Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (相似文献   

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P₂, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.David J. Gillooly and Camilla Raiborg contributed equally     

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.     

Role of Rab5 in the recruitment of hVps34/p150 to the early endosome

PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo . We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.     

Atg14 and UVRAG: Mutually exclusive subunits of mammalian Beclin 1-PI3K complexes

Vps34, a Class III phosphatidylinositol 3-kinase (PI3-kinase), produces phosphatidylinositol 3 phosphate (PI3P) and functions in various membrane traffic pathways including endocytosis, multivesicular body formation and autophagy. In mammalian cells, Vps34 forms a complex with Beclin 1, but it remains unclear how this Vps34 complex exerts its specific function on each membrane trafficking pathway. We recently identified mammalian Atg14, a new binding partner of the Vps34-Beclin 1 complex, using a computational approach. The Atg14 complex consists of Vps34, Beclin 1 and p150, but lacks UVRAG, which was previously reported to bind the Vps34-Beclin 1 complex. Atg14 localizes to isolation membrane/phagophore during starvation and is essential for autophagosome formation. In contrast, UVRAG primarily localizes to late endosomes. Since UVRAG shows homology with yeast Vps38, we speculate that it could be a mammalian Vps38 ortholog. These findings indicate that the Vps34-Beclin 1 complex has at least two distinct functions, which can be promoted by its binding partners Atg14 and UVRAG.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.     

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.     

Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations

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.      

Effect of 3-methyladenine on the fusion process of macropinosomes in EGF-stimulated A431 cells

In the process of receptor-mediated endocytosis, the fusion of endosomes in vitro is known to be inhibited by wortmannin or LY294002; inhibitors of phosphoinositide 3-kinase (PI3K), suggesting that the activity of PI3K is required for the fusion of early endosomes. In macropinocytosis, a process of bulk fluid-phase endocytosis, however, it remains unclear whether PI3K is required for the fusion of macropinosomes, since the macropinosome formation is inhibited by the PI3K inhibitors. In this study, we examined the effect of 3-methlyadenine (3-MA), which shows a distinct specificity to the PI3K classes from wortmannin and LY294002, on the macropinosome formation and fusion in EGF-stimulated A431 cells. Unlike wortmannin or LY294002, 3-MA did not inhibit the uptake of fluorescent dextran by macropinocytosis. However, the fusion of macropinosomes was inhibited by 3-MA. By imaging of live-cells expressing fluorescent protein-fused tandem FYVE domains, we found that PtdIns(3)P appeared on the macropinosomal membrane shortly after the closure of macropinocytic cups and remained on macropinosomes even at 60-min age. The production of PtdIns(3)P and the recruitment of EEA1 to macropinosomes were abolished by the 3-MA treatment. Therefore, it is likely that 3-MA impairs recruitment of EEA1 by inhibiting PtdIns(3)P production and resultantly blocks the fusion of macropinosomes. These results suggest that the local production of PtdIns(3)P implicates the fusion of macropinosomes via EEA1 as well as conventional early endosomes. However, the long association of PtdIns(3)P with macropinosomes may well be a cell-type specific feature of A431 cells.     

Calmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway

Membrane polarity is maintained by a complex intermingling of various trafficking pathways, including basolateral and apical endocytosis. The present work was undertaken to better define the role of basolateral endocytic transport in apical membrane homeostasis. When polarized HepG2 hepatoma cells were incubated with calmodulin antagonists, the cells lost their polarity, as reflected by an inhibition of lipid transport of a fluorescent sphingomyelin to the apical membrane and an impediment of its recycling to the basolateral membrane. Instead, an accumulation of the lipid in dilated early endosomal compartments was observed, presumably due to a frustration of vesiculation. Interestingly, lipid transport to the apical pole, lipid recycling to the basolateral membrane and cell polarity were reestablished, while dilated compartments disappeared, when the cells were simultaneously treated with specific inhibitors of protein kinase C (PKC). Consistently, following activation of PKC, extensive dilation/vacuolation of early sorting endosomes was observed, very similar as seen upon treatment with calmodulin antagonists. Thus, the results indicate that membrane trafficking at early steps of the basolateral endocytic pathway in HepG2 cells is regulated by an intricate interplay between calmodulin and PKC. This interference, although not affecting endocytosis as such, compromises cell polarity by impeding membrane trafficking from early endosomes to the apical membrane.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

Dissecting the localization and function of Atg18, Atg21 and Ygr223c

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p), a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and -Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time, that Ygr223cp affects an autophagic process, namely micronucleophagy.     

Cytoplasmic Signals Mediate Apical Early Endosomal Targeting of Endotubin in MDCK Cells

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin–Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.     

Class III Phosphoinositide 3‐Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling

Recycling is a limiting step for receptor‐mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan‐class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP‐2, causing surface down‐regulation. GFP‐(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002‐treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin‐GFP and dependence of dynamin‐GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K‐III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.     

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.     

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40^phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1''s function in endocytosis.     

Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.     

Infectious adenovirus type 2 transport through early but not late endosomes

Receptor-mediated endocytosis is a major gate for pathogens into cells. In this study, we analyzed the trafficking of human adenovirus type 2 and 5 (Ad2/5) and the escape-defective temperature-sensitive Ad2-ts1 mutant in epithelial cancer cells. Ad2/5 and Ad2-ts1 uptake into endosomes containing transferrin, major histocompatibility antigen 1 and the Rab5 effector early endosome antigen 1 (EEA1) involved dynamin, amphiphysin, clathrin and Eps15. Cointernalization experiments showed that most of the Ad2/5 and Ad2-ts1 visited the same EEA1-positive endosomes. In contrast to Ad2/5, Ad2-ts1 required functional Rab5 for endocytosis and lysosomal transport and was sensitive to the phosphatidyl-inositol-3 (PI3)-kinase inhibitor wortmannin or the ubiquitin-binding protein Hrs for sorting from early to late endosomes. Endosomal escape of Ad2 was not affected by incubation at 19 degrees C, which blocked membrane sorting in early endosomes and inhibited Ad2-ts1 transport to lysosomes. Unlike Semliki Forest Virus (SFV), sorting of Ad2-ts1 to late endosomes was independent of Rab7 and Ad2/5 infection independent of EEA1. The data indicate that Ad2/5 and Ad2-ts1 use an invariant machinery for clathrin-mediated uptake to early endosomes. We suggest that the infectious Ad2 particles are either directly released from early endosomes to the cytosol or sorted by a temperature-insensitive and PI3-kinase-independent mechanism to an escape compartment different from late endosomes or lysosomes.