Increased mean corpuscular haemoglobin concentration: artefact or pathological condition?

INTRODUCTION: In daily practice in haematology laboratories, spurious increased MCHC induces an analytical alarm and needs prompt corrective action to ensure delivery of the right results to the clinicians. The aim of this study was to establish a 'decision tree' using the new parameters red blood cells (RBC-O) and haemoglobin (HGB-O) from the Sysmex XN-10 RET obtained by flow cytometry to deliver appropriate results. METHODS: From 128 unknown patients with MCHC > 365 g/L, all erythrocyte parameters including reticulocyte parameters were measured and analysed in parallel with blood smears, chemistry index and osmolarity. Differences between optical parameters (RBC-O, HGB-O) and usual parameters (RBC, HGB) obtained by impedance and photometry were reported also. RESULTS: Four groups were defined from observations: -RBC agglutination (n = 22); -optical interference (n = 17); -RBC disease (n = 18); and -others (n = 71). The use of RBC-O and HGB-O permitted efficient correction of the abnormalities when RBC agglutination and/or optical interference were present in 36 of 39 patients. Reticulocyte parameters permitted to elaborate an RBC score that allowed a highly sensitive detection of RBC disease patients (17/18). CONCLUSION: Based on new parameters, we propose a 'decision tree' that delivers time savings and supports biological interpretation in case of elevated MCHC.

Results obtained with various antifungal susceptibility testing methods do not predict early clinical outcome in patients with cryptococcosis.

The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.

Src-dependent ezrin phosphorylation in adhesion-mediated signaling.

In addition to providing a regulated linkage between the membrane and the actin cytoskeleton, ezrin participates in signal transduction pathways. Here we describe that expression of the ezrin Y145F mutant delays epithelial cell spreading on fibronectin by inhibiting events leading to FAK activation. The defect in spreading was rescued by the overexpression of catalytically functional Src. We demonstrate that ezrin Y145 is phosphorylated in A431 cells stimulated with epidermal growth factor (EGF) and in v-Src-transformed cells. Moreover in cells devoid of Src, SYF-/- fibroblasts, ezrin Y145 phosphorylation could only be detected upon the introduction of an active form of Src. The phosphorylation of ezrin at Y145 required prior binding of the Src SH2 domain to ezrin. Our results further show that Src activity influences its binding to ezrin and a positive feedback mechanism for Src-mediated Y145 phosphorylation is implied. Interestingly, cells expressing ezrin Y145F did not proliferate when cultured in a 3D collagen gel. Collectively, our results demonstrate a key signaling input of Src-dependent ezrin phosphorylation in adhesion-mediated events in epithelial cells.

Na(+)/H(+) exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells.

Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

The membrane-microfilament linker ezrin is involved in the formation of the immunological synapse and in T cell activation.

Dynamic interactions between membrane and cytoskeleton components are crucial for T cell antigen recognition and subsequent cellular activation. We report here that the membrane-microfilament linker ezrin plays an important role in these processes. First, ezrin relocalizes to the contact area between T cells and stimulatory antigen-presenting cells (APCs), accumulating in F-actin-rich membrane protrusions at the periphery of the immunological synapse. Second, T cell receptor (TCR)-mediated intracellular signals are sufficient to induce ezrin relocalization, indicating that this protein is an effector of TCR signaling. Third, overexpression of the membrane binding domain of ezrin perturbs T cell receptor clustering in the T cell-APC contact area and inhibits the activation of nuclear factor for activated T cells (NF-AT).

Enteropathogenic Escherichia coli activates ezrin, which participates in disruption of tight junction barrier function.

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.

Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Ezrin-dependent promotion of glioma cell clonogenicity, motility, and invasion mediated by BCL-2 and transforming growth factor-beta2.

Ezrin belongs to the ezrin-radixin-moesin family proteins, which cross-link actin cytoskeleton and plasma membrane. Malignant glioma cells are paradigmatic for their strong migratory and invasive properties. Here, we report that the expression of dominant-negative ezrins inhibits clonogenicity, migration, and invasiveness of human malignant glioma cells. Furthermore, dominant-negative ezrins block hepatocyte growth factor (HGF)-mediated stimulation of clonogenicity and migration, without altering HGF-induced protein kinase B/Akt and focal adhesion kinase phosphorylation. Glioma cells expressing dominant-negative ezrins exhibit a shift of the BCL-2/BAX rheostat toward apoptosis, reduced alpha(V)beta(3) integrin expression and reduced matrix metalloproteinase (MMP) expression and activity. These changes are associated with a dramatic loss of transforming growth factor beta(2) (TGF-beta(2)) release. Exogenous supplementation of TGF-beta(2) overcomes the inhibitory effects of dominant-negative ezrins on migration and clonogenicity. A neutralizing TGF-beta(2) antibody mimics the effects of dominant-negative ezrins on clonogenicity and migration. Exogenous HGF markedly induces TGF-beta(2) protein levels, and a neutralizing TGF-beta(2) antibody abolishes the HGF-mediated increase in glioma cell motility. Finally, TGF-beta(2) does not modulate BCL-2 or BAX expression, but BCL-2 gene transfer increases the levels of latent and active TGF-beta(2). Intracranial xenografts of U87MG glioma cells transfected with the dominant-negative ezrins in athymic mice grow to significantly smaller volumes, and the median survival of these mice is 50 d compared with 28 d in the control group. These data define a novel pathway for HGF-induced glioma cell migration and invasion, which requires ezrin, changes in the BCL-2/BAX rheostat, and the induction of TGF-beta(2) expression in vitro, and underscore the important role of HGF signaling in vivo.

Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes.

The small G protein RhoA and its GDP/GTP exchange factors (GEFs) Net and Dbl can transform NIH 3T3 fibroblasts, dependent on the activity of the RhoA effector kinase ROCK. We investigated the role of the cytoskeletal linker protein ezrin in this process. RhoA effector loop mutants which can bind ROCK induce relocalization of ezrin to dorsal actin-containing cell surface protrusions, as do Net and Dbl. Both processes are inhibited by the ROCK inhibitor Y27632, which also inhibits association of ezrin with the cytoskeleton, and phosphorylation of T567, conserved between ezrin and its relatives radixin and moesin. ROCK can phosphorylate the ezrin C-terminus in vitro. The ezrin mutant T567A cannot be relocalized by activated RhoA, Net or Dbl or by ROCK itself, and also inhibits RhoA-mediated contractility and focal adhesion formation. Moreover, ezrin T567A, but not wild-type ezrin, restores contact inhibition to Net- and Dbl-transformed cells, and inhibits the activity of Net and Ras in focus formation assays. These results implicate ROCK-mediated ezrin C-terminal phosphorylation in transformation by RhoGEFs.

Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane.

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH(2)- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway.

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.

A functional role for ezrin during Shigella flexneri entry into epithelial cells.

Shigella flexneri is an enteroinvasive bacterium responsible for bacillary dysentery in humans. Bacterial entry into epithelial cells is a crucial step for the establishment of the infection. It is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment by a macropinocytic process. We show in this study that the membrane-cytoskeleton linker, ezrin, a member of the ERM (ezrin, radixin, moesin) family, plays an active role in the process of Shigella uptake. Ezrin is highly enriched in cellular protrusions induced by the bacterium and is found in close association with the plasma membrane. In addition, Shigella entry is significantly reduced in cells transfected with a dominant negative allele of ezrin with entry foci showing much shorter cellular protrusions. These results indicate that ezrin not only acts as a membrane-cytoskeleton linker, but may also mediate extension of cellular projections in the presence of signals such as those elicited by invading microorganisms.

Effect of hepatocyte growth factor on assembly of zonula occludens-1 protein at the plasma membrane.

Hepatocyte growth factor (HGF) is a paracrine cytokine that influences epithelial morphogenesis by modulating cell-cell adhesion and cell polarity. We have examined the role of HGF in the tight junction (TJ) formation. We followed the assembly and disassembly at the plasma membrane of the major component of the TJ, zonula occludens-1 (ZO-1) protein, after HGF treatment. We applied HGF to the basolateral compartment of MDCK cell monolayers grown on transwell filters to analyze the effect of HGF on polarized cells. Confocal laser scanning microscopy showed that HGF caused a marked reduction of ZO-1 at the lateral sites and a concomitant increase in the cytoplasm. We used the calcium switch assay to analyze the effect of HGF in early TJ development. In MDCK cells cultured in low calcium levels, ZO-1 is distributed intracellularly. The presence of HGF greatly retarded the movement of ZO-1 from the cytosol to the membrane after restoration of normal (1.8 mM) calcium levels for 1.5 and 3 hr. The presence of HGF during the calcium switch caused increased tyrosine phosphorylation of beta-catenin. The incubation of MDCK cells with vanadate, a potent tyrosine-specific phosphatase inhibitor, also affected the ZO-1 localization at the plasma membrane during the calcium switch. This was concomitant with increased tyrosine phosphorylation of beta-catenin. These results suggest that HGF affects the TJ assembly, and this phenomenon may be important in loosening of intercellular junctions and migration of epithelial cells during HGF-induced morphogenesis.

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane.

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.

Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells.

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

Differential addressing of 5-HT1A and 5-HT1B receptors in transfected LLC-PK1 epithelial cells: a model of receptor targeting in neurons.

Based on the finding that the mechanism of membrane protein sorting may be similar in neurons and epithelial cells, we have investigated the localization of the 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors in Eli Lilly and Company canine kidney (LLC-PK1) cells (epithelial cells from pig kidney) stably transfected with the corresponding cDNAs. Receptors were characterized by radioligand binding, measurement of coupled adenylate cyclase activity and immunofluorescene with specific antibodies. Confocal microscopy and cell surface biotinylation experiments showed that the 5-hydroxytryptamine1A receptor, which is at the somatodendritic level in neurons, was targeted to the basolateral surface whereas the 5-hydroxytryptamine1B receptor, which is localized on nerve endings, predominated intracellularly. Such a difference in the localization of these two receptors suggests that the signal for the differential addressing of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors is contained within their primary sequence and confirms that epithelial cells can be used as a suitable model to study receptor targeting in neurons.

Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells.

Shigella flexneri is an enteroinvasive bacterium which causes bacillary dysentery in humans. A major feature of its pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization process requiring polymerization of actin. Here we describe the cytoskeletal rearrangements during S. flexneri invasion of HeLa cells. After an initial contact of the bacterium with the cell surface, distinct nucleation zones of heavy chain actin polymerization appear in close proximity to the contact site underneath the parasite with long filaments being polymerized. These structures then push cellular protrusions that rise beside the entering bacterium, being sustained by tightly bundled long actin filaments organized in parallel orientation with their positive ends pointing to the cytoplasmic membrane. Finally, the cellular projections coalesce above the bacterial body, leading to its internalization. In addition, we found the actin-bundling protein plastin to be concentrated in these protrusions. Since plastin is known to bundle actin filaments in parallel orientation, colocalization of parallel actin filaments and plastin in the cellular protrusions strongly suggested a functional role of this protein in the architecture of parasite-induced cellular projections. Using transfection experiments, we show the differential recruitment of the two plastin isoforms (T- and L-) into Shigella entry zones. By transient expression of a truncated T-plastin which is deprived of one of its actin-binding sites, we also demonstrate the functional role of T-plastin in Shigella entry into HeLa cells.