Intracellular localization and degradation of diphtheria toxin

The internalization of surface-bound diphtheria toxin (DT) in BS-C-1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon preincubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomal fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the ligand appeared in the medium as TCA-soluble radioactivity. EGF degradation was partially leupeptin-sensitive and markedly NH4Cl-sensitive, indicating lysosomal degradation. In contrast, DT A-fragment degradation was resistant to these inhibitors, while B-fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.     

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

CD44 expression is developmentally regulated in the mouse lens and increases in the lens epithelium after injury

Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of α-smooth muscle actin expression typically used as a marker of epithelial–mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of α-smooth muscle actin.     

Role of CD44 in the organization of keratinocyte pericellular hyaluronan

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44−/− animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.     

A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices

A rat epidermal keratinocyte (REK) line which exhibits histodifferentiation nearly identical to the native epidermis when cultured at an air-liquid interface was used to study the metabolism of hyaluronan, the major intercellular macromolecule present in basal and spinous cell layers. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (approximately 75%) of the hyaluronan was retained in the epithelium when a basal lamina was present while most (approximately 80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of this macromolecule was much higher in the epithelium on the basal lamina (half-life approximately 1 day, similar to its half-life in native human epidermis). The formation of a true epidermal compartment in culture bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study epidermal metabolism.     

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.     

Synthesis and Assembly of the Hyaluronan-Containing Coats around Normal Human Mesothelial Cells

In this study we examined the capacity of normal human mesothelial (NHM) cells and human malignant mesothelioma cells to form hyaluronan-containing pericellular matrices or "coats." The assembly of the pericellular coats was visualized by a particle exclusion assay. We found that large hyaluronan-containing coats were formed around NHM cells whereas their transformed counterparts had no or very limited coats. The coats were removed by treatment with Streptomyces hyaluronidase, which specifically degrades hyaluronan. NHM cells exhibited hyaluronan-containing pericellular matrix within 5 h after seeding. The formation of the coats was stimulated by platelet-derived growth factor and epidermal growth factor. Interestingly, the assembly of the hyaluronan-dependent pericellular matrices was inhibited by the addition of hyaluronan dodecasaccharides. The inhibitory effect on the formation of the coats was due to a destabilization of pericellular matrix and not due to an inhibitory effect of hyaluronan dodecasaccharides on hyaluronan synthesis. In contrast, hyaluronan hexasaccharides, an inhibitor of the interaction between polymeric hyaluronan and its cell surface receptors, had no effect on the size of the coat. Thus, our results are compatible with the possibility that the pericellular matrix surrounding NHM cells consists of newly synthesized hyaluronan which is extruded from the cell and independent of hyaluronan receptors on the cell surface. The coat seems to be stabilized by interactions (hyaluronan-hyaluronan or hyaluronan-protein bridges) which can be prevented by hyaluronan dodecasaccharides.     

Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.     

Annexin A8 regulates late endosome organization and function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.     

Differences in the endosomal distributions of the two mannose 6- phosphate receptors

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI- MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.     

Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients

Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the β-subunit of β-hexosaminidase, that result in β-hexosaminidase A (αβ) and β-hexosaminidase B (ββ) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal–lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal–lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.     

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.     

Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Endocytosis is thought to play an essential role in the catabolism of HA due to the intracellular compartmentalization of the HA degrading hyaluronidase enzymes. Previous investigations have shown that keratinocytes, chondrocytes and breast tumor cell lines endocytose HA via the cell surface glycoprotein, CD44. However, other cell types endocytose HA using a CD44-independent mechanism that remains to be defined. The purpose of this study was to investigate HA endocytosis in B16-F10 melanoma cells. We found that B16-F10 melanoma cells expressed CD44 on their surfaces. Unexpectedly, CD44 did not play a role in the endocytosis of HA. Electron microscopy studies revealed that B16-F10 melanoma cells exhibited membrane ruffling, a characteristic feature of macropinocytosis, only after incubating the cells with the HA co-polymer. Moreover, B16-F10 melanoma cells endocytosed HA via macropinocytosis as assessed by drug inhibition studies and the co-localization of fluorescently labeled HA with fluorescent tracers under confocal microscopy. Based on these results, we conclude that induced macropinocytosis may provide a previously unrecognized avenue for HA endocytosis in some cell types.     

Hyaluronan export through plasma membranes depends on concurrent K+ efflux by K(ir) channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.     

Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver

The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hepatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosome compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocyte. In cell-free, postmitochondrial supernatants, we have previously observed that dense endosomes, but not light endosomes, interact with lysosomes. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabeled ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-ethylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomes did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomes was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethyl-maleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabeled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomes in a cell-free system.     

Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.