Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity.

Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

Molecular and functional characterization of a novel stefin analogue in large yellow croaker (Pseudosciaena crocea).

In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin). The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 11kDa, in which no signal peptide and potential N-glycoslation site are predicted. The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C1 cysteine peptidases: one glycine in the N-terminal region (G(6)) and Gln-Xaa-Val-Xaa-Gly motif (Q(48)LVAG(52)). It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines). Genomic analysis revealed that Lyccys gene, 757 nt long, consisted of three exons and two introns. The Lycstefin gene was constitutively expressed in various tissues examined although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, Lycstefin transcript was significantly up-regulated in spleen and head kidney while down-regulated in blood. Immuno-electron microscopy showed that Lycstefin was mainly localized in the cytoplasm of spleen cells of large yellow croaker, and also in the nucleus. Recombinant Lycstefin protein fused with glutathione S-transferase (rLycstefin) was shown to have strong inhibitory activity against papain with a K(i) of 1.3x10(-13)M. The in vivo experiments revealed that Lycstefin could not modulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and head kidney. To our knowledge, this is the first report on the molecular and functional identification of a stefin analogue in bony fish.

A new cell culture system for infection with hepatitis B virus that fuses HepG2 cells with primary human hepatocytes.

Hepatitis B virus (HBV) infection exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells, however none of the previously established experimental models can reproduce the natural process of HBV infection. In the present study, primary human hepatocytes were fused with HepG2 cells to establish the hybrid HepCHLine-4 cell line with high susceptibility to HBV. The HepCHLine-4 cells expressed HBV-specific antigen when co-incubated with HBV-positive serum from a hepatitis B patient. Post-infection, HBV relaxed circular DNA and covalently closed circular DNA were detected in HepCHLine-4 cells using a nested polymerase chain reaction, and HBV-specific particles were visualized by electron microscopy of the culture media of HepCHLine-4 cells. HepG2 cells were not susceptible to HBV infection under the same conditions. The HepCHLine-4 cells can be sub-cultured for > 12 months while maintaining susceptibility to HBV and may, therefore, be useful for studying HBV infection and the viral life cycle in human hepatocytes.

Dilated intercellular spaces as a marker of GERD.

Gastroesophageal reflux disease (GERD) is typically heralded by the substernal burning pain of heartburn. On endoscopic examination, about one third of GERD subjects with heartburn have erosive disease, and the remainder have nonerosive reflux disease (NERD). Unlike patients with erosive disease, those with NERD (approximately 50%) often do not respond to therapy with proton pump inhibitors (PPIs), raising the question of whether they have NERD and, if they do, whether the cause of their symptoms is similar to those who respond to PPIs. Recently, biopsies established that subjects with heartburn and PPI-responsive NERD, like those with erosive esophagitis, have lesions within the esophageal epithelium known as dilated intercellular space (DIS). In this article, we discuss the physicochemical basis for DIS in acid-injured esophageal epithelium and its significance in GERD. Although DIS is not pathognomic of GERD, it is a marker of a break in the epithelial (junctional) barrier reflecting an increase in paracellular permeability.

Evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment.

Human immunodeficiency virus type 1 (HIV-1) assembly occurs predominantly at the plasma membrane of infected cells. The targeting of assembly to intracellular compartments such as multivesicular bodies (MVBs) generally leads to a significant reduction in virus release efficiency, suggesting that MVBs are a nonproductive site for HIV-1 assembly. In the current study, we make use of an HIV-1 Gag-matrix mutant, 29/31KE, that is MVB targeted. We previously showed that this mutant is severely defective for virus particle production in HeLa cells but more modestly affected in primary macrophages. To more broadly examine the consequences of MVB targeting for virus production, we investigated 29/31KE particle production in a range of cell types. Surprisingly, this mutant supported highly efficient assembly and release in T cells despite its striking MVB Gag localization. Manipulation of cellular endocytic pathways revealed that unlike Vpu-defective HIV-1, which demonstrated intracellular Gag localization as a result of Gag endocytosis from the plasma membrane, 29/31KE mutant Gag was targeted directly to an MVB compartment. The 29/31KE mutant was unable to support multiple-round replication; however, this defect could be reversed by truncating the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 and by the acquisition of a 16EK change in matrix. The 16EK/29/31KE matrix mutant replicated efficiently in the MT-4 T-cell line despite maintaining an MVB-targeting phenotype. These results indicate that MVB-targeted Gag can be efficiently released from T cells and primary macrophages, suggesting that under some circumstances, late endosomal compartments can serve as productive sites for HIV-1 assembly in these physiologically relevant cell types.

Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning.

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production.

Salmeterol restores secretory functions in cystic fibrosis airway submucosal gland serous cells.

The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF.

Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by the progressive replacement of myocardial cells by fat and fibrous tissue. Here we describe the histopathological features of biopsied myocardium from a patient with ARVC. A large amount of adipose tissue was present in the biopsy specimen, and a group of myocardial cells were isolated as an island-like region in the adipose tissue. Electron microscopic examination of cardiomyocytes revealed a large number of intracellular lipid droplets, including some extremely large droplets. Disruptions of the plasma membrane and dissociation of intercellular junctions were associated with discharge of intracellular lipid droplets into the interstitial space. The high accumulation of intracellular lipid droplets may be involved in the pathogenesis of ARVC and may have played an important role in myocardial cell death and progressive replacement of cardiomyocytes by fatty tissue in the current case.

Probing cells with noble metal nanoparticle aggregates.

This review focuses on the integration of noble metal nanoparticle aggregates as tags and transport vessels in cellular applications. The natural tendency of nanoparticles to aggregate can be reduced through surface modification; however, this stabilization is often compromised in the cellular environment. The degree of nanoparticle aggregation has both positive and negative consequences. Nanoparticle aggregates are more efficiently removed by the organism compared with single nanoparticles, preventing delivery to their cellular target. In addition, these aggregates are recognized by cells in different ways versus isolated nanoparticles. Despite these negatives, aggregates exhibit enhancement for many detection and treatment techniques in comparison with single nanoparticles. In coming years, the role of aggregates and better control over the degree of aggregation in cellular studies will be required for the realization of medical applications.

Morphological changes in intracellular lipid droplets induced by different hepatitis C virus genotype core sequences and relationship with steatosis.

UNLABELLED: Hepatocellular steatosis is common in patients with chronic hepatitis C. Steatosis can be considered as a true cytopathic lesion induced by hepatitis C virus (HCV) genotype 3, suggesting that one or more viral proteins produced during genotype 3 infection are involved in the steatogenic process, while the same proteins produced during infection by other genotypes are not. We examined in vitro interactions between lipid droplets and full-length core protein isolated from patients with HCV genotype 3a infection, with and without steatosis, and from steatosis-free patients infected by HCV genotype 1b. We also examined morphological changes in the lipid droplets according to the HCV genotype and the presence of steatosis in vivo. Core protein processing by signal peptide peptidase was not affected by sequence differences between the variants. We showed that the core protein of both HCV genotypes 1b and 3a binds tightly to the surface of intracellular lipid droplets. However, cells transfected with genotype 3a contain more neutral lipids in lipid droplets, and more large lipid droplets, than cells transfected with genotype 1b sequences. This suggests that HCV core protein-lipid droplet interaction could play a role in virus-induced steatosis. Importantly, we found no genetic or functional differences between genotype 3a core proteins from patients with and without HCV-induced steatosis. CONCLUSION: This suggests that other viral proteins and/or host factors are involved in the development of hepatocellular steatosis in patients infected by HCV genotype 3a.

The role of chronic infection in children with otitis media with effusion: evidence for intracellular persistence of bacteria.

OBJECTIVE: Demonstrate mucosal bacterial infection in children with otitis media with effusion (OME). STUDY DESIGN AND SETTING: Middle ear mucosal biopsies from 11 children with OME were examined for bacteria utilizing transmission electron microscopy. This was correlated with standard culture and polymerase chain reaction (PCR) of middle ear effusions. RESULTS: Gram-positive coccal bacteria were demonstrated in middle ear mucosal epithelial cells of 4 of 11 (36%) children. Morphological appearance of bacteria and detection of pneumolysin DNA by PCR in middle ear fluid suggests a role for persistent intracellular infection with Streptococcus pneumoniae and other gram-positive cocci in some cases of OME. CONCLUSION: Intracellular bacterial infection of middle ear mucosal epithelial cells in children with OME may be an important mechanism for bacterial persistence, and contribute to inflammation and mucus production in the pathogenesis of this condition. SIGNIFICANCE: Persistent intracellular infection is a novel paradigm for OME pathogenesis in children and may influence antibiotic effectiveness in treatment of this condition.

Active migration into the subcellular space precedes Campylobacter jejuni invasion of epithelial cells.

The bacterial pathogen Campylobacter jejuni invades mucosal cells via largely undefined and rather inefficient (0.01-2 bacteria per cell) mechanisms. Here we report a novel, highly efficient C. jejuni infection pathway resulting in 10-15 intracellular bacteria per cell within 3 h of infection. Electron microscopy, pulse-chase infection assays and time-lapse multiphoton laser confocal microscopy demonstrated that the mechanism involved active and rapid migration of the pathogen into the subcellular space (termed 'subvasion'), followed by bacterial entry ('invasion') at the cell basis. Efficient subvasion was maximal after repeated rounds of selection for the subvasive phenotype. Targeted mutagenesis indicated that the CadF, JlpA or PEB1 adhesins were not required. Dissection of the selected and parental phenotypes by SDS-PAGE yielded comparable capsule polysaccharide and lipooligosaccharide profiles. Proteomics revealed reduced amounts of the chemotaxis protein CheW for the subvasive phenotype. Swarming assays confirmed that the selected phenotype exhibited altered migration behaviour. Introduction of a plasmid carrying chemotaxis genes into the subvasive strain yielded wild-type subvasion levels and migration behaviour. These results indicate that alterations in the bacterial migration machinery enable C. jejuni to actively penetrate the subcellular space and gain access to the cell interior with unprecedented efficiency.

Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways.

The exponential increase in the number of new nanomaterials that are being produced increases the likelihood of adverse biological effects in humans and the environment. In this study we compared the effects of cationic nanoparticles in five different cell lines that represent portal-of-entry or systemic cellular targets for engineered nanoparticles. Although 60 nm NH(2)-labeled polystyrene (PS) nanospheres were highly toxic in macrophage (RAW 264.7) and epithelial (BEAS-2B) cells, human microvascular endothelial (HMEC), hepatoma (HEPA-1), and pheochromocytoma (PC-12) cells were relatively resistant to particle injury. While the death pathway in RAW 264.7 cells involves caspase activation, the cytotoxic response in BEAS-2B cells is more necrotic in nature. Using fluorescent-labeled NH(2)-PS, we followed the routes of particle uptake. Confocal microscopy showed that the cationic particles entered a LAMP-1 positive lysosomal compartment in RAW 264.7 cells from where the particles could escape by lysosomal rupture. A proton pump inhibitor interfered in this pathway. Subsequent deposition of the particles in the cytosol induced an increase in mitochondrial Ca(2+) uptake and cell death that could be suppressed by cyclosporin A (CsA). In contrast, NH(2)-PS toxicity in BEAS-2B cells did not involve the LAMP-1 endosomal compartment, stimulation of proton pump activity, or an increase in mitochondrial Ca(2+). Particles were taken up by caveolae, and their toxicity could be disrupted by cholesterol extraction from the surface membrane. Although the particles induced mitochondrial damage and ATP depletion, CsA did not affect cytotoxicity. Cationic particles were taken up into HEPA-1, HMEC, and PC-12 cells, but this did not lead to lysosomal permeabilization, increased Ca(2+) flux, or mitochondrial damage. Taken together, the results of this study demonstrate the importance of cell-specific uptake mechanisms and pathways that could lead to sensitivity or resistance to cationic particle toxicity.

Dilation of intercellular spaces is associated with laryngo-pharyngeal reflux: an ultrastructural morphometric analysis of laryngeal epithelium.

Gastroesophageal reflux disease (GERD) can be associated with ear, nose, and throat signs and symptoms, a condition often referred to as laryngopharyngeal reflux (LPR). However, the morphologic alterations of laryngeal mucosa associated with LPR are currently poorly understood. Since the dilation of intercellular spaces (DIS) between squamous epithelial cells is considered a morphologic marker of acid damage to esophageal mucosa in GERD, we evaluated whether similar changes can be detected in the laryngeal epithelium of patients affected by LPR. The study group included 15 patients affected by LPR and 7 normal controls, who underwent laryngeal biopsies at the interarytenoid area. Specimens were routinely processed for light microscopic and ultrastructural examination. The intercellular spaces were measured in electron microscopy images using a computer assisted morphometric system. Ultrastructural analysis demonstrated an irregular intercellular space dilation in specimens from the group of patients with LPR. Another ultrastructural abnormality observed in a minority of patients was the presence of numerous cytoplasmic vacuoles. Computer assisted morphometric analysis demonstrated that the intercellular space between squamous cells was significantly wider in patients with LPR than in control subjects (411.7 nm +/- 188.6 SD vs. 155.8 nm +/- 56.4 SD, P = 0.003). These data indicate that ultrastructural evidence of DIS of epithelial cells may be a morphologic marker of acid reflux, as already described in esophageal mucosa. If this result will be confirmed in larger series it may provide a useful diagnostic tool for the identification of LPR.

Guanine nitration in idiopathic pulmonary fibrosis and its implication for carcinogenesis.

RATIONALE: Nitric oxide (NO)-induced nitrative stress of nucleic acids, as evidenced by guanine nitration, appears to be involved in inflammation-induced carcinogenesis. A high incidence of lung cancer in idiopathic pulmonary fibrosis (IPF) is the major reason for poor prognosis in patients with IPF. OBJECTIVES AND METHODS: We immunohistochemically analyzed the formation and localization of 8-nitroguanine in lung tissues from control subjects, patients with IPF, and patients with lung cancer. MAIN RESULTS: Immunohistochemical analysis of control smoker and nonsmoker lungs showed weak immunoreactivity for 8-nitroguanine, mainly in cytoplasm of bronchial epithelial cells. In addition to the bronchial epithelial cells, metaplastic regenerated epithelial cells overlying dense fibrotic lesions in IPF showed strong 8-nitroguanine staining in the cytoplasm. The staining in these metaplastic cells colocalized with staining of inducible and endothelial NO synthases and 8-oxodeoxyguanosine, as evidenced by double-immunostaining analysis. Confocal and immunoelectron microscopy revealed localization of 8-nitroguanine in metaplastic epithelial cytoplasm, mostly in mitochondria. Appreciable 8-nitroguanine immunostaining was also observed in both nuclei and cytoplasm of malignant epithelial cells in squamous cell carcinoma. No significant difference was found in the epithelial 8-nitroguanine formation between control smokers and nonsmokers, but much higher guanine nitration was observed in patients with IPF than in control subjects and patients with lung cancer, via a quantitative immunofluorescence image analysis. CONCLUSIONS: The present study indicates that not only oxidative stress but also nitrative stress induced by NO may participate in the pathogenesis of epithelial cell damage and aberrant regeneration occurring in IPF. Thus, guanine nitration may be a major risk factor for lung cancer development in IPF.

GEC1, a protein related to GABARAP, interacts with tubulin and GABA(A) receptor.

We have previously identified in uterine cells a novel estrogen-regulated gene called gec1. GEC1 presents 87% identity with GABARAP which, so far, was the only protein found to associate with tubulin and GABA(A) receptor. We demonstrated then that GEC1 interacts in vitro with tubulin and GABA(A) receptor, and promotes tubulin assembly and microtubule bundling. Since all polyclonal antibodies failed in discrimination of both proteins GEC1 and GABARAP, a GEC1-GFP fusion protein was used to specifically localize GEC1. GEC1-GFP was distributed over the cytoplasm in perinuclear vesicles with a scattered pattern. Overall, our data show that GEC1 could be a new member of the GABARAP family involved in the transport of GABA(A) receptor.

Intracellular collagen and fibronexus in fibromatosis and other fibroblastic tumors.

Myofibroblasts are mesenchymal cells with combined function and structure for contraction and collagen synthesis. They are found in reparative responses, nodular fasciitis, fibromatosis, and myofibroblastic sarcoma. Ultrastructurally, myofibroblasts are characterized by a specialized cell surface structure called the fibronexus (FNX). In addition, intracellular collagen fibers (ICF) have been described in nodular fasciitis and fibromatosis, but their origin and nature are still controversial. The aim of the present work was, first, to assess the frequency of FNX and ICF in proliferative myofibroblastic conditions compared to diverse mesenchymal tumors with spindle-shaped cells, and, second, to determine what kind of organelles contain ICF and if they are related to phagocytosis or cell synthesis. Forty-two cases of aggressive fibromatosis and 11 of nodular fasciitis (group A) were compared to 82 spindle-cell mesenchymal tumors of diverse nature (group B) by electron microscopy study. The presence and frequency of FNX and ICF was compared in both groups, and the organelles containing ICF were recorded. FNX and ICF were constantly found in group A (69.8 and 84.9%, respectively), and rarely in group B (0 and 5.12%, respectively). Most frequently ICF were contained in tunnels and phagolysosomes, but also were found in Golgi vesicles and cisternae of rough endoplasmic reticulum. In the majority of cases (75%), ICF were similar to collagen fibers of the extracellular space, but in some cases (22.5%), they were in dissimilar stages of fibrogenesis. Fibromatosis and nodular fasciitis are characterized by proliferation of myofibroblasts and constantly show FNX and ICF. These structures are rarely found in other mesenchymal tumors. The ICF are found in organelles of digestion and also in others related to synthesis and transport.

C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain.

In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as well as in JEG3 choriocarcinoma cells with absent expression in adult tissues. We show that the expression is restricted to first-trimester proliferative trophoblasts of the proximal column and show that C2360 is a nuclear protein. No detectable transactivation potential was observed for different domains of the protein. Secondary structure prediction showed that C2360 is a representative member of a eukaryotic family of proteins with a low conservation at the amino acid level, but with strong conservation at the structural level, sharing the general domain (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2), or CHCH domain. Each alpha-helix within this domain contains two cysteine amino acids, and these intrahelical cysteines are separated by nine amino acids (C-X(9)-C motif). The fixed position within each helix indicated that both helices could form a hairpin structure stabilized by two interhelical disulfide bonds. Other proteins belonging to the family include estrogen-induced gene 2 and the ethanol-induced 6 protein. The conserved motif was found in yeast, plant, Drosophila, Caenorhabditis elegans, mouse, and human proteins, indicating that the ancestor of this protein family is of eukaryotic origin. These results indicate that C2360 is a representative member of a multifamily of proteins, sharing a protein domain that is conserved in eukaryotes.

Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis-like mechanism and survives in a compartment that differs from that with extracellular phenotype.

Mycobacterium avium uptake by human macrophages differs between the phenotypes of bacterium grown in laboratory media (extracellular growth, EG) and bacterium grown within macrophages (intracellular growth, IG). Studies in vivo have confirmed that, when spreading, pathogenic mycobacteria enter macrophages by a complement receptor 3-independent pathway, in contrast to mycobacteria uptake in vitro. M. avium, grown in macrophages (IG) for 3 or more days, invade fresh macrophages by a macropinocytosis-like mechanism, in contrast to bacteria grown in media (EG), confirmed by the inhibitory effect of wortmannin, an inhibitor of phosphoinoside-3-kinase, on the uptake of IG, but not EG, by macrophages. The IG phenotype was seen in vacuoles with lower pH than those inhabited by the EG phenotype. Incubation of macrophages with bafilomycin A1, an inhibitor of vacuole acidification, decreased the viability of intracellular IG, but not EG, phenotype, suggesting the importance of an acidic environment for the regulation of IG genes. In addition, the percentage of vacuoles that incorporate and retain LAMP-1 is smaller with EG than with IG bacteria. The formation of M. avium macropinosomes was also shown to be independent of microtubules. These data suggest that uptake of extracellular fluid is part of M. avium IG phenotype uptake by macrophages, and that the IG phenotype inhabits a slightly different vacuole than that of EG.

CIN85 associates with multiple effectors controlling intracellular trafficking of epidermal growth factor receptors.

CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.