Role of endosomes in simian virus 40 entry and infection.

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

Host cell factors and functions involved in vesicular stomatitis virus entry.

Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although associated with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-associated proteins and cellular functions.

Ameobal pathogen mimivirus infects macrophages through phagocytosis.

Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae.

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

Synthesis of junctional proteins in metastasizing colon cancer cells.

Various authors have reported reduced synthesis of epithelial junctional proteins during dedifferentiation, tumorigenesis and metastasis in a great variety of tumors. Consequently, it is generally accepted that loss of adhesive molecules and adhesion structures is implicated in the development of an invasive phenotype and poor patient prognosis. Colon carcinomas, on the other hand, were shown to behave differently as synthesis of main adhesive proteins continues despite the development of an invasive phenotype. In this study we used cultured cells grown under conditions that inhibited intercellular adhesion (low Ca2+ concentration) and compared these results with data obtained from metastasizing colon cancer cells (signet ring cell carcinoma). Characterization of these proteins and their structures were performed by immunoprecipitations, Western blot analysis, immunohistochemistry, pre-embedding immuno-electron microscopy, and a new method to perform immuno-electron microscopy on paraffin-embedded material, which we present in this paper. We demonstrate that synthesis carries on for both, the desmosomal and the proteins of the zonula adhaerens. While, however, the assembly of desmosomal structures in the form of half-desmosomes at the cell surface continues, those of the zonula adhaerens did not. Instead E-cadherin was found, although associated with alpha-catenin, beta-catenin, and plakoglobin, evenly distributed at the plasma membrane of the cultured cells and also at the surface of the dissociated tumor cells. We conclude from our observations that continued expression and synthesis of junctional proteins do not necessarily contribute to the suppression of tumor invasion and metastasis of colon cancer.

Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.

The occurrence of extended tight junction (TJ) structures, including zonulae occludentes (ZO), and the spatial arrangement of TJ proteins in stratified mammalian epithelia has long been controversially discussed. Therefore, we have systematically examined the localization of TJ proteins in diverse stratified epithelial tissues (e.g., epidermis, heel pad, snout, gingiva, tongue, esophagus, exocervix, vagina, urothelium, cornea) of various species (human, bovine, rodents) as well as in human cell culture lines derived from stratified epithelia, by electron microscopy as well as by immunocytochemistry at both the light and the electron microscopic level, using antibodies to TJ proteins such as occludin, claudins 1 and 4, protein ZO-1, cingulin and symplekin. We have found an unexpected diversity of TJ-related structures of which only those showing colocalization with the most restricted transmembrane TJ marker protein, occludin, are presented here. While in epidermis and urothelium occludin is restricted to the uppermost living cell layer, TJ-related junctions are abundant in the upper third or even in the majority of the suprabasal cell layers in other stratified epithelia. Interfollicular epidermis contains, in the stratum granulosum, extended, probably continuous ZO-like structures which can also be traced at least through the Henle cell layer of hair follicles. Similar apical ZO-like structures have been seen in the upper living cell layers of all other stratified epithelia and cell cultures examined, but in most of them we have noticed, in addition, junctional regions showing relatively broad, ribbon-like membrane contacts which in cross-section often appear pentalaminar, with an electron-dense middle lamella ("lamellated TJs", coniunctiones laminosae). In suprabasal layers of several stratified epithelia we have further observed TJ protein-containing junctions of variable sizes which are characterized by a 10-30-nm dense lamina interposed between the two membranes ("sandwich junctions"; iuncturae structae). Moreover, we have often observed variously sized regions in which the intermembrane distance is rather regularly bridged by short rod-like elements ("cross-bridged cell walls"; parietes transtillati), often in close vicinity of TJ-related structures or desmosomes. The significance of these structures and their possible biological importance are discussed.

Wnt-signaling and apoptosis after neoadjuvant short-term radiotherapy for rectal cancer.

Recent surgical concepts for primary rectal cancer include the combination of surgery and short-term neoadjuvant radiotherapy (STNR). This is usually given in a dose of 25 Gy over five days in order to reduce local recurrence rates. Clinical studies have shown that local recurrence is found in some patients despite STNR. We identified molecular patterns of the Wnt- and apoptosis pathways as well as expression of junction-associated molecules in rectal cancer specimens of patients who received STNR and in those who did not. Expression patterns were examined by immunohistochemistry and molecular techniques such as LightCycler RT-PCR and Western blot analysis in 25 sporadic rectal adenocarcinoma specimens derived from STNR-patients or non-pretreated donors, respectively. The molecular pattern in response to STNR was heterogeneous and was reflected by responders who show activation of apoptosis and cellular remodeling, whereas the group of non-responders from STNR did not show such reaction and was very similar to untreated controls. Enhanced expression of beta-catenin was generally mediated by STNR, but exclusively in the responder group impaired expression of c-Myc and junction-associated molecules as well as cleavage of poly-ADP-ribose polymerase and of the caspase substrate cytokeratin 19 were found. The molecular profile suggests that STNR interferes with Wnt-signaling and c-Myc expression. STNR in its present form is not suitable to fully complete the sequence of apoptosis in all rectal adenocarcinomas.

Impaired expression of acyl-CoA-synthetase 5 in epithelial tumors of the small intestine.

Fatty acids are implicated in tumorigenesis, but data are limited concerning endogenous fatty acid metabolism of tumor cells in adenomas and adenocarcinomas of the small intestine. The recently cloned human acyl-CoA-synthetase 5 (ACS5) is predominantly found in the small intestine and represents a key enzyme in providing cytosolic acyl-CoA thioesters. Protein synthesis and mRNA expression of ACS5 were studied in human intestinal tissues using different methods, including a newly established monoclonal antibody. In the healthy small intestine, expression of ACS5 was restricted to the villus surface epithelium but was not detectable in enterocytes lining crypts. ACS5 protein and mRNA were progressively diminished in epithelial cells of adenomas and adenocarcinomas of the small intestine. In conclusion, altered expression of ACS5 is probably related to the adenoma-carcinoma sequence of small intestinal epithelial tumors due to an impaired acyl-CoA thioester synthesis.

GM1 structure determines SV40-induced membrane invagination and infection.

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

Staining patterns of keratins in the human urinary tract.

The keratins, members of the intermediate filament family, are characteristically expressed in epithelial cells. In the various types of epithelia, the keratin expression pattern is characterized by cell-type specific combinations of the keratin isotypes with a plain pattern in monolayered (simple) epithelia and more complex patterns in stratified and pseudostratified epithelia. Here we demonstrate that the transitional epithelium of the human urinary tract holds an exceptional position between the pseudostratified and stratified epithelia. We show that the simple epithelia keratins 7, 8, 18 and 19 are expressed throughout the whole epithelium as known from pseudostratified epithelia. In addition, we demonstrate expression of keratins 5, 14 and 17, otherwise present in basal cells of multilayered epithelia, and keratins 4 and 13, present in suprabasal areas of non cornified multilayered epithelia. Moreover, we report differences in expression in the various morphological parts of the urinary tract which might be related to their specific functions. Keratin 20, a typical component of the simple epithelia of the digestive tract, is present in bladder and ureter but not in the renal pelvis. Keratin 6, typical for stratified epithelia, is found only in parts of the renal pelvis. We further show that changes in keratin pattern occur during the development from embryonic to adult bladder urothelium. In contrast to adult tissue, the simple type keratins 7, 8 and 18 are not synthesized in basal embryonic cells. Further, keratin 20, present in cells facing the bladder lumen in adult urothelium, is expressed in all but the basal cells in embryonic bladder.

Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes.

The endocytic entry of lymphocytic choriomeningitis virus (LCMV) into host cells was compared to the entry of viruses known to exploit clathrin or caveolae/raft-dependent pathways. Pharmacological inhibitors, expression of pathway-specific dominant-negative constructs, and siRNA silencing of clathrin together with electron and light microscopy provided evidence that although a minority population followed a classical clathrin-mediated mechanism of entry, the majority of these enveloped RNA viruses used a novel endocytic route to late endosomes. The pathway was clathrin, dynamin-2, actin, Arf6, flotillin-1, caveolae, and lipid raft independent but required membrane cholesterol. Unaffected by perturbation of Rab5 or Rab7 and apparently without passing through Rab5/EEA1-positive early endosomes, the viruses reached late endosomes and underwent acid-induced penetration. This membrane trafficking route between the plasma membrane and late endosomes may function in the turnover of a select group of surface glycoproteins such as the dystroglycan complex, which serves as the receptor of LCMV.

Characterization of a novel type of adherens junction in meningiomas and the derived cell line HBL-52.

Adhering junctions are generally grouped into desmosomes and adherens junctions based on their ultrastructural appearance and molecular composition. The armadillo-protein plakoglobin is common to both types of junctions, which are otherwise composed of mutually exclusive proteins. This view is based on observations in epithelial tissues but cannot easily be transferred to other cell types and tissues, as has become apparent during the last decade with the identification of new junctional proteins and the investigation of further non-epithelial junctions. Using a broad array of well-characterized specific antibodies against key junctional proteins in immunoblot reactions, high-resolution double-label laser scanning confocal microscopy, and immunoelectron microscopy, we describe a new type of adherens junction in human meningiomas and the human meningioma cell line HBL-52. This novel junction has a unique composition of proteins not found in any other tissue; it contains the desmosomal armadillo-protein plakophilin 2 together with the classic proteins of "epithelial" adherens junctions, i.e., E-cadherin (in some instances replaced by N-cadherin), alpha-catenin, beta-catenin, plakoglobin, and p120(ctn). Ultrastructurally, it is formed between two or three neighboring cells. For pragmatic reasons, we suggest the name "meningeal junction" for this new structure.

NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

E-cadherin is a selective and strongly dominant prognostic factor in squamous cell carcinoma: a comparison of E-cadherin with desmosomal components.

E-cadherin-mediated and desmosomal cell-cell adhesion have been implicated in the suppression of invasive and metastatic behavior of squamous cell carcinomas. Whether the adhaerens junction represented by E-cadherin and the desmosomes interplay or have distinct and separate roles in squamous cell cancer progression is still unclear. We have studied a cohort of 200 primary tumors and 56 lymph node metastases from different anatomic sites of the head and neck region for changes in synthesis of E-cadherin, desmoplakin and desmoglein by immunohistochemistry (IHC). Selected cases were studied by indirect immunofluorescence (IIF) and electron microscopy (EM). Only frozen sections were evaluated since they gave stronger and reproducible staining results. IHC data obtained were compared to clinical parameters. While some reduction in immunostaining was found in virtually all invasive tumors, at least partial expression, including that of E-cadherin, persisted in most late stage tumors and in lymph node metastases. Reduced desmosomal staining correlated with desmosomes reduced in numbers, size or in structural defects by EM analysis. By univariate analysis, reduction in synthesis of both E-cadherin and the desmosomal components that were generally linked (i.e., they showed positive rank correlations) were significantly associated with clinical parameters including overall and disease-free survival. However, by multivariate analysis including a Cox proportional hazards regression model (backward selection), the desmosomal components were not significant as independent prognostic factors. By contrast, E-cadherin was strongly associated with patient prognosis. In line with the highly significant association of reduced E-cadherin synthesis with an increased relative risk of follow up events, i.e., regional lymph node (p = 0.0007) and distant metastasis (p < 0.0001), as well as local recurrences (p < 0.0001), the prognostic strength of E-cadherin was independent of and stronger than histological grading, N stage, tumor site, and even stronger than the TNM stage. Based on these results, evaluation of E-cadherin in squamous cell carcinomas by immunostaining is recommended as a significant prognostic marker.

Release and intercellular transfer of cell surface CD81 via microparticles.

The human tetraspan molecule CD81 is a coreceptor in B and T cell activation and a candidate receptor for hepatitis C virus infection. We examined the surface expression of CD81 on B and T lymphocytes by quantitative flow cytometry. Upon cellular activation, CD81 surface levels were rapidly reduced. This reduction occurred as early as 1 h after activation and was linked to the release of CD81-positive microparticles into the cell culture medium. CD81 mRNA levels were not affected early after activation, but the release of CD81-positive microparticles was rapidly enhanced. In addition, intercellular transfer of CD81 was observed upon coculture of CD81-positive donor cells (Jurkat T cell line) with CD81-negative acceptor cells (U937 promonocytic cell line). This transfer was rapidly increased upon T cell activation, coinciding with enhanced CD81 release from activated Jurkat cells. We propose that the release and intercellular trafficking of CD81-positive microparticles regulate the expression of CD81 surface receptors in lymphocytes and play a role in the immune response during infections.

Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol.

Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 alpha and beta kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway.

Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus.

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

Molecular characterization of desmosomes in meningiomas and arachnoidal tissue.

Intercellular junctions morphologically identical to epithelial desmosomes are known structures in meningiomas and arachnoidal tissue. Desmoplakin as one of the desmosomal plaque components has proven to be a reliable marker for diagnosis of meningeal tumors. Here we demonstrate by immunofluorescence microscopy, immunoblot and reverse transcription-PCR reactions that cells of arachnoidal tissue, of diverse meningioma subtypes and of a meningioma-derived cell line contain the full complement of the typical desmosomal proteins desmoplakin (DP), plakophilin 2 (PP2), desmocollin 2 (Dsc2) and desmoglein 2 (Dsg2). Consequently, all these molecules are suitable for diagnostic applications of meningioma tumors. In addition to these constitutive desmosomal components, representative for single-layered (simple) epithelia, the dural border cells of the arachnoid and about 60% of the meningiomas tested were positive for desmocollin 3 (Dsc3), a protein in epithelia taken as an indicator for differentiation.

Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif.

The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in core-expressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.