Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division

The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is a unique archaeal protein that interacts with the membrane, while CdvB is homologous to the eukaryal Vps24 and forms helical filaments. Most Crenarcheota contain additional CdvB paralogs. In Sulfolobus acidocaldarius these are termed CdvB1–3. We have used a gene inactivation approach to determine the impact of these additional cdvB genes on cell division. Independent deletion mutants of these genes were analyzed for growth and protein localization. One of the deletion strains (ΔcdvB3) showed a severe growth defect on plates and delayed growth on liquid medium. It showed the formation of enlarged cells and a defect in DNA segregation. Since these defects are accompanied with an aberrant localization of CdvA and CdvB, we conclude that CdvB3 fulfills an important accessory role in cell division.     

古菌ESCRT系统研究进展

内体分拣转运复合体（ESCRT,endosomal sorting complex required for transport）曾被认为是真核生物特有的系统,涉及膜重塑、泛素化蛋白质分拣等重要细胞生命过程。近年的研究显示,TACK（包括Thaumarchaeota、Aigarchaeota、Crenarchaeota和Korarchaeota门）古菌超门中存在着一类与分泌膜囊泡、古菌病毒出胞以及细胞分裂过程等膜重塑过程相关的细胞分裂（Cdv,cell division）系统,该系统中的CdvB和CdvC是真核生物ESCRT-III和Vps4的同源蛋白,提示真核生物ESCRT系统可能起源自古菌。然而,由于TACK古菌中缺少真核生物ESCRT系统的其他关键成分,这一假设仍有争议。最近发现的阿斯加德（Asgard）古菌是一类被认为与真核生物最近缘的古菌,其基因组具有较完整的ESCRT相关蛋白的编码基因,提示真核生物的ESCRT很可能起源于阿斯加德古菌。本文首先简要介绍真核生物ESCRT系统的组成及生物学功能,然后分别总结TACK古菌的Cdv系统和阿斯加德古菌的ESCRT系统的研究进展,重点讨论它们的组成及生物学功能,为进一步了解古菌ESCRT系统与真核生物起源的关系提供参考。     

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background

Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites.

Methodology/Principal Findings

We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro.

Conclusions/Significance

Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.     

The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

Background and Aims

Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.

Methods

Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.

Key Results

The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.

Conclusions

The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.     

Analysis of Vascular Development in the hydra Sterol Biosynthetic Mutants of Arabidopsis

Background

The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning.

Methodology/Principal Findings

Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development.

Conclusions

The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.     

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment

Background

Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions.

Methodology/Principal Findings

To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata''s oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions.

Conclusions/Significance

The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.     

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division

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Highlights? A conserved protein, CdvA, recruits ESCRT-III to membranes during cell division ? Recruitment is mediated by a peptide-winged helix domain interaction ? We have determined the structure of this complex ? CdvA and a single ESCRT-III protein can drive membrane deformation in vitro     

D-ribose induces cellular protein glycation and impairs mouse spatial cognition

Background

D-Ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation.

Methodology/Principal Findings

Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test.

Conclusions/Significance

These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition.     

Refolding process of cysteine-rich proteins:Chitinase as a model

Background:

Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.

Methods:

Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.

Results:

After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.

Conclusions:

By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.Key Words: Chitinase, Cysteine-rich proteins, Protein refolding, Protein solubilization     

Polymers for improving the in vivo transduction efficiency of AAV2 vectors

Background

Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency.

Methodology

Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2.

Conclusions

Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.     

Extracellular HCV-Core Protein Induces an Immature Regulatory Phenotype in NK Cells: Implications for Outcome of Acute Infection

Background

Hepatitis C viral (HCV) proteins, including core, demonstrate immuno-modulatory properties; however, the effect of extracellular core on natural killer (NK) cells has not previously been investigated.

Aims

To characterise NKs in acute HCV infection over time, and, to examine the effect of exogenous HCV-core protein on NK cell phenotype and function.

Methods

Acute HCV patients (n = 22), including 10 subjects who spontaneously recovered, were prospectively studied. Flow-cytometry was used to measure natural cytotoxicity and to phenotype NKs directly ex vivo and after culture with HCV-core protein. Microarray analysis was used to identify pathways involved in the NK cell response to exogenous HCV-core.

Results

Direct ex vivo analysis demonstrated an increased frequency of immature/regulatory CD56^bright NKs early in acute HCV infection per se which normalized with viral clearance. Natural cytotoxicity was reduced and did not recover after viral clearance. There was a statistically significant correlation between the frequency of CD56^bright NKs and circulating serum levels of HCV core protein. In vitro culture of purified CD56^bright NK cells with HCV-core protein in the presence of IL-15 maintained a significant proportion of NKs in the CD56^bright state. The in vitro effect of core closely correlates with NK characteristics measured directly ex vivo in acute HCV infection. Pathway analysis suggests that HCV-core protein attenuates NK interferon type I responses.

Conclusions

Our data suggest that HCV-core protein alters NK cell maturation and may influence the outcome of acute infection.     

Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium

Background

Supplementation of broiler chicken diets with probiotics may improve carcass characteristics and meat quality. However, the underlying molecular mechanism remains unclear. In the present study, 2D-DIGE-based proteomics was employed to investigate the proteome changes associated with improved carcass traits and meat quality of Arbor Acres broilers (Gallus gallus) fed the probiotic Enterococcus faecium.

Results

The probiotic significantly increased meat colour, water holding capacity and pH of pectoral muscle but decreased abdominal fat content. These meat quality changes were related to the altered abundance of 22 proteins in the pectoral muscle following E. faecium feeding. Of these, 17 proteins have central roles in regulating meat quality due to their biological interaction network. Altered cytoskeletal and chaperon protein expression also contribute to improved water holding capacity and colour of meat, which suggests that upregulation of chaperon proteins maintains cell integrity and prevents moisture loss by enhancing folding and recovery of the membrane and cytoskeletal proteins. The down-regulation of β-enolase and pyruvate kinase muscle isozymes suggests roles in increasing the pH of meat by decreasing the production of lactic acid. The validity of the proteomics results was further confirmed by qPCR.

Conclusions

This study reveals that improved meat quality of broilers fed probiotics is triggered by proteome alterations (especially the glycolytic proteins), and provides a new insight into the mechanism by which probiotics improve poultry production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1167) contains supplementary material, which is available to authorized users.     

Proteomic analysis of the cyst stage of Entamoeba histolytica

Background

The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools.

Aims

Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools.

Methods

Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins.

Results

A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets.

Major Conclusions

The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.     

LRCH Proteins: A Novel Family of Cytoskeletal Regulators

Background

Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs.

Principal Findings

Taking advantage of the existence of a single LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures.

Conclusion/Significance

These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations.     

Amino Acid Accumulation Limits the Overexpression of Proteins in Lactococcus lactis

Background

Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media.

Methodology/Principal Findings

The proteomic profiles of cells growing in media in which the proteins were expressed to high or low levels suggested a limitation in the availability of branched-chain amino acids, more specifically a too limited capacity to accumulate these nutrients. By supplying the cells with an alternative path for accumulation of Ile, Leu and/or Val, i.e., a medium supplement of the appropriate dipeptides, or by engineering the transport capacity for branched-chain amino acids, the expression levels could be increased several fold.

Conclusions

We show that the availability of branched chain amino acids is a critical factor for the (over)expression of proteins in L. lactis. The forward engineering of cells for functional protein production required fine-tuning of co-expression of the branched chain amino acid transporter.