Hepatocytes traffic and export hepatitis B virus basolaterally by polarity-dependent mechanisms

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.     

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.     

Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent role in the biogenesis of the canalicular membrane, reflected by its ability to sort and redistribute apical and basolateral sphingolipids. Here we show that SAC appears to be a crucial target for a cytokine-induced signal transduction pathway, which stimulates membrane transport exiting from this compartment promoting apical membrane biogenesis. Thus, oncostatin M, an IL-6-type cytokine, stimulates membrane polarity development in HepG2 cells via the gp130 receptor unit, which activates a protein kinase A-dependent and sphingomyelin-marked membrane transport pathway from SAC to the apical membrane. To exert its signal transducing function, gp130 is recruited into detergent-resistant membrane microdomains at the basolateral membrane. These data provide a clue for a molecular mechanism that couples the biogenesis of an apical plasma membrane domain to the regulation of intracellular transport in response to an extracellular, basolaterally localized stimulus.     

Hepatitis A virus-specific immunoglobulin A mediates infection of hepatocytes with hepatitis A virus via the asialoglycoprotein receptor

The mechanisms underlying the hepatotropism of hepatitis A virus (HAV) and the relapsing courses of HAV infections are unknown. In this report, we show for a mouse hepatocyte model that HAV-specific immunoglobulin A (IgA) mediates infection of hepatocytes with HAV via the asialoglycoprotein receptor, which binds and internalizes IgA molecules. Proof of HAV infection was obtained by detection of HAV minus-strand RNA, which is indicative for virus replication, and quantification of infectious virions. We demonstrate that human hepatocytes also ingest HAV-anti-HAV IgA complexes by the same mechanism, resulting in infection of the cells, by using the HepG2 cell line and primary hepatocytes. The relevance of this surrogate receptor mechanism in HAV pathogenesis lies in the fact that HAV, IgA, and antigen-IgA complexes use the same pathway within the organism, leading from the gastrointestinal tract to the liver via blood and back to the gastrointestinal tract via bile fluid. Therefore, HAV-specific IgA antibodies produced by gastrointestinal mucosa-associated lymphoid tissue may serve as carrier and targeting molecules, enabling and supporting HAV infection of IgA receptor-positive hepatocytes and, in the case of relapsing courses, allowing reinfection of the liver in the presence of otherwise neutralizing antibodies, resulting in exacerbation of liver disease.     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

(Glyco)sphingolipids Are Sorted in Sub-Apical Compartments in HepG2 Cells: A Role for Non-Golgi–Related Intracellular Sites in the Polarized Distribution of (Glyco)sphingolipids

In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C₆-NBD-GlcCer) and sphingomyelin (C₆-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van IJzendoorn, S.C.D., M.M.P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell Biol. 137:347–457). In the present study we have identified a non-Golgi–related, sub-apical compartment (SAC), in which sorting of the lipids occurs. Thus, in the apical to basolateral transcytotic pathway both C₆-NBD-GlcCer and C₆-NBD-SM accumulate in SAC at 18°C. At this temperature, transcytosing IgA also accumulates, and colocalizes with the lipids. Upon rewarming the cells to 37°C, the lipids are transported from the SAC to their preferred membrane domain. Kinetic evidence is presented that shows in a direct manner that after leaving SAC, sphingomyelin disappears from the apical region of the cell, whereas GlcCer is transferred to the apical, bile canalicular membrane. The sorting event is very specific, as the GlcCer epimer C₆-NBD-galactosylceramide, like C₆-NBD-SM, is sorted in the SAC and directed to the basolateral surface. It is demonstrated that transport of the lipids to and from SAC is accomplished by a vesicular mechanism, and is in part microtubule dependent. Furthermore, the SAC in HepG2 bear analogy to the apical recycling compartments, previously described in MDCK cells. However, in contrast to the latter, the structural integrity of SAC does not depend on an intact microtubule system. Taken together, we have identified a non-Golgi–related compartment, acting as a “traffic center” in apical to basolateral trafficking and vice versa, and directing the polarized distribution of sphingolipids in hepatic cells.     

Basolateral Internalization of GPI-anchored Proteins Occurs via a Clathrin-independent Flotillin-dependent Pathway in Polarized Hepatic Cells

In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2–positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.     

Aminopeptidase N is directly sorted to the apical domain in MDCK cells

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.     

Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model

Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.     

Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.     

Steady state analysis and experimental validation of a model for hepatic high-density lipoprotein transport

Transport of high-density lipoprotein (HDL) in the hepatocyte plays a fundamental role in reverse cholesterol transport and regulation of plasma HDL levels. On the basis of a recently developed kinetic model, the steady state distribution of HDL was analyzed. Fractional fluorescence of labeled HDL in the basolateral membrane, sorting endosomes (SE), the subapical compartment/ apical recycling compartment, the biliary canaliculus and in late endosomes and lysosomes (LE/LYS) including expected standard deviation is predicted. Improved parameter estimation was obtained by including kinetic data of apical endocytosis of fluorescent markers for LE/LYS, asialoorosomucoid and Rhodamine-dextran, in the regression. Predicted values using the refined kinetic parameters are in good agreement with experimental values of compartmental steady state fluorescence of Alexa488-HDL in polarized hepatic HepG2 cells. From calculated steady state fluxes, it is suggested that export of HDL from basolateral SE is the key step for determining the transport of HDL through the hepatocyte. The analysis provides testable predictions for high-throughput fluorescence microscopy screening experiments on potential inhibitors of hepatic HDL processing. By quantitative fluorescence imaging and model analysis, it is shown that the phosphoinositide kinase inhibitor wortmannin prevents apical transport of fluorescent HDL from basolateral SE. The results support that endosomes of polarized hepatic cells have different sorting functions and that apical endocytosis is an integrative trafficking step in hepatocytes.     

WIF-B cells: an in vitro model for studies of hepatocyte polarity

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction- associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC- dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC- dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.     

Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Mechanical compression attenuates normal human bronchial epithelial wound healing

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.     

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.     

Vasopressin-induced morphological changes in polarized rat hepatocyte multiplets: dual calcium-dependent effects

Calcium-mobilizing hormones and neurotransmitters are known to affect cell morphology and function including cell differentiation or division. In this study, we examined vasopressin (AVP)-induced morphological changes in a polarized system of rat hepatocytes. Light and electron microscope observations showed that AVP induced microvilli formation and a remodeling of the isolated hepatocyte F-actin submembrane cytoskeleton, these two events being correlated. We showed that these effects were rapid, reversible, observed at nanomolar AVP concentration and mediated by the V(1a) receptor. On polarized multicellular systems of hepatocytes, we observed a rapid reduction of the bile canaliculi lumen at the apical pole and micovilli formation at the basolateral domain with an enlarged F-actin cytoskeleton. Neither activation of protein kinase C nor A via phorbol ester or dibutyryl cAMP induced such rapid morphological changes, at variance with ionomycin, suggesting that AVP-induced intracellular calcium rise plays a crucial role in those effects. By using spectrofluorimetry and cytochemistry, we showed that calcium release from intracellular stores was involved in bile canaliculus contraction, while calcium entry from the extracellular space controlled microvilli formation. Taken together, AVP and calcium-mobilizing agonists differentially regulate physiological hepatocyte plasma membrane events at the basal and the apical domains via topographically specialized calcium-dependent mechanisms.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells

Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (CoV) known as SARS-CoV, is a contagious and life-threatening respiratory illness with pneumocytes as its main target. A full understanding of how SARS-CoV would interact with lung epithelial cells will be vital for advancing our knowledge of SARS pathogenesis. However, an in vitro model of SARS-CoV infection using relevant lung epithelial cells is not yet available, making it difficult to dissect the pathogenesis of SARS-CoV in the lungs. Here, we report that SARS-CoV can productively infect human bronchial epithelial Calu-3 cells, causing cytopathic effects, a process reflective of its natural course of infection in the lungs. Indirect immunofluorescence studies revealed a preferential expression of angiotensin-converting enzyme 2 (ACE-2), the functional receptor of SARS-CoV, on the apical surface. Importantly, both ACE-2 and viral antigen appeared to preferentially colocalize at the apical domain of infected cells. In highly polarized Calu-3 cells grown on the membrane inserts, we found that cells exposed to virus through the apical rather than the basolateral surface showed high levels of viral replication. Progeny virus was released into the apical chamber at titers up to 5 logs higher than those recovered from the basolateral chambers of polarized cultures. Taken together, these results indicate that SARS-CoV almost exclusively entered and was released from the apical domain of polarized Calu-3 cells, which might provide important insight into the mechanism of transmission and pathogenesis of SARS-CoV.