Cytoplasmic Membrane Structures Research Articles

Antibacterial activity and biosynthetic potential of Streptomyces sp. PBR19, isolated from forest rhizosphere soil of Assam.

An Actinomycetia isolate, designated as PBR19, was derived from the rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The isolate, identified as Streptomyces sp., shares a sequence similarity of 93.96% with its nearest type strain, Streptomyces atrovirens. This finding indicates the potential classification of PBR19 as a new taxon within the Actinomycetota phylum. PBR19 displayed notable antibacterial action against some ESKAPE pathogens. The ethyl acetate extract of PBR19 (EtAc-PBR19) showed the lowest minimum inhibitory concentration (MIC) of ≥ 0.195µg/mL against Acinetobacter baumannii ATCC BAA-1705. A lower MIC indicates higher potency against the tested pathogen. Scanning electron microscope (SEM) findings revealed significant changes in the cytoplasmic membrane structure of the pathogen. This suggests that the antibacterial activity may be linked to the disruption of the microbial membrane. The predominant chemical compound detected in the EtAc-PBR19 was identified as phenol, 3,5-bis(1,1-dimethylethyl), comprising 48.59% of the area percentage. Additionally, PBR19 was found to contain the type II polyketide synthases (PKS type II) gene associated with antibiotic synthesis. The predicted gene product of PKSII was identified as the macrolide antibiotic Megalomicin A. The taxonomic distinctiveness, potent antibacterial effects, and the presence of a gene associated with antibiotic synthesis suggest that PBR19 could be a valuable candidate for further exploration in drug development and synthetic biology. The study contributes to the broader understanding of microbial diversity and the potential for discovering bioactive compounds in less-explored environments.

Phospholipase C-γ1 potentially facilitates subcellular localization of activated β-catenin, p-β-catenin(S552), during bovine herpesvirus 1 productive infection in MDBK cells

Bovine herpesvirus 1 (BoHV-1) is a significant risk factor for the bovine respiratory disease complex (BRDC), a severe disease causing great economic losses to the cattle industry worldwide. Previous studies have reported that both phospholipase C-γ1 (PLC-γ1) and β-catenin are activated during BoHV-1 infection for efficient replication. However, the interplay between PLC-γ1 and β-catenin as a consequence of virus infection remains to be elucidated. Here, we reported that PLC-γ1 interacted with β-catenin, which was enhanced following virus infection. PLC-γ1-specific inhibitor, U73122, significantly reduced the mRNA levels of β-catenin in BoHV-1-infected cells; however, the steady-state protein levels were not affected due to the virus infection. Interestingly, the treatment of virus-infected cells with U73122 reduced the accumulation of activated β-catenin [p-β-catenin(S552)] in fractions of the cytoplasmic membrane as that observed with the treatment of methyl-β-cyclodextrin (MβCD), which can disrupt cytoplasmic membrane structure via sequestering cholesterol. Nucleus accumulation of p-β-catenin(S552) was increased following U73122 treatment in virus-infected cells. In addition, the association of p-β-catenin(S552) with cytoplasmic membrane induced by the virus infection was significantly disrupted by the treatment of U73122 and MβCD. These data indicated that the PLC-γ1 signaling is potentially involved in the regulation of β-catenin signaling stimulated by BoHV-1 infection partially via affecting the subcellular localization of p-β-catenin(S552).

Disruption of the Cytoplasmic Membrane Structure and Barrier Function Underlies the Potent Antiseptic Activity of Octenidine in Gram-Positive Bacteria.

ABSTRACTThe antimicrobial killing mechanism of octenidine (OCT), a well-known antiseptic is poorly understood. We recently reported its interaction with Gram-negative bacteria by insertion of OCT into the outer and cytoplasmic membrane of Escherichia coli, resulting in a chaotic lipid rearrangement and rapid disruption of the cell envelope. Its action primarily disturbs the packing order of the hydrophobic moiety of a lipid, which consequently might result in a cascade of multiple effects at a cellular level. Here, we investigated OCT’s impact on two different Gram-positive bacteria, Enterococcus hirae and Bacillus subtilis, and their respective model membranes. In accordance with our previous results, OCT induced membrane disorder in all investigated model systems. Electron and fluorescence microscopy clearly demonstrated changes in cellular structure and membrane integrity. These changes were accompanied by neutralization of the surface charge in both E. hirae and B. subtilis and membrane disturbances associated with permeabilization. Similar permeabilization and disordering of the lipid bilayer was also observed in model membranes. Furthermore, experiments performed on strongly versus partly anionic membranes showed that the lipid disordering effect induced by OCT is a result of maximized hydrophobic over electrostatic forces without distinct neutralization of the surface charge or discrimination between the lipid head groups. Indeed, mutants lacking specific lipid head groups were also susceptible to OCT to a similar extent as the wild type. The observed unspecific mode of action of OCT underlines its broad antimicrobial profile and renders the development of bacterial resistance to this molecule less likely.IMPORTANCE OCT is a well-established antiseptic molecule routinely used in a large field of clinical applications. Since the spread of antimicrobial resistance has restricted the use of antibiotics worldwide, topically applied antiseptics like OCT, with a broad spectrum of antimicrobial activity and high safety profile, gain increasing importance for effective infection prevention and therapy. To eliminate a wide spectrum of disease-causing microorganisms, a compound’s antiseptic activity should be unspecific or multitarget. Our results demonstrate an unspecific mechanism of action for OCT, which remained largely unknown for years. OCT disturbs the barrier function of a bacterial cell, a function that is absolutely fundamental for survival. Because OCT does not distinguish between lipids, the building blocks of bacterial membranes, its mode of action might be attributed to all bacteria, including (multi)drug-resistant isolates. Our results underpin OCT’s potent antiseptic activity for successful patient outcome.

The ultrastructural damage caused by Eugenia zeyheri and Syzygium legatii acetone leaf extracts on pathogenic Escherichia coli

BackgroundAntibiotics are commonly added to livestock feeds in sub-therapeutic doses as growth promoters and for prophylaxis against pathogenic microbes, especially those implicated in diarrhoea. While this practice has improved livestock production, it is a major cause of antimicrobial resistance in microbes affecting livestock and humans. This has led to the banning of prophylactic antibiotic use in animals in many countries. To compensate for this, alternatives have been sought from natural sources such as plants. While many studies have reported the antimicrobial activity of medicinal plants with potential for use as phytogenic/botanical feed additives, little information exists on their mode of action. This study is based on our earlier work and describes ultrastructural damage induced by acetone crude leaf extracts of Syzygium legatii and Eugenia zeyheri (Myrtaceae) active against diarrhoeagenic E. coli of swine origin using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescent microscopy (FM). Gas chromatography/mass spectrometry (GC-MS) was used to investigate the chemical composition of plant extracts.ResultsThe extracts damaged the internal and external anatomy of the cytoplasmic membrane and inner structure at a concentration of 0.04 mg/mL. Extracts also led to an increased influx of propidium iodide into treated bacterial cells suggesting compromised cellular integrity and cellular damage. Non-polar compounds such as α-amyrin, friedelan-3-one, lupeol, and β-sitosterol were abundant in the extracts.ConclusionsThe extracts of S. legatii and E. zeyheri caused ultrastructural damage to E. coli cells characterized by altered external and internal morphology. These observations may assist in elucidating the mode of action of the extracts.

Escherichia coli as a Model for the Description of the Antimicrobial Mechanism of a Cationic Polymer Surface: Cellular Target and Bacterial Contrast Response.

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.

Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6

BackgroundCystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli.ResultsUsing transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles.ConclusionsOur results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

Morphologic and Molecular Characterization of a Strain of Zika Virus Imported into Guangdong, China.

The recent outbreaks of Zika virus (ZIKV) disease have caused worldwide concerns. Guangdong province is one of the commercial centers in China and communicates frequently with the epidemic areas. To date, 65.2% of the ZIKV infection cases in China were imported via port of entry in Guangdong. The continuous surveillance of imported cases is crucial for the prevention and control of potential ZIKV infection outbreak in China. In this study, a strain of ZIKV was isolated from the serum of a 6-year-old child returning from Venezuela. The morphology of the ZIKV was analyzed in vivo and in vitro by electron microscopy, and clusters of virus particles were found in the loose cytoplasmic membrane structures. The genomic sequence of the isolated ZIKV was determined, and the alignment and phylogenetic analysis identified one unique amino acid substitution occurring in the non-structural protein 4B (NS4B), and the isolated virus belonged to the Asian lineage.

Structure elucidation and biological activity of antibacterial compound from Micromonospora auratinigra, a soil Actinomycetes.

The aim of this study was to isolate and characterize the bioactive compound of Micromonospora auratinigra, HK-10 and its antibacterial inhibitory mechanism. An oily bioactive compound was extracted from HK-10 (GenBank accession no. JN381554) and found to have promising antibacterial activity. The compound was characterized as 2-methylheptylisonicotinate (1) by (1) H, (13) C NMR and mass spectroscopy. Minimum inhibitory concentration (MIC) of this molecule was tested by micro broth dilution method and was found to be 70, 40, 80, 60, 60 and 50μg for Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Echerichia coli, Pseudomonas aeruginosa and Mycobacterium abscessus respectively. The effects of compound 1 were studied on bacterial membrane structure using scanning electron microscopy. The results indicated a membrane-disrupting mechanism, resulting in the dysfunction of the cytoplasmic membrane structure and cell death of the pathogenic bacterial strains. Kinetics of growth of the test organisms was also analysed and indicated 2-methylheptylisonicotinate 1 as a bactericidal agent. Furthermore, we have studied the binding affinity of 1 towards different membrane proteins of pathogenic bacteria by in silico analysis. 2-methylheptylisonicotinate was isolated from M.auratinigra, a rare actinobacterial strain possessing antibacterial activity through a membrane-disrupting mechanism, and has MICs similar to standard antibiotic neomycin sulphate. It is the first report about a strain of M.auratinigra, isolated from Indo-Burma biodiversity hotspot of North-east India with new antimicrobial activities. In silico studies have also supported these results performed on various membrane targets of pathogenic bacteria. The antibacterial potential of M.auratinigra is reported for the first time. The results indicate the possible use of 2-methylheptylisonicotinate as a source of antibacterial agent against dreaded human pathogens.

Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy.

As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry.

Studies on the kinetics of killing and the proposed mechanism of action of microemulsions against fungi

Microemulsions are physically stable oil/water clear dispersions, spontaneously formed and thermodynamically stable. They are composed in most cases of water, oil, surfactant and cosurfactant. Microemulsions are stable, self-preserving antimicrobial agents in their own right. The observed levels of antimicrobial activity associated with microemulsions may be due to the direct effect of the microemulsions themselves on the bacterial cytoplasmic membrane. The aim of this work is to study the growth behaviour of different microbes in presence of certain prepared physically stable microemulsion formulae over extended periods of time. An experiment was designed to study the kinetics of killing of a microemulsion preparation (17.3% Tween-80, 8.5% n-pentanol, 5% isopropyl myristate and 69.2% sterile distilled water) against selected test microorganisms (Candida albicans, Aspergillus niger, Schizosaccharomyces pombe and Rhodotorula spp.). Secondly, an experiment was designed to study the effects of the microemulsion preparation on the cytoplasmic membrane structure and function of selected fungal species by observation of 260 nm component leakage. Finally, the effects of the microemulsion on the fungal membrane structure and function using S. pombe were studied using transmission electron microscopy. The results showed that the prepared microemulsions are stable, effective antimicrobial systems with effective killing rates against C. albicans, A. niger, S. pombe and Rhodotorula spp. The results indicate a proposed mechanism of action of significant anti-membrane activity, resulting in the gross disturbance and dysfunction of the cytoplasmic membrane structure which is followed by cell wall modifications, cytoplasmic coagulation, disruption of intracellular metabolism and cell death.

Crinivirus replication and host interactions.

Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense single-stranded RNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV) is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was developed. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as Beet yellows virus (BYV)-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA 1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA-binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP (major coat protein), CPm (minor coat protein), Hsp70h, and P59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5′ end of RNA 2 as ORF 1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the endoplasmic reticulum as a Type III integral membrane protein. The other small protein, P9, is encoded by ORF 4 overlaps with ORF 3 that encodes the structural protein, P59. P9 seems to be unique to viruses in the genus Crinivirus, as no similar protein has been detected in viruses of the other two genera of the Closteroviridae.

Isolation and characterization of a novel alphanodavirus

BackgroundNodaviridae is a family of non-enveloped isometric viruses with bipartite positive-sense RNA genomes. The Nodaviridae family consists of two genera: alpha- and beta-nodavirus. Alphanodaviruses usually infect insect cells. Some commercially available insect cell lines have been latently infected by Alphanodaviruses.ResultsA non-enveloped small virus of approximately 30 nm in diameter was discovered co-existing with a recombinant Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) in Hz-AM1 cells. Genome sequencing and phylogenetic assays indicate that this novel virus belongs to the genus of alphanodavirus in the family Nodaviridae and was designated HzNV. HzNV possesses a RNA genome that contains two segments. RNA1 is 3038 nt long and encodes a 110 kDa viral protein termed protein A. The 1404 nt long RNA2 encodes a 44 kDa protein, which exhibits a high homology with coat protein precursors of other alphanodaviruses. HzNV virions were located in the cytoplasm, in association with cytoplasmic membrane structures. The host susceptibility test demonstrated that HzNV was able to infect various cell lines ranging from insect cells to mammalian cells. However, only Hz-AM1 appeared to be fully permissive for HzNV, as the mature viral coat protein essential for HzNV particle formation was limited to Hz-AM1 cells.ConclusionA novel alphanodavirus, which is 30 nm in diameter and with a limited host range, was discovered in Hz-AM1 cells.

Role of Lipids in Virus Replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.

4-epi-Pimaric acid: a phytomolecule as a potent antibacterial and anti-biofilm agent for oral cavity pathogens

The present study focused on the antibacterial and biofilm inhibitory potential of 4-epi-pimaric acid isolated from aerial parts (stem and leaves) of Aralia cachemirica L. (Araliaceae) against oral cavity pathogens. 4-epi-Pimaric acid exhibited minimum inhibitory concentration (MIC) in the range of 4-16μg/ml and minimum bactericidal concentration (MBC) two- to four-folds higher than MIC. There was significant inhibition in the biofilm formation by Streptococcus mutans on the saliva coated surface (P < 0.05), and confocal microscopy revealed that 4-epi-pimaric acid inhibited the clumping and attachment of S. mutans. At 8 × MIC concentration, it significantly prevented the pH drop and reduced S. mutans biofilms (P < 0.05). Increased propidium iodide staining and leakage of 260- and 280-nm absorbing material by 4-epi-pimaric acid treated cells of S. mutans suggested that it probably causes disruption of the cytoplasmic membrane structure. It also exhibited significant suppression of TNF-α expression in human neutrophils, suggestive of its anti-inflammatory activity. Furthermore, the compound was found to be significantly safe (IC(50) >100μg/ml) in the MTT assay on AML-12 cell lines. In conclusion, 4-epi-pimaric acid showed promising antibacterial, anti-biofilm and anti-inflammatory potency and this compound can be exploited for therapeutic application in oral microbial infections.

Environmental pollution changes in membrane lipids, antioxidants and vitality of Scots pine (Pinus sylvestris L.) pollen

Investigations were carried out on pollen grains of Scots pine (&lt;em&gt;Pinus sylvestris &lt;/em&gt;L.) collected from trees at 1.5, 3, 4 km and control, 20 km from the Luboń factory producing mineral fertilisers. The percentage of germination of pollen formed close to the pollution source was ca 20% lower compared to the control pollen. Lowered vitality of the pollen was effected in changes of the structure of cytoplasmic membranes. Pollen from the polluted area contained ca 15% less total phospholipids, mainly phosphatidylcholine and phosphatytidylinositol and had a lower content of soluble proteins and less of low molecular antioxidants, such as thiols and ascorbic acid. Composition of total fatty acid in phospholipids fractions showed a significant reduction in the degree of unsaturation of fatty acids. Pollen originating from the polluted area and stored at -30°C showed considerably stronger degradation of cytoplasmic membranes than control.

Vacuolar myopathy in a dog resembling human sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is the most common myopathy in people over the age of 50 years. While immune-mediated inflammatory myopathies are well documented in dogs, sIBM has not been described. An 11-year-old dog with chronic and progressive neuromuscular dysfunction was evaluated for evidence of sIBM using current pathologic, immunohistochemical and electron microscopic diagnostic criteria. Vacuoles and congophilic intracellular inclusions were identified in cryostat sections of multiple muscle biopsies and immunostained with antibodies against amyloid-β peptide, amyloid-β precursor protein, and proteosome 20S of the ubiquitin–proteosome system. Cellular infiltration and increased expression of MHC Class I antigen were observed. Cytoplasmic filamentous inclusions, membranous structures, and myeloid bodies were identified ultrastructurally. These observations constitute the first evidence that both the inflammatory and degenerative features of human sIBM can occur in a non-human species.

Localization of gene products using a chromosomally tagged GFP‐fusion library in the fission yeast Schizosaccharomyces pombe

We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3'-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP-fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm.

Autophagy Regulates Membrane Homeostasis in T Lymphocytes

Autophagy Regulates Membrane Homeostasis in T Lymphocytes Autophagy is a highly conserved intracellular process targeting cytoplasmic components for bulk degradation that has been extensively characterized in Saccharomyces cerevisiae. Recent work from our lab has shown this process to be indispensable for the survival of peripheral T lymphocytes in vivo. In addition to bulk cytoplasmic targeting, autophagy has been proposed to regulate other cellular components including mitochondria and endoplasmic reticulum. In T lymphocytes lacking the essential autophagy protein Atg7, there is an expansion of cytoplasmic membranous structures. These vesicles occupy more than twice the cytoplasmic volume of vesicles in wild type T lymphocytes. Although the massive onset of death is not incurred until after thymic egress, thymocytes lacking Atg7 already have expanded cytoplasmic membranes. A microarray performed on naïve T lymphocytes shows the transcriptional upregulation of many vesicle trafficking and lipid particle formation proteins. Also upregulated are several components of a mitochondrial shuttle pathway for long fatty acid chains, the acyl-carnitine pathway. Analysis of the acyl-chain intermediates of this pathway by LC/MS showed an upregulation in the β-oxidation of several lipids, most notably stearic acid. This pathway has been shown to be upregulated in rat hepatocytes to adjust to high fat diets and skeletal muscles of exercising individuals to maintain healthy mitochodria. These data suggest another function of autophagy in T lymphocytes is to regulate the expansion of vesicles in order to reduce lipid toxicity.

West Nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral MxA protein

The human MxA protein is a type I and III interferon (IFN)-induced protein with proven antiviral activity against RNA viruses. In this study, we investigated the effect of MxA expression on the replication of West Nile Virus strain Kunjin (WNV(KUN)). Pretreatment of A549 cells with IFN-alpha lead to increased expression of MxA, which contributed to inhibition of WNV(KUN) replication and secretion. However, in Vero cells stably expressing the MxA protein, WNV(KUN) replication, maturation and secretion was not inhibited. Biochemical and subcellular localization studies of WNV(KUN) proteins and MxA suggest that the MxA activity was not compromised by a flavivirus-encoded antagonist. Instead, we show that characteristic membranous structures induced during WNV(KUN) replication provide partial protection from MxA, possibly by 'hiding' WNV(KUN) replication components. This distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can hide from host surveillance.

Ultrastructure and localization of a visual Gq protein in ied epitoke ocelli of Perinereis brevicirris (Polychaeta, Annelida)

Functional ultrastructural changes in the rhabdomeric photoreceptors of the cerebral ocelli are described for normal and sexually mature (epitoke) Perinereis brevicirris (Polychaeta, Annelida). With sexual maturation, the cerebral ocelli hypertrophied, increasing in volume to 5.5 times that of ocelli in the normal state, and the thickness of the retinal layer increased up to 10 times. Perinereis ocelli have a pigmented retinal layer consisting of at least two cell types: photoreceptor cell (PR) and pigmented supporting cells (PS). In epitoke ocelli, PR bear well-developed rhabdomeric microvilli, multilamellar bodies, and numerous cytoplasmic membranous structures, including vesicles, smooth endoplasmic reticulum, and secondary lysosomes. Localization of a visual Gq protein in the ocelli was studied with anti-GqC antibody. The antibody strongly labeled not only microvilli and multilamellar bodies throughout the retinal layer, but also secondary lysosomes and vesicles in the cytoplasm of the PR in the epitoke ocelli, although labeling was observed only in the microvilli and multilamellar bodies in normal ocelli. Reverse transcription/polymerase chain reaction analysis revealed that the amount of G protein alpha subunit mRNA in the epitoke head increased by roughly twice that of the normal head. Since Gq protein is essential for phototransduction in Perinereis ocelli, these results suggest that the sites are involved in photoreceptive membrane turnover, which occurs much more extensively in epitoke ocelli. Thus, epitoke ocelli may represent a model system for studying rhabdomeric photoreceptive membrane turnover.