Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles

Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.     

Endocytosis and exocytosis of protein in capillary endothelium

The transport of proteins across continuous capillary endothelium is believed to be mediated by micropinocytic vesicles which shuttle molecules between the lumenal and abluminal plasma membrane. We have studied the ability of capillary endothelial cells isolated from rat epididymal fat to endocytose fluorescently labelled ovalbumin within micropinocytic vesicles. Net association of fluorescent ovalbumin with endothelial cells reaches an equilibrium after 40 minutes of incubation. This equilibrium is presumably due to a balance between endocytosis and subsequent exocytosis of this protein. Capillaries equilibrated with fluorescent ovalbumin exhibited rapid exocytosis of this protein when it was removed from the external medium. The rate of endocytosis was concentration dependent and obeyed the kinetics expected for adsorptive phase endocytosis. High concentrations of ovalbumin stimulated the ingestion of 14C-sucrose, a marker of fluid endocytosis, suggesting that protein can affect the movement of vesicles within the endothelial cytoplasm. These results imply that capillary endothelium isolated from rat epididymal fat exhibits the ability to endocytose and subsequently exocytose protein. This demonstrates that the two components of endothelial vesicular transport or transcytosis can be observed and studied in a system of isolated capillary endothelium.     

From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcgamma receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn(+)) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4(+)Rab11(+) or Rab11(+) compartments. For Rab4(+)Rab11(+) compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11(+)FcRn(+) vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.     

Placental alkaline phosphatase, a GPI-anchored protein, is clustered in clathrin-coated vesicles.

Pure clathrin-coated vesicles were prepared from a fresh human placenta. The analysis of their content revealed the presence of transferrin, low density lipoproteins, IgG and placental alkaline phosphatase. Since the latter is a membrane protein anchored by a glycan-phosphatidyl inositol (GPI) moiety, its presence in coated vesicles was unexpected. Placental alkaline phosphatase is neither adsorbed to the surface of the vesicles, nor appearing due to plasma membrane contaminants, but is located in the lumen of the vesicles. The presence of alkaline phosphatase in coated vesicles strengthens its postulated physiological role in the transcytosis of IgG molecules in placenta.     

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

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.     

Ectoplasmic insertion of a glycosylphosphatidylinositol-anchored protein in glycosphingolipid- and cholesterol-containing phosphatidylcholine vesicles.

Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.     

Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes

The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern. Horseradish peroxidase uptake was extensively inhibited by incubation at 0-4 degrees C consistent with an endocytic mechanism. To further characterize the process, the fate of labeled protein was investigated. Following uptake [3H] hemoglobin A was extensively degraded (40-50%) to acid-soluble products within 10 h. Degradation displayed a biphasic pattern with a rapid early phase followed by a much slower second phase. The decreasing rate of proteolysis can be accounted for, in part, by a simultaneous exocytosis of a substantial fraction (25-30%) of acid-insoluble label from myotubes. The lysosomotropic agents methylamine, monensin, and chloroquine significantly inhibited (23-75%) proteolysis, indicating a lysosomal site of degradation. Part of the inhibitory effect results from an increase in exocytosis in the presence of these agents. Degradation of endocytosed [3H]hemoglobin A was not inhibited by insulin. In contrast degradation of endogenous myotube proteins was inhibited (40%) by insulin and blocked by methylamine. These results suggest that cultured myotubes possess a coupled endocytic/exocytic pathway for macromolecules and that a fraction of the internalized substrate is degraded by an insulin-insensitive lysosomal pathway.     

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, DeltaS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical DeltaS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, mu1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.     

Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells

Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of 20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K⁺ and 2 mM Ca²⁺) provoking intense exocytosis (51 ± 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 ± 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots (0.7 µm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions. endocytosis; mouse chromaffin cells; calcium signal; FM1-43; ADVASEP-7; bromophenol blue     

Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in Rab11-positive endosomes

Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.     

Nerve growth factor-dependent sorting of synaptotagmin IV protein to mature dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598-5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca(2+)-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.     

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.     

Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This “burst phase” lasts about 3–5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol.40, 245–255; D. Epel, 1978, Curr. Top. Dev. Biol.12, 185–246) that initiation of endocytosis is most likely a Ca²⁺-dependent event.     

Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus

Adaptins are the major components of adaptors, the protein complexes that link clathrin to transmembrane proteins (e.g., receptors) in coated pits and vesicles. The plasma membrane adaptor contains an alpha- adaptin subunit and a beta-adaptin subunit, while the Golgi adaptor contains a gamma-adaptin subunit and a beta'-adaptin subunit. A partial cDNA clone encoding gamma-adaptin was isolated from a bovine brain expression library by screening with antibodies, and was used to obtain a cDNA clone from a mouse brain library containing the full coding sequence. The identity of the clones was confirmed by protein sequencing. The deduced amino acid sequence of gamma-adaptin was found to be homologous to that of alpha-adaptin, with several stretches of identical amino acids or conservative substitutions in the first approximately 70 kD, and 25% identity overall. Weaker homology was seen between gamma- and beta-adaptins. Like both alpha- and beta-adaptins, gamma-adaptin has a proline and glycine-rich hinge region, dividing it into NH2- and COOH-terminal domains. A chimeric gamma-adaptin was constructed from the mouse and bovine cDNAs and transfected into Rat 1 fibroblasts. Immunofluorescence microscopy was carried out using an mAb which recognizes an epitope present on the chimera but not found on the rodent protein. The construct was found to have a distribution typical of endogenous gamma-adaptin. Using this transfection system, it should now be possible to exchange domains between alpha- and gamma-adaptins, to try to find out how adaptors are targeted to the appropriate membrane compartment of the cell, and how they recruit the appropriate receptors into the coated vesicle.     

An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.     

Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system

In the flagellated protozoon Trypanosoma brucei, endo- and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D-fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol-anchored glycoproteins, because 10(7) molecules of the variant surface glycoprotein form a dense coat at the cell's surface. Because the endocytic rate varies widely at different stages in the parasite's life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.     

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.