A three-dimensional study of organelle interrelationships in regenerating rat liver. 4. Multivesicular bodies.

Double-membraned plasma-membrane-loops are formed from the plasma membrane and released into the cytoplasm. The vesiculation of mainly their inner membrane may transform them into MVB. The so-formed internal vesicles contain cytosol. In many MVB the surface of all their internal vesicles to gether corresponds well with the surface of their bordering membrane. This correlation may be the result of a dynamic equilibrium whereby degradation (and partial recyclage) of internal vesicles is compensated for by the formation of new internal vesicles. When this equilibrium becomes disturbed, MVB may show much less internal vesicles. Such 'depleted' MVB are often producing peribiliary vesicles and may themselves transform into lacunate emptying into the bile canaliculi.     

体外冲击波碎石术对兔肝酶活性的影响

采用超微组织化学方法,观察了体外冲击波碎石术(ESWL)对家免肝脏酶活性的影响。实施 ESWL 后,肝细胞的琥珀酸脱氢酶(SDH)和毛细胆管壁上的碱性磷酸酶(ALP)、焦磷酸硫胺素酶(TPPase)反应活性减弱或消失。TPPase 从损伤的肝细胞高尔基体分泌面扁囊、溶酶体样小泡和毛细胆管内溢出,并伴有肝细胞面的质膜上出现了 TPPase 反应产物和形成膜包内凹小泡。结果提示 ESWL 可对肝细胞及毛细胆管的功能和结构有损伤作用。     

Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells

We investigated the uptake of surfactant by isolated alveolar type II cells by using native pulmonary surfactant complexed with colloidal gold. Internalization to lamellar bodies (LB) occurred via vesicles (mainly coated) and the endosomal system. The highest labeling density was found in the endosomal system: vacuoles and the electron-lucent multivesicular bodies (MVB), which were labeled within 10 min. The labeling of electron-dense MVB (D-MVB) and LB was time dependent, reaching a plateau after 120 min, at which time approximately 30% and 70% of the LB and D-MVB were labeled, respectively. Internalization of surfactant-gold was inhibited by the addition of native surfactant or treatment of the gold complex with antibody against surfactant apoproteins. The internalization pathway of lectin from Macula pomifera (MPA) complexed with gold was compared to that of surfactant. Both pathways were found to be similar, except that mainly smooth vesicles rather than coated ones were involved in the process of MPA-G internalization. The partial labeling of the LB, the possible routing to lysosomes, and the endosomes as junction between the biosynthetic and endocytic pathways are discussed.     

Microtubule disruption interferes with the structural and functional integrity of the apical pole in primary cultures of rat hepatocytes.

The effect of microtubule disruption on the development and maintenance of cell polarity was studied in rat hepatocytes cultured as primary monolayers in the presence of colchicine or nocodazole. Addition of colchicine immediately after plating did not inhibit the generation of bile canaliculi (the apical pole) after 1 day of culture, as judged by electron microscopic examination, and did not allow penetration of Ruthenium Red through the tight junctions. However, the bile canaliculi developed in the presence of colchicine or nocodazole were not fully normal since they were not able to concentrate fluorescein diacetate in their lumina, and did not enrich with proteins of the apical plasma membrane domain, as control cells did. When the drugs were added after 1 or 2 days of culture, the new bile canaliculi appeared to be unaffected when examined by electron microscopy, but many of them did not concentrate fluorescein and were not enriched with apical membrane proteins within 4 to 24 h after drug addition. Whenever the drugs were added, the proteins that would normally concentrate on the membrane of the bile canaliculi accumulated intracellularly in endocytic vesicles after 2 to 4 h of drug treatment, and in vacuoles resembling lysosomes when the drugs were maintained for 24 h or more. These results show that microtubule disruption does not inhibit the structural reconstitution of bile canaliculi, but impairs their normal function and the transport of proteins of the apical plasma membrane domain.     

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.     

Intracellular lumen in cultured hepatocytes of rats. Study of an original cytoplasmic infrastructure

A few hours after plating, isolated rat hepatocytes reassociate into clusters and differentiate intercellular cavities bordered by junctional complexes. These structures show a great resemblance to bile canaliculi seen in vivo. Intracellular lumens surrounded by microvilli are observed in the cytoplasm of some cultured hepatocytes. The formation of these structures, which contain an osmiophilic substance, probably results from modifications in the functioning of the secretory apparatus. It can be speculated that these intracellular lumens may contribute to the formation of new canaliculi differentiated between reassociated cells.     

Observations on the ultrastructure of the carp liver (Cyprinus carpio L.)

The paper presents information about the fine structure of the sinusoids, the space of Dissé, the development of the bile canaliculi and some compartments of the hepatocytes in the liver of the carp. The sinusoids are covered completely by flat endothel cells. The plasma of these cells contains numerous vesicles. The endothelial cells possess plasmatic processes, which extend into the space of Dissé. The fine structure of the space of Dissé corresponds to that of mammals, the existence of fibrebundles included. The bile canaliculi don't develop intracellularly as described by other authors. They run intercellularly as in mammals, but they form diverticles which reach into the plasma sideways. The rough ER was found in two types. Outwardly the liver is limited by a layer of connective tissue existing in two different layers.     

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum

The absorptive cell of the suckling rat ileum is specialized for the uptake and digestion of milk macromolecules from the intestinal lumen. The apical cytoplasm contains an extensive tubulocisternal system, a variety of vesicles and multivesicular bodies (MVB), and a giant phagolysosomal vacuole where digestion is completed. To determine if sorting of membrane-bound and fluid-phase macromolecules occurs in this elaborate endocytic system, we infused adsorptive and soluble tracers into ligated intestinal loops in vivo and examined their fates. Lysosomal compartments were identified by acid phosphatase histochemistry. Native ferritin and two ferritin-lectin conjugates that do not bind to ileal membranes (Con A, UEAI) served as soluble tracers. Horseradish peroxidase binds to ileal membranes and thus was not useful as a fluid-phase tracer in this system. Cationized ferritin and a lectin that binds to terminal B-D-galactosyl sites on ileal membranes (Ricinus communis agglutinin [RCAI]-ferritin) were used as tracer ligands. All tracers entered the wide apical invaginations of the luminal cell surface and were transported intracellularly. Membrane-bound tracers were found in coated pits and vesicles, and throughout the tubulocisternal system (where cationized ferritin is released from the membrane) and later, in large clear vesicles and MVB. In contrast, fluid-phase tracers appeared within 5 min in vesicles of various sizes and were not transported through the tubulocisternae, rather, they were concentrated in a separate population of vesicles of increasing size that contained amorphous dense material. Large clear vesicles, large dense vesicles, and MVB eventually fused with the giant supranuclear vacuole. Acid phosphatase activity was present in MVB and in the giant vacuole but was not present in most large vesicles or in the tubulocisternae. These results demonstrate that membrane-bound and soluble protein are transported to a common lysosomal destination via separate intracellular routes involving several distinct prelysosomal compartments.     

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

Multivesicular bodies isolated from rat hepatocytes. Cytochemical evidence for transformation into secondary lysosomes by fusion with primary lysosomes

Plasma lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ. Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70-120 nm in diameter, which stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive arc or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, "intact" Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatocytes in situ. These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartments.     

Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway

The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.     

Ultrastructural studies of a vesicle system associated with endoplasmic reticulum in exo-erythrocytic forms of Plasmodium berghei

Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exoerythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. IV. Disintegration of bile canaliculi and disturbance of tight junction formation caused by vinblastine

Treatment of cultured hepatocytes with vinblastine or colchicine caused striking perturbations of the structural organization of the biliary pole and of the junctional complexes. During the early hours of cultivation the reassociation of the bile canaliculi was impaired by the drug, whereas at later times in culture preformed canaliculi were disintegrated to small vesicular remnants lacking microvilli. Vinblastine did not impair tight junction formation per se. However, under the influence of the drug, tight junctional strands associated in an atypic manner perpendicular to the upper surface of the hepatocytes, whereas those strands lining the canaliculi were decomposed to smaller entities and dislocated within the lateral membrane. Concomitantly to the structural disintegration of the biliary pole an accumulation of vesicles in the pericanalicular cytoplasm was noted. As indicated by numerous filipin-induced lesions, they were characterized by a high content of membrane cholesterol. The apical pole and the contiguous membrane on the other hand contained only very few filipin-cholesterol lesions. These findings suggest that antimicrotubular drugs impair the fusion of pericanalicular vesicles with the luminal membranes of the canaliculi, thus interrupting the delivery of membraneous material to the apical pole. In addition, microtubules seem to play an important role in the coordinated development and the structural fixation of the biliary pole of cultured hepatocytes.     

Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies

Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.     

Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation of receptors

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for D-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, glactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Disé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2-5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Biochimica et biophysica acta,vol. 646 (1981)

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for d-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, galactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Dissé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2–5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Receptor-mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Evidence for intracellular ligand sorting and identification of distinct endosomal compartments

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor-mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30-45 min. GalBSA was essentially digested in lysosomes. By electron microscopy using ligand-peroxidase conjugates, both ligands were internalized through coated pits/coated vesicles into similar electron-lucent vesicles and tubules. Subsequently, pIgA remained mostly associated with small vesicles clustering around and fusing with bile canaliculi, while GalBSA was predominantly found in large, heterogeneous endocytic structures and in lysosomes. By subcellular fractionation, they were associated at 3 min after injection with structures that similarly sedimented in the P fraction (250 000 - 3 X 10(6) X g X min) and equilibrated at densities of about 1.13 g/ml in sucrose gradients. At 10 min and 20 min, pIgA distribution remained mostly in the P fraction at the same equilibrium density. A minor component of the pIgA distribution was found at the density of lysosomes, but contrary to lysosomal enzymes, its distribution was not affected by Triton WR 1339. In contrast to pIgA, GalBSA was progressively recovered in the L fraction (33 000 - 250 000 X g X min) with organelles equilibrating around 1.11 g/ml, and, by 20-45 min, was found in the ML fraction (10 000 - 250 000 X g X min), around 1.20 g/ml, i.e. in lysosomes. Chloroquine did not reduce the efficiency but delayed the secretion of pIgA into bile. Similarly, it did not affect the uptake of GalBSA but apparently delayed GalBSA transfer along successive populations of host organelles. The low density, GalBSA-containing structures were devoid of proteolytic activity. Anti-secretory components IgG and F(ab')2 were selectively excreted into bile, partially or totally as compounds of lower molecular mass. These antibody fragments probably result from a disulfide reduction activity along the pIgA pathway. In conclusion, our data (a) strongly suggest that pIgA and GalBSA are sorted between 3 min and 10 min after injection in non-lysosomal acidic organelles, (b) identify two successive and physically distinct endosomal populations containing GalBSA, and (c) provide the first evidence for a disulfide reduction activity along the transcytotic pathway of rat hepatocytes.     

Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.     

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.     

Ultrastructural Studies of a Vesicle System Associated with Endoplasmic Reticulum in Exo-Erythrocytic Forms of Plasmodium berghei1

ABSTRACT. Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.