αVβ3-integrin relocalizes nectin1 and routes herpes simplex virus to lipid rafts

Herpes simplex virus (HSV) enters cells by fusion at plasma membranes or endosomes. Cellular factors route the virus to different pathways. αVβ3-integrin directs HSV to a lipid raft and acidic endosome pathway. We report that infection mediated by nectin1 plus αVβ3-integrin exhibits the same characteristics as entry mediated by raft-located forms of nectin. αVβ3-integrin relocalizes nectin1 to lipid rafts, independently of virus. Thus, HSV routing to the lipid raft-dependent pathway is consequent to the integrin-induced relocalization of nectin1. Inhibition by the Na+/H+ exchanger 5-(N-ethyl-N-isopropyl)amirolide suggests that αVβ3-integrin overexpression favors HSV macropinocytic uptake in some cells but not in others.     

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.     

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.     

Distinct cellular responses differentiating alcohol- and hepatitis C virus-induced liver cirrhosis

Background

Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway.

Results

Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH-independent entry at the plasma membrane. In addition, CHO-nectin-2 cells supported pH-dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid (t_1/2 of 5–10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO.

Conclusion

HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.     

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.     

Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes

After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of endosomes in simian virus 40 entry and infection

After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was especially sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.     

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca(2+) binding sites (sites 1, 2 and 3), with Ca(2+) at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptor-ligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca(2+) and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca(2+) binding sites, site 3 is low affinity, and that Ca(2+) ions bind to sites 1 and 2 cooperatively. The pH and Ca(2+) concentration dependences of Ca(2+) binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca(2+) makes it possible for ASGP-R to be more sensitive to Ca(2+) concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca(2+) and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.     

Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.     

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

While severe acute respiratory syndrome coronavirus （SARS-CoV）~as initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that yirus entry may also involve endocytosis. We have found that SARS-CoV enters cells viapH- and receptor-dependent endocytosis. Treatment of cells with either SARS-COV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 （ACE2）, the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic path- way inhibitors and dominant-negative Epsl5 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.     

Acidification of morphologically distinct endosomes in mutant and wild- type Chinese hamster ovary cells

In the preceding paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2713-2721), we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wild-type Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5- 1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308), our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para- Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled alpha 2-macroglobulin or fluorescein- isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH greater than 6.5 in the CHO mutants when compared with wild-type cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.     

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.     

Differential sorting and fate of endocytosed GPI-anchored proteins

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI- APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway.     

Entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes

Feline calicivirus is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of Caliciviridae. We have examined the entry process of feline calicivirus (FCV). An earlier study demonstrated that acidification in endosomes may be required. We have confirmed this observation and expanded upon it, demonstrating, using drugs to inhibit the various endocytic pathways and dominant-negative mutants, that FCV infects cells via clathrin-mediated endocytosis. We have also observed that FCV permeabilizes cell membranes early during infection to allow the co-entry of toxins such as alpha-sarcin. Inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked this permeabilization event, demonstrating that acidification is required for uncoating of the genome and access to the cytoplasm.