Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.     

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D- labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.     

Endocytosis in filter-grown Madin-Darby canine kidney cells

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three- dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.     

Tubular early endosomal networks in AtT20 and other cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.     

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.     

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.     

Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat

Summary The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome. However, unlike transferrin, native ferritin at the late time intervals appeared in dense multivesicular bodies and secondary lysosomes. When the adsorptive marker cationic ferritin and the fluid-phase marker albumin-gold were coinjected, again both proteins often shared the same endocytic pit and vesicle, endosome, pale and dense multivesicular body and secondary lysosomes. However, several endocytic vesicles labeled only with cationic ferritin appeared to bypass the endosomal and lysosomal compartments and to reach the lateral intercellular space and areas of the basement membrane. The rete epithelial cells, therefore, appear to be internalizing proteins and ligands by receptor-mediated and non-specific endocytosis which, after having shared the same endocytic vesicle and endosome, appear to be capable of being segregated and routed to different destinations.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

Meeting of the apical and basolateral endocytic pathways of the Madin- Darby canine kidney cell in late endosomes

Electron microscopic approaches have been used to study the endocytic pathways from the apical and basolateral surface domains of the polarized epithelial cell, MDCK strain I, grown on polycarbonate filters. The cells were incubated at 37 degrees C in the presence of two distinguishable markers administered separately to the apical or the basolateral domain. Initially each marker was visualized within distinct apical or basolateral peripheral endosomes. However, after 15 min at 37 degrees C, both markers were observed within common perinuclear structures. The compartment in which meeting first occurred was shown to be a late endosome (prelysosome) that labeled extensively with antibodies against the cation-independent mannose-6-phosphate receptor (MPR) on cryosections. With increasing incubation times, markers passed from these MPR-positive structures into a common set of MPR-negative lysosomes that were mainly located in the apical half of the cell. A detailed quantitative analysis of the endocytic pathways was carried out using stereological techniques in conjunction with horseradish peroxidase and acid phosphatase cytochemistry. This enabled us to estimate the absolute volumes and membrane surface areas of the endocytic organelles involved in apical and basolateral endocytosis.     

Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis

We studied with morphometric methods the endocytosis by pheochromocytoma cells of a conjugate of wheat germ agglutinin with ferritin (WGA-Ft) and of horseradish peroxidase (HRP). Quantitative studies indicated that WGA-Ft was cleared slowly from cell surfaces and that it was not recycled to the surface. Cells labeled with WGA-Ft for 15 min at room temperature were washed and incubated in medium containing HRP for 15 or 30 min at 37 degrees C. The greatest proportion of labeled vesicles and tubules contained only WGA-Ft (83.4% at 15 min and 85.3% at 30 min). A very small fraction of labeled vesicles and tubules contained only HRP (0.2% at 15 min and 0.9% at 30 min). Vesicles and tubules at the Golgi apparatus were labeled almost exclusively with WGA-Ft (97% at 15 min and 30 min); the rest had both labels. Most labeled lysosomes contained both labels (80.1% at 15 min and 80.8% at 30 min). Of the remainder more contained WGA-Ft alone (20% at 15 min and 10.9% at 30 min), then HRP alone (none at 15 min and 8.2% at 30 min). In contrast to the various and varying patterns of labeling with WGA-Ft and HRP of the other organelles studied, the vast majority of endosomes contained both markers (94.1% at 15 min and 100% at 30 min); the rest contained WGA-Ft only. These results demonstrate that endosomes are recipients of both fluid phase and adsorptive endocytosis markers; these findings are consistent with the hypothesis that endosomes mediate the sorting out and subsequent intracellular traffic of membrane bound and fluid phase markers. Cisterns of the Golgi apparatus did not contain WGA-Ft; in sharp contrast, when WGA-HRP was used, the cisterns of the Golgi apparatus consistently contained HRP.     

Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells.

To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway.     

Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes

After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.     

Intracellular distribution of a biotin-labeled ganglioside, GM1, by immunoelectron microscopy after endocytosis in fibroblasts.

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 micrometer in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.     

"Synchronized" endocytosis and intracellular sorting in alveolar macrophages: the early sorting endosome is a transient organelle

Incubation of alveolar macrophages in hypoosmotic K(+)-containing buffers results in persistent cell swelling and an inability to undergo regulatory volume decrease. We demonstrate that cells incubated in hypo- K+ show an inhibition of endocytosis without any observed alteration in recycling. The inhibition of endocytosis affected all forms of membrane internalization, receptor and fluid phase. Both increased cell volume and the inhibition of endocytosis could be released upon return of cells to iso-Na+ buffers. The ability to synchronize the endocytic apparatus allowed us to examine hypotheses regarding the origin and maturation of endocytic vesicles. Incubation in hypo-K+ buffers had no effect on the delivery of ligands to degradative compartments or on the return of previously internalized receptors to the cell surface. Thus, membrane recycling and movement of internalized components to lysosomes occurred in the absence of continued membrane influx. We also demonstrate that fluorescent lipids, that had been incorporated into early endosomes, returned to the cell surface upon exposure of cells to hypo-K+ buffers. These results indicate that the early sorting endosome is a transient structure, whose existence depends upon continued membrane internalization. Our data supports the hypothesis that the transfer of material to lysosomes can best be explained by the continuous maturation of endosomes.     

Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.     

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.     

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.     

Identification of a membrane glycoprotein found primarily in the prelysosomal endosome compartment

Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes.