Hormonal regulation of sulfated glycoprotein-1 synthesis by nonciliated cells of the efferent ducts of adult rats

The objective of this study was to define the factors regulating the endogenous production of sulfated glycoprotein-1 (SGP-1) in nonciliated cells of the efferent ducts. To this end we examined five different groups of animals undergoing the following experimental procedures: (1) hypophysectomized animals at 7, 14, and 28 days, (2) 7-day hypophysectomized rats receiving testosterone implants given at various time intervals thereafter, (3) castration at various time intervals up to 7 days, (4) 7-day castrated rats receiving testosterone implants at various time intervals thereafter, and (5) castrated rats given testosterone implants immediately after castration and sacrificed at different time intervals thereafter. Efferent ducts were fixed by perfusion with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for quantitative immunocytochemical analysis at the level of the electron microscope. For each experimental condition and their controls, the number of gold particles/μm² within the endosomal and lysosomal compartments was calculated taking into account the changes in both the volume of the cell and organelles being quantified and expressed as labeling content. The results revealed that hypophysectomy (up to 4 weeks) caused a marked significant decrease in the SGP-1 labeling content of the endosomal and lysosomal compartments. The labeling content of the lysosomal compartment of efferent ducts from rats castrated for up to 1 week did not change significantly. However, there was a significant decrease in the labeling content of endosomes. This decrease is due to SGP-1, which is secreted by Sertoli cells, not being available for uptake in the efferent aucts. These results suggested that testosterone is not required for maintaining the high labeling content of SGP-1 within lysosomes of nonciliated cells, but that a pituitary factor appears to be needed. The administration of testosterone at different intervals to 7-day castrated animals resulted in a significant decrease of lysosomal SGP-1, suggesting that testosterone under these experimental conditions inhibits the production of a pituitary factor that maintains the high labeling content of SGP-1 within lysosomes of the nonciliated cells. Testosterone administered to 7-day hypophysectomized animals over a 24-hr period had no effect on the labeling content of SGP-1 within lysosomes. However, the administration of testosterone to animals immediately following castration showed no differences in the labeling content of SGP-1 within compared to controls. Together these results suggest that the labeling content of SGP-1 within lysosomes of nonciliated cells of the efferent ducts is not dependent on luminal or circulating androgens, nor is it dependent on a testicular factor entering the lumen of the ducts. It does appear, however, that SGP-1 synthesis and targeting to secondary lysosomes is dependent on a pituitary factor that may have a direct or an indirect effect on the nonciliated cells. © 1995 Wiley-Liss, Inc.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Role of sialic acid in the endocytosis of prosaposin by the nonciliated cells of the rat efferent ducts

The present study examines the mechanism of endocytosis of testicular prosaposin by the nonciliated cells of the efferent ducts. Testicular prosaposin is secreted by Sertoli cells into the lumen of the seminiferous tubules as a 70 kDa isomer where it binds to the tail of spermatozoa. In the efferent ducts, after dissociating from the plasma membrane of the spermatozoa, prosaposin is endocytosed by the nonciliated cells, presumably by receptor-mediated endocytosis. The initial step of receptor-mediated endocytosis usually results from the binding of a ligand's terminal oligosaccharide to a receptor on the cell surface. Thus, in the present study, several monosaccharides were injected in the lumen of the efferent ducts to compete with the binding and endocytosis of prosaposin. A quantitative electron microscopic approach was utilized and the number of gold particles, indicating anti-prosaposin immunoreactive sites, were scored over the various cell compartments including the plasma membrane, endocytic vesicles, early endosomes, and late endosomes. The length of the plasma membrane and the areas of endocytic vesicles, early endosomes, and late endosomes were measured with an image analyzer and the number of grains expressed per μm (plasma membrane) and μm² (endocytic vesicles/endosomes) respectively. The quantitative analysis was performed in untreated animals (controls) and animals treated with various sugars (i.e., glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine and N-acetylgalactosamine) injected into the lumen of the efferent ducts at a concentration of 20 mM. Sialic acid caused the greatest decrease in the labeling density of the endocytic elements. Mannose 6-phosphate also caused a decrease in labeling but to a lesser extent. Various amounts of sialic acid (0.02 mM, 0.2 mM, 2 mM, 20 mM, and 200 mM) showed that most of these concentrations produced a significant decrease in the labeling density of endocytic vesicles and endosomes. Moreover, Western blots of prosaposin isolated from seminiferous tubular fluids followed by glycan analysis with Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), revealed that this protein has sialic acid residues that are terminally linked to galactose and/or N-acetylgalactosamine ( α-NeuNAc-[2->6]-Gal and α-NeuNAc-[2->6]-GalNAc). These data indicate that testicular prosaposin is removed from the lumen of the efferent ducts by the noncialiated cells via a receptor that recognizes prosaposin's terminal sialic acid residues. Mol. Reprod. Dev. 51:156–166, 1998. © 1998 Wiley-Liss, Inc.     

The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM₂AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Expression and regulation of LRP‐2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development

Low density lipoprotein receptor‐related protein‐2/megalin (LRP‐2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP‐2. LRP‐2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP‐2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP‐2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7–90 and in 15‐day‐old efferent‐duct‐ligated animals sacrificed at later ages. In the efferent ducts, LRP‐2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli‐cell‐derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP‐2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP‐2 expression from 7–15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP‐2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP‐2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP‐2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP‐2 expression is under the influence of both stimulatory and region‐specific inhibitory factors. Analysis of 15‐day‐old efferent‐duct‐ligated animals at all later ages examined revealed that there was no change in LRP‐2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP‐2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP‐2 expression and its ligand, Apo J. In summary, LRP‐2 expression in the epididymis undergoes region‐specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors. Mol. Reprod. Dev. 53:282–293, 1999. © 1999 Wiley‐Liss, Inc.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.     

Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

Ferlins are an ancient family of C2 domain-containing proteins, with emerging roles in vesicular trafficking and human disease. Dysferlin mutations cause inherited muscular dystrophy, and dysferlin also shows abnormal plasma membrane expression in other forms of muscular dystrophy. We establish dysferlin as a short-lived (protein half-life ∼4–6 h) and transitory transmembrane protein (plasma membrane half-life ∼3 h), with a propensity for rapid endocytosis when mutated, and an association with a syntaxin-4 endocytic route. Dysferlin plasma membrane expression and endocytic rate is regulated by the C2B-FerI-C2C motif, with a critical role identified for C2C. Disruption of C2C dramatically reduces plasma membrane dysferlin (by 2.5-fold), due largely to accelerated endocytosis (by 2.5-fold). These properties of reduced efficiency of plasma membrane expression due to accelerated endocytosis are also a feature of patient missense mutant L344P (within FerI, adjacent to C2C). Importantly, dysferlin mutants that demonstrate accelerated endocytosis also display increased protein lability via endosomal proteolysis, implicating endosomal-mediated proteolytic degradation as a novel basis for dysferlin-deficiency in patients with single missense mutations. Vesicular labeling studies establish that dysferlin mutants rapidly transit from EEA1-positive early endosomes through to dextran-positive lysosomes, co-labeled by syntaxin-4 at multiple stages of endosomal transit. In summary, our studies define a transient biology for dysferlin, relevant to emerging patient therapeutics targeting dysferlin replacement. We introduce accelerated endosomal-directed degradation as a basis for lability of dysferlin missense mutants in dysferlinopathy, and show that dysferlin and syntaxin-4 similarly transit a common endosomal pathway in skeletal muscle cells.     

GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment

Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 - sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL[LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.     

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.     

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.     

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

Transformational process of the endosomal compartment in nephrocytes of Drosophila melanogaster

Summary This study investigates by electron microscopy the transformational process of the endosomal compartment of the Drosophila nephrocyte, the garland cell, which occurs during endocytotic processing of internalized material. The endosomal compartment of the garland cell consists of a prominent tubular/vacuolar complex in the cortical cytoplasm. When internalization of coated pits is blocked at 29°C using the endocytosis mutant, shibire ^ts, the tubules gradually disappear after 7 min at 29°C. By 12 min at 29°C, the vauoles also disappear. Thus, the endosomal compartment appears to constantly undergo a transformational process that necessitates continuous replenishment by coated vesicles. The data suggest that the tubular component of the endosomal compartment gradually transforms into vacuoles by the expansion of the tubular membrane. The vacuoles then transform by invaginating into themselves, creating flattened cisternae. The electron-lucent substance in the lumina of the vacuoles appears to be extruded into the cytoplasm through the invaginating membrane. No shuttle vehicles such as vesicles or tubules could be identified that might have been involved in the transporting of endocytosed materials and membrane from the endosomal compartment to lysosomes or back to the plasma membrane.     

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

The essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71

Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 μm chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.     

Low pH-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.     

Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.     

Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi''s sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells.