Major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms

Intercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111-123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.     

Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.     

Uptake of poliovirus into the endosomal system of HeLa cells

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.     

Children''s emerging regulation of conduct: restraint, compliance, and internalization from infancy to the second year

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.     

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.     

Free-flow electrophoretic analysis of endosome subpopulations of rat hepatocytes

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.     

Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.     

On the putative mechanistic basis for intraoperative propofol-induced penile erections

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.     

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.     

Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes

In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.     

Endocytosis during postnatal differentiation in superficial cells of the mouse urinary bladder epithelium

This electron microscopical study was performed in order to follow the endocytic pathway of horseradish peroxidase and colloidal gold tracers and to determine the involvement of endocytosis in postnatal differentiation in superficial cells of the mouse urinary bladder epithelium. Morphometric analyses of late endosomes/multivesicular bodies from day of birth to day 25 were performed. The internalisation and intracellular transport of luminal plasmalemma to multivesicular bodies via endocytic vesicles, early endosomes and pleomorphic compartments was established. Dynamic changes in endocytic activity took place within the first few days of postnatal differentiation. During this period the number of multivesicular bodies changed in an inverse ratio to their size. After the third day endocytic activity gradually approached the low rate of adult urothelium.     

Studies of small GTPase Rab7 association with endosomes of cells expressing normal and mutant forms of epidermal growth factor receptors

A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.     

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.     

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.     

Calmodulin regulates endosome fusion

Calmodulin (CaM) has previously been implicated in regulated exocytosis, transcytosis, and receptor recycling. We have investigated the role of CaM in endocytic transport by examining the effects of several CaM antagonists in intact cells. We present evidence indicating that the mixing of sequentially internalized ligands is inhibited by CaM antagonists, indicating that CaM may play a general role in regulating endosomal membrane trafficking. To address the specific events that are affected by CaM we studied its role in an in vitro assay that reconstitutes fusion among endosomes. CaM antagonists inhibited endosome fusion, and the inhibition was reversed by the addition of CaM. Moreover, we found that Ca2+ stimulates fusion among endosomes and that addition of CaM stimulates fusion beyond that produced by Ca2+ alone. Our data indicate that one of the possible targets for CaM in endosome fusion is the CaM-dependent kinase II. We propose that CaM regulates endocytic transport by modulating an essential component(s) of the membrane traffic machinery.     

Influenza virus M2 protein slows traffic along the secretory pathway. pH perturbation of acidified compartments affects early Golgi transport steps

M2, an acid-activated ion channel, is an influenza A virus membrane protein required for efficient nucleocapsid release after viral fusion with the endosomal membrane. Recombinant M2 slows protein traffic through the Golgi complex (Sakaguchi, T., Leser, G. P)., and Lamb, R. A. (1996) J. Cell Biol. 133, 733-47). Despite its critical role in viral infection, little is known about the subcellular distribution of M2 or its fate following delivery to the plasma membrane (PM). We measured the kinetics of M2 transport in HeLa cells, and found that active M2 reached the PM considerably more slowly than inactive M2. In addition, M2 delayed intra-Golgi transport and cell surface delivery of influenza hemagglutinin (HA). We hypothesized that the effects of M2 on transport through non-acidified compartments are due to inefficient retrieval from the trans-Golgi of machinery required for intra-Golgi transport. In support of this, acutely activated M2 had no effect on intra-Golgi transport of HA, but still slowed HA delivery to the PM. Thus, M2 has an indirect effect on early transport steps, but a direct effect on late steps in PM delivery. These findings may help explain the conflicting and unexplained effects on protein traffic observed with other perturbants of intraorganelle pH such as weak bases and inhibitors of V-type ATPases.     

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.     

Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7

Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTP-binding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.     

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.     

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.