To gain a better understanding of intracellular processing of nanomedicines, we employed quantitative live-cell fluorescence colocalization microscopy to study endosomal trafficking of polyplexes in retinal pigment epithelium cells. A new, dynamic colocalization algorithm was developed, based on particle tracking and trajectory correlation, allowing for spatiotemporal characterization of internalized polyplexes in comparison with endosomal compartments labeled with EGFP constructs. This revealed early trafficking of the polyplexes specifically to Rab5- and flotillin-2-positive vesicles and subsequent delivery to Rab7 and LAMP1-labeled late endolysosomes where the major fraction of the polyplexes remains entrapped for days, suggesting the functional loss of these nanomedicines. Colocalization of polyplexes with the autophagy marker LC3 suggests for the first time that the process of xenophagy could play an important role in the persistent endosomal entrapment of nanomedicines.

Rab GTPases play an important regulatory role in early endocytosis. We recently demonstrated that epitope-tagged Rab15 (HArab15) co-localizes with Rab4, -5, and -11 on early endosomal membranes in CHO cells (Zuk, P. A., and Elferink, L. A. (1999) J. Biol. Chem. 274, 22303-22312). To characterize the role of Rab15 in endocytosis, we prepared functional mutants of HArab15 and examined their effects on early endocytic trafficking. Wild-type HArab15 and its constitutively active, GTP-bound mutant (Q67L) reduce fluid phase and receptor-mediated endocytosis without affecting the rate of recycling from early endosomal compartments. Inhibition of early endocytosis appears to be due to a reduction in the rate of homotypic early endosome fusion. Conversely, mutations that constitutively inactivate HArab15 stimulate early endocytosis and the homotypic fusion of early endosomes in vitro. Unlike active forms of HArab15, constitutively inactive HArab15 mutants also affect recycling from early endosomal compartments. Moreover, the two constitutively inactive mutants, GDP-bound HArab15-T22N and the non-nucleotide binding mutant HArab15-N121I, differentially regulate the transit of fluid phase and receptor-mediated endocytic tracers through early/sorting endosomes. Together, these data suggest that HArab15 may counteract the reported stimulatory effect of Rab5 on early endocytosis. Consistent with this, overexpression of constitutively active HArab15-Q67L attenuates Rab5-stimulated endocytosis, whereas Rab5-stimulated endocytosis is augmented in cells overexpressing a constitutively inactive HArab15 mutant defective in guanine nucleotide binding (N121I). Our data indicate that HArab15 differentially regulates distinct steps in membrane trafficking through early/sorting and pericentriolar recycling endosomes.

Fusion of intracellular membrane-bound compartments is a common step in the transport of macromolecules along the endocytic and secretory pathways. Previous work has shown that GTP gamma S stimulates endosome fusion in the presence of low concentrations of cytosol. In this study, we have characterized the effect of rab5:Q79L, a mutant with reduced GTPase activity, on endosome fusion in a cell-free assay. rab5:Q79L stimulates in vitro endosome fusion. The stimulatory effects required ATP, were blocked by N-ethylmaleimide (NEM) and anti-NEM-sensitive fusion (NSF) protein antibody, but could proceed in the absence of cytosol. Stimulation of fusion with rab5:Q79L led to rapid inactivation of the vesicles when tested in a second incubation for fusogenic activity. By electron microscopy, endosomes connected by tubular structures were frequently observed in the presence of rab5:Q79L. Rab5:Q79L promoted fusion only among early endosomes; when the ligands were chased into more mature endocytic compartments, fusion was not observed. Phospholipase A2 inhibitors blocked rab5:Q79L-stimulated fusion. The results indicate that rab5:Q79L promotes fusion by activating factors already present in the membranes and that NSF and phospholipase A2 activities are required downstream of rab5.

BACKGROUND

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.

RESULTS

In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.

CONCLUSIONS

Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.

Neurite outgrowth and its maintenance are essential aspects of neuronal cells for their connectivity and communication with other neurons. Recent studies showed that over-expression of either leucine-rich repeat kinase 2 (LRRK2), whose mutations are associated with familial Parkinson's disease (PD), or Rab5b, an early endosomal marker protein, induces reduction in neurite length. Based on our recent findings that LRRK2 co-localizes and interacts with Rab5, we tested the hypothesis that LRRK2 and Rab5 may functionally interplay while controlling neurite outgrowth. Firstly, we confirmed previous reports that over-expression of either the LRRK2 PD-specific G2019S mutant or the Rab5 constitutively active Q79L mutant, but not of dominant negative N133I mutant, significantly reduces neurite outgrowth. Secondly, when over-expression of either LRRK2 wild type (WT) or G2019S was accompanied with over-expression of one of the Rab5 variants (WT, Q79L and N133I), or with down-regulation of Rab5, the reduction extent of its neurite length was similar to that of cells over-expressing LRRK2 alone, regardless of Rab5's status. Finally, we observed similar patterns of neurite length regulation in embryonic rat hippocampal neuron cultures. Taken together, our results suggest that LRRK2 and Rab5 functionally coordinate their regulation of neurite outgrowth and that LRRK2 is a more critical factor than Rab5.

Exoenzyme S (ExoS) ADP-ribosylates multiple low-molecular-mass G- (LMMG-) proteins in vitro. Identification of the in vivo substrate specificity of ExoS has been hindered by its bacterial contact delivery into eukaryotic cells and difficulties in identifying ADP-ribosylated proteins within cells. Two-dimensional electrophoresis comparisons of substrate modifications by ExoS in vitro to that following bacterial translocation into HT-29 epithelial cells identified Ras, Ral, and Rab proteins and Rac1 as in vivo substrates of ExoS ADPRT activity. Cellular fractionation studies identified a relationship between membrane association and efficiency of substrate modification. Moreover, Rac and Cdc42 relocalized to the membrane in response to ExoS. Comparisons of substrate modification to time of exposure to ExoS identified a progression of substrate modification, with Ras, RalA, and Rab5 modified first, followed by Rab8 and 11, then Rab7 and Rac1. The data support that intrinsic properties of LMMG-proteins and their subcellular localization are determinants of bacterially translocated ExoS substrate selectivity.

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.

The porcine reproductive and respiratory syndrome virus (PRRSV) enters its target cell via clathrin-mediated endocytosis. Using dominant-negative Rab5 and Rab7 mutants, we show that upon internalization, PRRSV enters early endosomes but does not continue through the endocytic pathway to late endosomes. This was confirmed via colocalization experiments visualizing PRRSV and markers for different compartments of the endocytic pathway. Furthermore, it was shown that PRRSV colocalizes with its internalization receptor, sialoadhesin, on the cell surface and beneath the plasma membrane, while CD163 and PRRSV only meet in early endosomes.

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain-containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

Endosomes and lysosomes receive cargo via vesicular carriers that arrive along multiple trafficking routes. On both organelles, tethering proteins have been identified that interact specifically with Rab5 on endosomes and Rab7 on late endosomes/lysosomes and that facilitate the SNARE-driven membrane fusion. Even though the structure and stoichiometry of the involved proteins and protein complexes differ strongly, they may operate by similar principles. Within this review, we will provide insights into their common functions and discuss the open questions in the field.

Generating and maintaining proper lumen size and shape in tubular organs is essential for organ function. Here, we demonstrate a novel role for p21-activated kinase 1 (Pak1) in defining the size and shape of the Drosophila embryonic salivary gland lumen by regulating the size and elongation of the apical domain of individual cells. Pak1 mediates these effects by decreasing and increasing E-cadherin levels at the adherens junctions and basolateral membrane, respectively, through Rab5- and Dynamin-dependent endocytosis. We also demonstrate that Cdc42 and Merlin act together with Pak1 to control lumen size. A role for Pak1 in E-cadherin endocytosis is supported by our studies of constitutively active Pak1, which induces the formation of multiple intercellular lumens in the salivary gland in a manner dependent on Rab5, Dynamin and Merlin. These studies demonstrate a novel and crucial role for Pak1 and E-cadherin endocytosis in determining lumen size and shape, and also identify a mechanism for multiple lumen formation, a poorly understood process that occurs in normal embryonic development and pathological conditions.

The polypeptides of three highly purified endosomal fractions isolated from the livers of oestradiol-treated rats were analysed by Western blotting, and the amount and distribution of intrinsic and cytoskeletal-associated proteins were quantified and studied. The 'late' endosomes [multivesicular bodies (MVBs)] had the lowest content of cytoskeletal-associated proteins, the most significant being the presence of 25% of the total dynein found in endosomes. The 'early' endosome [compartment of uncoupling receptors and ligands (CURL)] fraction contained kinesin (40% of the total in endosomes), dynein (23%), actin (15%) and tubulin (10%). The receptor-recycling compartment (RRC), also demonstrated to be involved in transcytosis, contained the largest number and enrichment of cytoskeletal proteins: actin (84% of the total in endosomes), alpha-actinin (90%), dynein (52%), tubulin (91%) and kinesin (45%). We also analysed and compared the presence of different endosomal markers such as Rab4, Rab5 and cellubrevin (vesicle soluble NSF attachment protein receptor) in CURL (41%, 15% and 60%) and in RRC (44%, 75% and 30% respectively). Finally, the expression of annexins I, II, IV and VI was studied: annexin I was equally distributed between MVBs and CURL; annexin II was highly enriched in RRC (95%), annexin IV was equally distributed between CURL and RRC, and annexin VI was enriched in CURL (57%). The results indicate that isolated rat liver endosomes contain all the required molecular machinery for the achievement of their role in intracellular trafficking.

Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.

Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoid cortisol. This hormone upregulates the serum- and glucocorticoid-inducible kinase (SGK)1, which in turn stimulates I(Ks), a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in I(Ks) channel alpha (KCNQ1, KvLQT1, Kv7.1) or beta (KCNE1, IsK, minK) subunits cause long QT syndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together with the GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show altered SGK1-dependent regulation of LQTS-associated mutant I(Ks) channels. Whereas some mutant KCNQ1 channels had reduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P) were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and the LQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disrupted RAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of I(Ks) channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the I(Ks) recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.

ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. We used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. We found that the IAC undergoes turnover in MTJs and that this process is mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. We propose that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.

Dopamine (DA) transporter (DAT) functions at the surface of dopaminergic neurons to clear extracellular DA. DAT surface levels are regulated by endocytosis. However, the endosome-lysosome system is not well characterized in dopaminergic neurons and the endocytic trafficking of endogenous DAT is poorly studied. Hence we analyzed the distribution of endocytic compartments and DAT localization in cultured rat embryonic and postnatal neurons using fluorescence microscopy. Early Rab5 and EEA.1 containing endosomes were mostly found in somatodendritic regions of neurons, whereas endosomes containing recycling markers were primarily found in axons. In axons, DAT was located mainly in recycling endosomes and plasma membrane whereas in cell bodies and dendrites DAT was detected in early, late and recycling endosomal compartments. Subcellular fractionation of adult rat striatal synaptosomes demonstrated that DAT is enriched in fractions containing plasma membrane and recycling endosomes. This pattern of DAT distribution was not altered upon activation of protein kinase C in postnatal DA neurons. Altogether, our data suggest that axonal DAT mainly shuttles between the plasma membrane and recycling endosomes, whereas in the somatodendritic region of neurons DAT traffics through all conventional endosomal pathways.

Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr(14)), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.

Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a "dead-end" route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection.

The hepatitis B virus (HBV) core particle serves as a protective capsid shell for the viral genome and is highly immunogenic. Recombinant capsid-like core particles are used as effective carriers of foreign T and B cell epitopes and as delivery vehicles for oligonucleotides. The core monomer contains an arginine-rich C terminus that directs core particle attachment to cells via membrane heparan sulfate proteoglycans. Here we investigated the mechanism of recombinant core particle uptake and its intracellular fate following heparan sulfate binding. We found that the core particles are internalized in an energy-dependent manner. Core particle uptake is inhibited by chlorpromazine and by cytosol acidification known to block clathrin-mediated endocytosis but not by nystatin, which blocks lipid raft endocytosis. Particle uptake is abolished by expression of dominant negative forms of eps15 and Rab5, adaptors involved in clathrin-mediated endocytosis and early endosome transport, respectively. Endocytosed particles are transported to lysosomes where the core monomer is endoproteolytically cleaved into its distinct domains. Using protease inhibitors, cathepsin B was identified as the enzyme responsible for core monomer cleavage. Finally we found that monomer cleavage promotes particle dissociation within cells. Together, our results show that HBV capsid-like core particles are internalized through clathrin-mediated endocytosis, leading to lysosomal cleavage of the core monomer and particle dissociation.

Inherited disorders of elastic tissue represent a complex and heterogeneous group of diseases, characterized often by sagging skin and occasionally by life-threatening visceral complications. In the present study, we report on an autosomal-recessive disorder that we have termed MACS syndrome (macrocephaly, alopecia, cutis laxa, and scoliosis). The disorder was mapped to chromosome 20p11.21-p11.23, and a homozygous frameshift mutation in RIN2 was found to segregate with the disease phenotype in a large consanguineous kindred. The mutation identified results in decreased expression of RIN2, a ubiquitously expressed protein that interacts with Rab5 and is involved in the regulation of endocytic trafficking. RIN2 deficiency was found to be associated with paucity of dermal microfibrils and deficiency of fibulin-5, which may underlie the abnormal skin phenotype displayed by the patients.