacuoles in and near the Golgi apparatus and in the vicinity of the sinusoidal border was seen, both after palmitate-³H and glycerol-³H. It is proposed that these particles represent lipoproteins which are formed in the endoplasmic reticulum, "processed" in the Golgi apparatus, and transported in vacuoles to the sinusoid surface to be discharged into the circulation.     

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.     

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.     

The intracellular movement and cycling of ricin

The binding, internalization and recycling of the plant toxin ricin, was studied using electron microscopy and biochemical techniques. For the electron microscope study, ricin was visualized using a gold-labeled second antibody, in the cells of the EJ human bladder carcinoma line growing in monolayer culture. The labeled antibody/toxin complex was found to enter the cell in coated pits and to accumulate in endosomes and to a lesser extent in vesicles associated with the Golgi system. The complex recycled to the cell surface partly in uncoated vesicles, but largely in multivesicular bodies which appeared to exocytose their contents to the extracellular space. Twenty hours after the initial contact with ricin as much as 50% of the cellular label was found on the cell surface mainly associated with shed vesicles. When cells were treated with unlabeled ricin holotoxin and then after 20 h stained post-fixation, ricin molecules, partly associated with vesicles, were present on the cell surface. Biochemical studies showed that ricin was internalized by cells and then released in an intact form to the extracellular space. It was found that less than 10% of the released material had been degraded during its passage through the cells, which is in accord with the low level of label found in the lysosomal system during the morphological study.     

Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella.

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.     

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.     

An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage

Summary The synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.Supported by grants CA 05196-04S1 and GM-K3-13, 979-C1-A from the United States Public Health Service.The results reported in this paper were presented at the second annual meeting of the American Society for Cell Biology, November 6, 1962.     

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.