Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Endocytosis in nonciliated epithelial cells of the ductuli efferentes in the rat

The nonciliated cells lining the ductuli efferentes presented three distinct cytoplasmic regions. The apical region contained, in addition to cisternae of endoplasmic reticulum and mitochondria, two distinct membranous elements. The tubulovesicular system consisted of dilated tubules connected to the apical plasma membrane and subjacent distended vesicular profiles. The apical tubules, not connected to the cell surface, consisted of numerous densely stained tubules of small size which contain a compact, finely granulated material. The supranuclear region, in addition to a Golgi apparatus and ER cisternae, contained dilated vacuoles, pale and dense multivesicular bodies, as well as numerous dense granules identified cytochemically as lysosomes. The basal region contained the nucleus and many lipid droplets. The endocytic activity of these cells was investigated using cationic ferritin (CF) and concanavalin-A-ferritin (Con-A-ferritin) as markers of adsorptive endocytosis; and native ferritin (NF), concanavalin-A-ferritin in the presence of alpha-methyl mannoside, and horseradish peroxidase or albumin bound to colloidal gold for demonstrating fluid-phase endocytosis. These tracers were injected separately into the rete testis, and animals were sacrificed at various time intervals after injection. At 1 min, CF or Con-A-ferritin were seen bound to the apical plasma membrane, to the membrane of microvilli, and to the membrane delimiting elements of the tubulovesicular system. Between 2 and 5 min, these tracers accumulated in the densely stained apical tubules and at 15 min in the dilated vacuoles. Between 30 min and 1 hr, the tracers appeared in multivesicular bodies of progressively increasing density, whereas at 2 hr and later time intervals, many dense lysosomal elements became labeled. The tracers for fluid-phase endocytosis showed a distribution similar to that for CF or Con-A-ferritin except that they did not bind to the apical plasma membrane, microvilli, or membrane delimiting the tubulovesicular system. At no time interval were any of the tracers observed in the abluminal spaces. Thus, the nonciliated epithelial cells of the ductuli efferentes are actively involved in fluid-phase and adsorptive endocytosis, both of which result in the sequestration of endocytosed material within the lysosomal apparatus of the cell.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Vesicular transport of cationized ferritin by the epithelium of the rat choroid plexus

We have studied the transport of ferritin that was internalized by coated micropinocytic vesicles at the apical surface of the choroid plexus epithelium in situ. After ventriculocisternal perfusion of native ferritin (NF) or cationized ferritin (CF), three routes followed by the tracers are revealed: (a) to lysosomes, (b) to cisternal compartments, and (c) to the basolateral cell surface. (a) NF is micropinocytosed to a very limited degree and appears in a few lysosomal elements whereas CF is taken up in large amounts and can be followed, via endocytic vacuoles and light multivesicular bodies, to dark multivesicular bodies and dense bodies. (b) Occasionally, CF particles are found in cisterns that may represent GERL or trans-Golgi elements, whereas stacked Golgi cisterns never contain CF. (c) Transepithelial vesicular transport of CF is distinctly revealed. The intercellular spaces of the epithelium, below the apical tight junctions, contain numerous clusters of CF particles, often associated with surface-connected, coated vesicles. Vesicles in the process of exocytosis of CF are also present at the basal epithelial surface, whereas connective tissue elements below the epithelium are unlabeled. Our conclusion is that fluid and solutes removed from the cerebrospinal fluid by endocytosis either become sequestered in the lysosomal apparatus of the choroidal epithelium or are transported to the basolateral surface. However, our results do not indicate any significant recycling via Golgi complexes of internalized apical cell membrane.     

Internalization of cationized ferritin by isolated pancreatic acinar cells

The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.     

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.     

Endocytosis in secretory cells

Membranes of secretion granules inserted during exocytosis into the luminal plasma membranes of glandular cells are retrieved by endocytosis as revealed by electron dense tracers applied selectively to the apical cell surfaces. Two major pathways that endocytic vesicles may take are described: (1) a direct route to the Golgi complex (e.g. in parotid and exocrine pancreas) with later appearance of the tracer in the periphery of mature secretion granules; (2) an indirect route with lysosomes as a first station and the subsequent appearance of tracer in stacked Golgi cisternae. It is presumed that some of the retrieved membrane follows the same pathways and is reutilized in the secretory cycle.     

The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Endocytosis of native and cationized ferritin by intralobular duct cells of the rat parotid gland

Summary The ability of the intralobular ducts of the rat parotid gland to take up protein from the lumen was examined after retrograde infusion of native and cationized ferritin. At high concentrations (3–10 mg/ml), cells of both intercalated- and striated ducts avidly internalized the tracers. No differences were noted in the mode of uptake or fate of native or cationized ferritin. Large, apical ferritin-containing vacuoles up to 5 m in size were present in cells of the intercalated ducts after infusion for 15 min. Small, smooth-surfaced spherical or flattened vesicles and tubules containing ferritin were also observed, often in association with the large vacuoles. Ferritin uptake increased with increasing infusion time, up to 1 h. Uptake by the striated ducts was less consistent than by the intercalated ducts, and occurred mainly in small vesicles and tubules. Secondary lysosomes became labeled with ferritin in both cell types. Ferritin was not observed in the Golgi saccules, nor was it discharged from the cells at the basolateral surfaces. At low concentrations (0.3–1 mg/ml), uptake was reduced, especially by cells of intercalated ducts, and differences were noted in the behavior of the two tracers. Cationized ferritin was internalized mainly into vesicles and tubules of cells of striated ducts; little uptake of native ferritin occurred at low concentrations. These results demonstrate that the ductal cells of the salivary glands are capable of luminal endocytosis of foreign proteins. They also suggest that in addition to modifying the primary saliva by electrolyte reabsorption and secretion, and secretion of various glycoproteins, the ductal cells are able to reabsorb proteins secreted by the acinar cells.     

Intracellular fate of ferritin in HeLa cells following microinjection

It is known that following iron overload newly synthesized ferritin molecules accumulate in lysosomes. However, the way in which these molecules enter the lysosomes has not been clarified. In order to assess if these molecules can be taken up by lysosomes from the cell sap, i.e., by way of autophagy, ferritin was introduced into HeLa cells through microinjection with a glass capillary. The fate of the ferritin was studied after varying intervals with the electron microscope. Shortly after microinjection ferritin molecules could be observed in the cell sap. After both 1 and 2 h, they were found in clusters and still mainly in the cell sap. After 4 h, ferritin molecules were present not only in the cell sap and in autophagic vacuoles but also in occasional secondary lysosomes. After 12 h, they were seen mainly in lysosomes, undergoing degradation. In no instance were ferritin molecules translocated into other organelles such as mitochondria, Golgi apparatus, or endoplasmic reticulum. The present study demonstrates that ferritin can be introduced into cells by glass capillary microinjection without cell damage. From its initial location in the cell sap ferritin is taken up into the lysosomal vacuome. Autophagy is considered to be the principal mechanism for the transfer of the ferritin molecules into lysosomes.     

Membrane modification during secretory granule formation in rat somatotrophs

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.     

Transitional cells at the junction of seminiferous tubules with the rete testis of the rat: their fine structure, endocytic activity, and basement membrane

Transitional cells line the intermediate region of rat seminiferous tubules situated between the rete testis and the seminiferous epithelium proper. These tall elongated cells orient themselves in a downstream direction and converge on one another distally in the lumen of the rete testis where they form a distinct papillalike structure through which a narrow patent lumen is apparent. In addition to widely dispersed Golgi apparatus and mitochondria, these cells contain an abundance of microtubules, cisternae of endoplasmic reticulum, and a distinct lobulated nucleus showing clumps of chromatin and a prominent nucleolus. The endocytic activity of these cells was examined by employing adsorptive (cationic ferritin, concanavalin A ferritin) and fluid-phase tracers (native ferritin, horseradish peroxidase-colloidal gold complex, and concanavalin A ferritin in presence of alpha methyl-D-mannoside). Such tracers were injected separately into the lumen of the rete testis, and the animals were killed at 2, 5, 15, and 30 min and 1, 2, and 6 hr after injection. At 2 min, both adsorptive and fluid-phase tracers were found within coated and uncoated pits of the apical plasma membrane of these cells as well as in large, subsurface, uncoated spherical, C-shaped, and tubular membranous elements. At 5 min the tracers were seen in endosomes of different sizes; while at 15 min and 30 min, pale and dense multivesicular bodies of small and large sizes, respectively, were labeled. At 1-hr and longer time intervals secondary lysosomes became labeled. While both fluid-phase and adsorptive tracers followed the same pathway and fate, binding to the apical and lateral plasma membranes of the transitional cells and to the membrane delimiting coated and uncoated pits was observed only with the adsorptive tracers. These results demonstrate that the transitional cells are actively involved in both fluid-phase and adsorptive endocytosis, which may play an important role in modifying the composition of the luminal fluid. The transitional cells of the distal zone of the intermediate region rest on an elaborate basement membrane (BM) complex which includes a thin BM immediately underlying these cells, a thick distal layer of BM, and strands of BM spanning the distance between the two in the form of a loose anastomotic network. Use of antisera against heparan sulfate proteoglycan, laminin, and type IV collagen revealed the presence of all three components within all areas of the BM complex. In the meshes of the anastomotic BM network, extracellular vesicles were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cortical granule exocytosis is coupled with membrane retrieval in the egg of Brachydanio

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.     

Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion

Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan- sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.     

Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes

We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors

Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.