Analysis of post-lysosomal compartments

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.     

Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.     

Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments

Recently we demonstrated that low-pH compartments can be visualized with the electron microscope using a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), which concentrates in acidic compartments and can be detected by immunocytochemistry with a monoclonal anti-dinitrophenol antibody. We now report that DAMP also accumulates in cisternae and vesicles associated with the trans face of the Golgi apparatus. DAMP rapidly leaves this compartment when cells are incubated with the ionophore monensin, which indicates that accumulation is due to the acidic pH in this compartment. Using indirect protein A-gold immunocytochemistry, we localized fibronectin, a major secretory protein in fibroblasts, to the trans Golgi vesicles that took up DAMP. Therefore, the trans cisternae of the Golgi apparatus and forming secretory vesicles have an acidic pH.     

Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes

Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.     

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.     

Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker

A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.0, with that of lysosomal hydrolases bearing the mannose 6-phosphate recognition marker (Man-6-P proteins), which dissociate around pH 5.8. Chinese hamster ovary cells were pulsed for 2 min with 125I-insulin, acid-washed to remove surface binding, and chased. During a 40-min period, about 50% of the internalized 125I-insulin was released intact via a retrocytotic pathway. Retrocytosis was not inhibited by monensin, suggesting that the release was not dependent on acidic endosomes. The remaining insulin dissociated from its receptor in an acidification-sensitive manner and was eventually degraded. Dissociation was 70% complete within 5 min of internalization. When cells were similarly incubated with 125I-Man-6-P proteins, about 35% of the internalized radioactivity was released during a 1-h chase, reflecting proteolytic maturation of the Man-6-P proteins. Dissociation of Man-6-P proteins was acidification-dependent (i.e. inhibited by monensin), and was 50% complete after about 11 min. The results indicate that acidification-dependent dissociation of ligands does not occur in a single step and suggest that multiple endocytic compartments are involved in receptor/ligand sorting.     

Molecular probes for sensing the cholesterol composition of subcellular organelle membranes

Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic beta-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.     

Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling

Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules.     

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.     

SILICON DEPOSITION IN DIATOMS: CONTROL BY THE pH INSIDE THE SILICON DEPOSITION VESICLE

To test the hypothesis that silicification occurs under acid conditions in the silicon deposition vesicle (SDV), the acidity of the SDV of the pennate diatoms Navicula pelliculosa (Brébisson et Kützing) Hilse, N. salinarum (Grunow) Hustedt, and Nitzschia sigma (Kützing) Smith was determined during development of new frustule valves. Cells were incubated with the weak base 3-(2,4-dinitroanilino)-3′-amino-N-methylpropylamine (DAMP) followed by immunocytochemical localization in whole cells and on ultrathin sections. After resupplying silicate to cells synchronized by silicon depletion, the uptake of this nutrient from the medium was the same with or without DAMP; new valves developed without morphological aberrations that could conceivably have been caused by the probe. DAMP was found in cellular compartments known to be acidic, such as vacuoles active as lysosomes, the lumen of thylakoids, and microbodies. In the nucleus and mitochondria, which are circumneutral and basic compartments, the probe did not appear. Besides its presence in acidic compartments, DAMP was specifically accumulated within the SDV during formation of new valves; during the process of valve maturation, the SDV seemed to become increasingly acidic. In control experiments using the ionophores chloroquine, valinomycin, and nigericin, the compartmental location of DAMP was clearly disturbed, resulting in a random intracellular distribution. Accumulation of the fluorescent probe rhodamine 123, which can be translocated over membranes by a reducing potential, confirmed that the SDV can translocate weak bases. The results with DAMP suggest that the pH of the SDV is important in the silicification of diatoms: It facilitates a fast nucleation and aggregation of silica particles, thus increasing the rate of formation of the mature frustules. In addition, the acidic environment might protect the newly formed valves against dissolution before completion and coverage by the organic casing prior to their secretion.     

EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments

Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.     

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.     

Fluorescence lifetime characterization of novel low-pH probes.

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH.     

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4- dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti- proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.     

A role for small GTPase RhoA in regulating intracellular membrane traffic of lysosomes in invasive rat hepatoma cells

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.     

Studies in vivo and in vitro on the uptake and degradation of soluble collagen alpha 1(I) chains in rat liver endothelial and Kupffer cells.

Intravenously administered 125I-labelled monomeric alpha 1 chains (125I-alpha 1) of collagen type I were rapidly cleared and degraded by the liver of rats. Isolation of the liver cells after injection of the label revealed that the uptake per liver endothelial cell equalled the uptake per Kupffer cell, whereas the amount taken up per hepatocyte was negligible. The uptake of 125I-alpha 1 in cultured cells was 10 times higher per liver endothelial cell than per Kupffer cell. The ligand was efficiently degraded by cultures of both cell types. However, spent medium from cultures of Kupffer cells, unlike that from cultures of other cells, contained gelatinolytic activity which degraded 125I-alpha 1. The presence of hyaluronic acid, chondroitin sulphate or mannose/N-acetylglucosamine-terminal glycoproteins, which are endocytosed by the liver endothelial cells via specific receptors, did not interfere with binding, uptake or degradation of 125I-alpha 1 by these cells. Unlabelled alpha 1 and heat-denatured collagen inhibited the binding to a much greater extent than did native collagen. The presence of fibronectin or F(ab')2 fragments of anti-fibronectin antibodies did not affect the interaction of the liver endothelial cells, or of other types of liver cells, with 125I-alpha 1. The accumulation of fluorescein-labelled heat-denatured collagen in vesicles of cultured liver endothelial cells is evidence that the protein is internalized. Moreover, chloroquine, 5-dimethylaminonaphthalene-1-sulphonylcadaverine (dansylcadaverine), monensin and cytochalasin B, which impede one or more steps of the endocytic process, inhibited the uptake of 125I-alpha 1 by the liver endothelial cells. Leupeptin, an inhibitor of cathepsin B and 'collagenolytic cathepsins', inhibited the intralysosomal degradation of 125I-alpha 1, but had no effect on the rate of uptake of the ligand. The current data are interpreted as follows. (1) The ability of the liver endothelial cells and the Kupffer cells to sequester circulating 125I-alpha 1 efficiently may indicate a physiological pathway for the breakdown of connective-tissue collagen. (2) The liver endothelial cells express receptors that specifically recognize and mediate the endocytosis of collagen alpha 1(I) monomers. (3) The receptors also recognize denatured collagen (gelatin). (4) Fibronectin is not involved in the binding of alpha 1 to the receptors. (5) Degradation occurs intralysosomally by leupeptin-inhibitable cathepsins.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.     

PKC and PKA Regulate AChR Dynamics at the Neuromuscular Junction of Living Mice

The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.