Perinuclear Area Research Articles

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

Hypoxia-Inducible Lipid Droplet-Associated Interacts with DGAT1 to Promote Lipid Storage in Hepatocytes

Lipid droplets (LD) are dynamic organelles that can expand and shrink, driven by fluctuations in the rate of triglyceride synthesis and degradation. Triglyceride synthesis, storage in LD, and degradation are governed by a complex set of LD-associated proteins. One of these LD-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair LD breakdown by inhibiting adipose triglyceride lipase. Here we characterized the physiological role and mechanism of action of HILPDA in hepatocytes. Expression of HILPDA was induced by fatty acids in several hepatoma cell lines. Fluorescence microscopy showed that HILPDA partly colocalizes with LD and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging revealed that HILPDA preferentially localizes to LD that are being remodelled. Deficiency of HILPDA in mouse precision-cut liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labelled fatty acids in LD, respectively, which was independent of adipose triglyceride lipase. Confocal microscopy and Forster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA colocalizes and physically interacts with DGAT1. In human hepatoma HepG2 cells, HILPDA overexpression increased lipid storage concomitant with an increase in DGAT activity and DGAT1 protein levels. Finally, hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglyceride and plasma alanine aminotransferase levels in mice with non-alcoholic fatty liver disease. Overall, our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity and DGAT1 protein levels. These findings suggest a novel mechanism in hepatocytes that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage.

Lysyl oxidase-variant 2 functions as an amine oxidase toward histone H2A

Lysyl oxidase-variant 2 (LOX-v2) is a novel variant of lysyl oxidase (LOX) that functions as an amine oxidase for the formation of lysine-mediated crosslinks found in collagen and elastin fibrils. In addition to the amine oxidase activity in the extracellular matrix, several novel functions, such as tumor suppression, tumor progression, chemotaxis, cellular senescence, and modification of histones, have been assigned to LOX. In recent years, it has been reported that LOX is also present in nuclear locations, suggesting a novel functional role of LOX in the nucleus. To test the amine oxidase activity of LOX and LOX-v2 to nuclear histone proteins, we expressed and purified LOX and LOX-v2 as recombinant forms and then assessed the amine oxidase activity toward histone H2A in in vitro peroxidase-coupled fluorometric assays. Both LOX and LOX-v2 proteins showed significant levels of amine oxidase activity toward histone H2A in a β-aminopropionitrile-inhibitable manner. In immunofluorescence staining after ectopic expression in cultured cells, LOX was observed in the perinuclear, cytoplasmic, and extracellular areas, whereas LOX-v2 was predominantly detected in the nucleoplasm with a punctuate pattern. These findings suggest that LOX-v2 may play a novel functional role in the nucleus through the amine oxidase activity to the nuclear histone proteins. Elucidation of the specific functional roles of LOX-v2, such as substrate specificity toward different types of nuclear proteins and detailed analysis on subnuclear localization, will provide a significant clue in understanding the diverse functional roles currently assigned to a single enzyme, LOX.

Different localization of lysosomal-associated membrane protein 1 (LAMP1) in mammalian cultured cell lines.

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells.

PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity.

The trafficking chaperone PDE6D (also referred to as PDEδ) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a “chemical spring” into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block stemness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future.

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth.

Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella–Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24–48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.

P.178A novel mutation in TNPO3 causes congenital limb girdle myopathy with slow progression

We report a second family with autosomal dominant TNPO3/transportinopathy presenting with congenital or early-onset myopathy and slow progression, causing proximal and less pronounced distal muscle weakness. Patients of this Swedish family had clinical examinations, muscle MRI and EMG. Muscle biopsies were used for histopathological and protein expression studies, and TNPO3 constructs were used to study the effect of the mutations in transfected cells. By using the MYOcap targeted exome sequencing gene panel, we identified a novel heterozygous mutation, c.2757delC, in the last part of the Transportin-3/TNPO3 gene in the affected family members. The mutation causes an almost identical frameshift affecting the stop codon and elongating the C-term protein product of the TNPO3 transcript as was previously reported in the first large Spanish-Italian LGMD1F kindred. TNPO3 belongs to the importin beta superfamily, facilitating the nuclear import of Ser/Arg-rich (SR) proteins. TNPO3 plays a role in HIV infection, however, little is known of its normal functions in muscle. We found that the TNPO3 protein was increased in the patient muscle, and accumulated in the subsarcolemmal and perinuclear areas in muscle biopsy. One of the cargo proteins, the splicing factor SRRM2 was normally located in the nucleus, whereas RBM4 showed perinuclear accumulation. In addition, transiently transfected mutant TNPO3 constructs failed to localize to cytoplasmic annulate lamellae pore complexes in cultured cells. We report the clinical, molecular genetic and histopathological features of the second transportinopathy family. The variability of the clinical phenotype together with histopathological findings suggest that several molecular pathways may be involved in the disease pathomechanism, such as nucleo-cytoplasmic shuttling, protein aggregation, and defective protein turnover.

Depletion of Host Cell Focal Adhesion Kinase Increases the Susceptibility to Invasion by Trypanosoma cruzi Metacyclic Forms.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.

Toxic cardiac effect of cobalt chloride excessive chronic consumption in male Wistar rats

Cobalt is widespread in the natural environment and can be formed as an effect of anthropogenic activity. The ionic cobalt can be transported by blood in the body, causing adverse effects by the generation of reactive oxygen species. The aim of this research was to describe the changes of myocardial cells during experimental cobalt induced cardiomyopathy. Summarizing the in vivo experiments, it can be stated that severe histotoxic cardiomyopathy occurred in male rats treated with cobalt chloride. Long-term oral administration of cobalt resulted in diminished dietary intake and growth inhibition in the exposed rats compared to the control group. The primary morphological alteration of cardiomyocytes is mitochondrial damage that possibly reflects an enzymatic block of oxidative decarboxylation. Due to that myofibrils of the myocardial cells were affected highlighting that the main cause of myofibril reduction could be a lower oxygen intake in the perinuclear area. The reduction of the contractile support of myocardial cells can explain the possible myocardial dysfunction. Knowing structural changes in cardiomyocytes could explain the pathophysiology of the disease and allow a correct therapeutic approach.

Novel mutation in TNPO3 causes congenital limb-girdle myopathy with slow progression.

ObjectiveWe report a second family with autosomal dominant transportinopathy presenting with congenital or early-onset myopathy and slow progression, causing proximal and less pronounced distal muscle weakness.MethodsPatients had clinical examinations, muscle MRI, EMG, and muscle biopsy studies. The MYOcap gene panel was used to identify the gene defect in the family. Muscle biopsies were used for histopathologic and protein expression studies, and TNPO3 constructs were used to study the effect of the mutations in transfected cells.ResultsWe identified a novel heterozygous mutation, c.2757delC, in the last part of the transportin-3 (TNPO3) gene in the affected family members. The mutation causes an almost identical frameshift affecting the stop codon and elongating the C-term protein product of the TNPO3 transcript, as was previously reported in the first large Spanish-Italian LGMD1F kindred. TNPO3 protein was increased in the patient muscle and accumulated in the subsarcolemmal and perinuclear areas. At least one of the cargo proteins, the splicing factor SRRM2 was normally located in the nucleus. Transiently transfected mutant TNPO3 constructs failed to localize to cytoplasmic annulate lamellae pore complexes in cells.ConclusionsWe report the clinical, molecular genetic, and histopathologic features of the second transportinopathy family. The variability of the clinical phenotype together with histopathologic findings suggests that several molecular pathways may be involved in the disease pathomechanism, such as nucleocytoplasmic shuttling, protein aggregation, and defective protein turnover.

Interleukin-1β Induces Intracellular Serum Amyloid A1 Expression in Human Coronary Artery Endothelial Cells and Promotes its Intercellular Exchange.

Serum amyloid A (SAA) is an acute-phase protein with important, pathogenic role in the development of atherosclerosis. Since dysfunctional endothelium represents a key early step in atherogenesis, we aimed to determine whether induced human coronary artery endothelial cells (HCAEC) modulate SAA1/2/4 expression and influence intracellular location and intercellular transport of SAA1. HCAEC were stimulated with 1ng/ml IL-1β, 10ng/ml IL-6, and/or 1μM dexamethasone for 24h. QPCR, Western blots, ELISA, and immunofluorescent labeling were performed for detection of SAA1/2/4 mRNA and protein levels, respectively. In SAA1 transport experiments, FITC- or Cy3-labeled SAA1 were added to HCAEC separately, for 24h, followed by a combined incubation of SAA1-FITC and SAA1-Cy3 positive cells, with IL-1β and analysis by flow cytometry. IL-1β upregulated SAA1 (119.9-fold, p < 0.01) and SAA2 (9.3-fold; p < 0.05) mRNA expression levels, while mRNA expression of SAA4 was not affected. Intracellular SAA1 was found mainly as a monomer, while SAA2 and SAA4 formed octamers as analyzed by Western blots. Within HCAEC, SAA1/2/4 located mostly to the perinuclear area and tunneling membrane nanotubes. Co-culturing of SAA1-FITC and SAA1-Cy3 positive cells for 48h showed a significantly higher percentage of double positive cells in IL-1β-stimulated (mean ± SD; 60 ± 4%) vs. non-stimulated cells (48 ± 2%; p < 0.05). IL-1β induces SAA1 expression in HCAEC and promotes its intercellular exchange, suggesting that direct communication between cells in inflammatory conditions could ultimately lead to faster development of atherosclerosis in coronary arteries.

Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression in primary mouse tumorigenic cells.

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

The BOLF1 gene is necessary for effective Epstein–Barr viral infectivity

The Epstein–Barr virus (EBV) is a causative agent of infectious mononucleosis and several malignancies. Here, we focused on an EBV lytic protein, BOLF1, which is conserved throughout the herpesvirus family and is reported to be a virion tegument protein. We first constructed BOLF1-deficient viruses using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although the loss of BOLF1 had almost no effect on viral protein expression, DNA synthesis, or extracellular progeny release, EBV infectivity was significantly reduced. Further analysis showed that nuclear transportation of the incoming virus was decreased by the disruption of BOLF1. Our results indicate that BOLF1enhances the infectious potential of progeny virions, at least partly by increasing nuclear transportation of incoming nucleocapsids. We also found that BOLF1 interacted with BKRF4, and the BOLF1 and BKRF4 proteins were localized in the nucleus and perinuclear area, during the viral lytic cycle.

Feedback-Driven Mechanisms between Microtubules and the Endoplasmic Reticulum Instruct Neuronal Polarity

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity.

KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named ‘traffic-induced degradation response for secretion’ (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system.

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.

The role of microtubules and the dynein/dynactin motor complex of host cells in the biogenesis of the Coxiella burnetii-containing vacuole.

Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies

Nuclear envelopathies are recognized genetic disorders affecting individuals with mutations in their genes encoding members of the lamin family of nuclear envelope proteins that are responsible for maintaining the architectural structure of the nucleus. Irregularity in shape and size of the nuclei, nuclear membrane rupture, and appearance of micronuclei in the cytoplasm are among the pathological features of the syndrome. Here, we demonstrate that Bcl2-associated anthanogene-3 (BAG3), a stress-induced co-chaperone protein that by association with heat-shock protein 70 (HSP70) participates in regulation of autophagy, plays a critical role in the integrity of the nuclear membrane in cardiomyocytes. Cells subjected to proteotoxic stress or BAG3 downregulation show perinuclear accumulation of the aberrant ubiquitinated proteins that are often associated with the appearance of misshapen, enlarged, and elongated nuclei. There were dense accumulations of lamin B in the perinuclear area and distribution of lamin B-positive micronuclei in the cytoplasmic space, indicative of nuclear envelope rupture. Overexpression of BAG3 in cells under proteotoxic stress ameliorated pathological nuclear morphology and reduced cytoplasmic distribution of the micronuclei particles. Subcellular co-localization and co-immunoprecipitation demonstrated interaction of lamin B with the BAG domain of BAG3 and HSP70, suggesting the importance of BAG3 in the selective clearance of a surplus of aggregated lamin B that is generated during stress conditions. Our findings define a novel role for BAG3 in nuclear protein quality control and suggest an alternative pathogenetic pathway that contributes to the development of nuclear envelopathies.

Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells

ABSTRACT Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.