Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.     

Trophozoites of Giardia lamblia may have a Golgi-like structure

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgi complex. Using C(6)-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of the perinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thin sections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling the Golgi complex.     

Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.     

Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Cell wall structure and deposition in Glaucocystis

Events leading to cell wall formation in the ellipsoidal unicellular alga Glaucocystis are described. The wall is deposited in three phases: (a) a thin nonfibrillar layer, (b) cellulosic microfibrils arranged in helically crossed polylamellate fashion, and (c) matrix substances. At poles of cells, microfibrils do not terminate but pass around three equilaterally arranged points, resulting in microfibril continuity between the twelve helically wound wall layers. These findings were demonstrated in walls of both mother cells and freeze-fractured growing cells, and models of the wall structure are presented. Cellular extension results in spreading apart, and in rupture, of microfibrils. On freeze-fractured plasma membranes, there were 35 nm X 550 nm structures associated with the ends of microfibrils. These are interpreted as representing microfibril-synthesizing centers (terminal complexes) in transit upon the membrane. These terminal complexes are localized in a zone, or zones. The plasma membrane is subtended by flattened sacs, termed shields, which become cross-linked to the plasma membrane after completion of wall deposition. During wall deposition, microtubules lie beneath the shields, and polarized filaments lie between shields and plasma membrane. The significance of these findings in relation to understanding the process of cellulose deposition is discussed, and comparisons are made with the alga Oocystis.     

The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia

The connecting cilium of rat retinal rods was studied by freeze-fracture and thin-sectioning techniques. Transverse strands of intramembranous particles could be observed on fracture face B on the ciliary plasma membrane. The strands were essentially similar to those found at the transitional zone of motile cilia ("ciliary necklace"). The larger number of intramembranous particles obscured the pattern on fracture face A of the membrane. On longitudinal sections of the cilia, beads showing a periodicity similar to the necklace strands were observed. Each bead consisted of two structures apposed to both sides of the plasma membrane. Transverse sections of the cilia revealed radial Y-shaped structures that connected each ciliary doublet with the plasma membrane. Axial tubules, central sheath, radial spokes and dynein arms were missing in the connecting cilium. Comparing the fine structure of the retinal cilia with that of motile cilia it becomes evident that the connecting cilium is analogous in structure with the transitional zone of motile cilia. The present observations suggest that periodic membrane beads along the plasma membrane on thin sections correspond to strands of necklace particles as observed on freeze-fractured membranes. The arrangement of the particles in transverse strands is probably ensured by the radial connecting structures.     

Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.     

CILIARY MEMBRANE DIFFERENTIATIONS IN TETRAHYMENA PYRIFORMIS : Tetrahymena Has Four Types of Cilia

We have examined thin sections and replicas of freeze-fractured cilia of Tetrahymena pyriformis. The ciliary necklace located at the base of all freeze-fractured oral and somatic cilia has been studied in thin sections. Since electron-dense linkers have been found to connect both microtubule doublets and triplets to the ciliary membrane at the level of the necklace, the linkers and the associated necklace seem to be related to the transition region between the doublets and triplets of a cilium. Plaque structures, consisting of small rectangular patches of particles located distal to the ciliary necklace, are found in strain GL, but are absent in other strains examined in this study. In freeze-cleaved material, additional structural differentiations are observed in the distal region of the ciliary membranes of somatic and oral cilia. Somatic cilia contain many randomly distributed particles within their membrane. Oral cilia can be divided into three categories on the basis of the morphology of their freeze-fractured membranes: (a) undifferentiated cilia with very few randomly distributed particles: (b) cilia with particles arranged in parallel longitudinal rows spaced at intervals of 810–1080 Å that are located on one side of the cilium; and (c) cilia with patches of particles arranged in short rows oriented obliquely to the main axis of the cilium. The latter particles, found on one side of the cilium, seem to serve as attachment sites for bristles 375–750 Å long and 100 Å wide which extend into the surrounding medium. The particles with bristles are located at the tips of cilia in the outermost membranelle and may be used to detect food particles and/or to modify currents in the oral region so that food particles are propelled more efficiently into the buccal cavity. Examination of thin-sectioned material indicates that the particles in oral cilia which form the longitudinal rows could be linked to microtubule doublets. Linkage between microtubule doublets and adjacent membrane areas on one side of the cilium could modify the form of ciliary beat by restricting the sliding of the microtubules. It is suggested that membrane-microtubule interactions may form the basis for the various forms of ciliary beat observed in different organisms.     

Intracytoplasmic membrane formation and increased oxidation of glycerol growth of Gluconobacter oxydans.

Gluconobacter oxydans is well known for the limited oxidation of compounds and rapid excretion of industrially important oxidation products. The dehydrogenases responsible for these oxidations are reportedly bound to the cell's plasma membrane. This report demonstrates that fully viable G. oxydans differentiates at the end of exponential growth by forming dense regions at the end of each cell observed with the light microscope. When these cells were thin sectioned, their polar regions contained accumulations of intracytoplasmic membranes and ribosomes not found in undifferentiated exponentially growing cells. Both freeze-fracture-etched whole cells and thin sections through broken-cell envelopes of differentiated cells demonstrate that intracytoplasmic membranes occur as a polar accumulation of vesicles that are attached to the plasma membrane. When cells were tested for the activity of the plasma membrane-associated glycerol dehydrogenase, those containing intracytoplasmic membranes were 100% more active than cells lacking these membranes. These results suggest that intracytoplasmic membranes are formed by continued plasma membrane synthesis at the end of active cell division.     

Ultrastructural observations on synaptic ribbons in the pineal organ of the goldfish

Summary Synaptic ribbons in the pineal organ of the goldfish were examined electron microscopically with particular attention to their topography. These structures were formed of parallel membranes, which were poorly preserved with OsO₄ fixation and could be extracted from thin sections with pronase indicating their proteinaceous nature. Synaptic ribbons were closely apposed to the plasma membrane bordering dendrites of ganglion cells, but were also related to processes of both photoreceptor and supportive cells. Their close proximity to invaginations of the plasma membrane and portions of the endoplasmic reticulum suggest that they are involved in the turnover of cytoplasmic membranes. Tubular and spherical organelles of unknown function are also described.     

Structure of plasma membrane in radicles from cotton seeds

Summary Cortical cells in the apical region of excised radicles from cotton (Gossypium hirsutum L. var. M-8) seeds of differing moisture content (6, 12, or 16%) were examined in thin sections of vapor-fixed tissue and in freeze-etched replicas of unfixed tissue with transmission electron microscopy (TEM). The structure of the plasma membrane, as revealed in chemically-fixed tissue, had the tripartite feature characteristic of unit membranes. Regions of the plasma membrane appearing as discontinuities or breaks were shown by stereo tilting to represent an optical artifact caused by image superposition of curved regions of the plasma membrane contained within the thickness of a thin section. In freeze-etched preparations two complementary-type images of the plasma membrane were consistently observed. The asymmetric distribution of membrane-associated particles (MAPS) in the P and E fracture faces suggests that the structure of plasma membranes in cells of dry seeds resembles that of plasma membranes in germinating seeds. The physiological significance of these findings is that leaching of ions and small molecules during seed imbibition does not appear to involve passage through disorganized membranes in the radicle.     

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.     

Freeze-fracture of sperm of Plumbago zeylanica L. in pollen and in vitro

 Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm. Received: 14 January 1997 / Revision accepted: 6 June 1997     

A freeze-fracture study of the tegumental membrane of Schistosoma mansoni (Platyhelminthes:Trematoda).

Two fracture faces in each half of the freeze-fractured tegumental membrane of adult Schistosoma mansoni indicate the presence of two trilaminate membranes. This result is compatible with the heptalaminate appearance of the tegumental membrane in ultrathin sections. Intramembranous particles are located mainly in the outermost leaflet of the outer membrane and in the cytoplasmic leaflet of the inner membrane. The tegumental membrane of the cercaria (infective larva) has a single fracture plane, which conforms with its trilaminate appearance in sections. Intramembranous particles are extremely numerous and are almost all located in the cytoplasmic leaflet.     

Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol- digitonin structural complexes; (b) distinguishes cholesterol-rich and - poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.     

MEMBRANE SYNTHESIS IN BACILLUS SUBTILIS : III. The Morphological Localization of the Sites of Membrane Synthesis

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.     

EFFECTS OF PHOSPHOTUNGSTATE NEGATIVE STAINING ON THE MORPHOLOGY OF THE ISOLATED GOLGI APPARATUS

Isolated Golgi complexes can be recognized in phosphotungstate (PTA) negative stain as stacks of membranous plates surrounded by a complex anastomosing network of tubules and vesicles. The extent of this tubular network is, however, much greater than can be observed in thin sections of whole cells. To determine which of the steps leading to the final negatively stained image may produce the observed changes, we have monitored each of the steps by other electron microscope and biochemical methods. The first damage to the membranes seems to occur during the initial isolation procedure as judged by the appearance of smooth patches on the freeze-fractured membrane faces that are normally covered with particles. Subsequent suspension of the Golgi fraction in water, to dilute the sucrose for negative staining, leads to the disappearnce of the stacking, to some tubulation and some vesiculation of the membranes as judged by thin section and freeze-cleave microscopy. The latter technique also reveals an increase in smooth-cleaving membrane faces. Application of the negative stain to the water-washed Golgi fraction, finally, produces extensive tubular arrays and a simultaneous decrease in the remaining large membranous vesicles. The freeze-cleaved tubular membranes appear essentially smooth except for small patches of aggregated particles. Parallel gel electrophoresis studies of the membranes and of the water and negative stain wash extracts indicate that protein extraction is involved in these morphological changes. PTA seems to be a particularly effective solvent for certain membrane proteins that are not removed by the water wash. These observations suggest that removal of membrane proteins alters structural restraints on the membrane lipids so that they behave semiautonomously like myelinics and form new artificial structures. This does not eliminate the possibility, however, that some tubules also exist in the Golgi apparatus in vivo.     

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.     

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.