Gap junctions in the ovary of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels

Background  

In the Drosophila ovary, germ-line and soma cells are interconnected via gap junctions. The main gap-junction proteins in invertebrates are members of the innexin family. In order to reveal the role that innexins play in cell-cell communication during oogenesis, we investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and analyzed follicle development following channel blockade.     

Molecular Characterization,Localization, and Distribution of Innexins in the Silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Gap junctions that allow for a direct exchange of second messenger and ions are the most conserved cellular structures in multicellular organisms. We have isolated and characterized a Bombyx mori gene innexin3 that encodes a new member of the innexin family required for the early embryonic development. The BmINX3 mRNA was 1,814 nucleotide residues in length, and the deduced amino acid sequence of BmInx3 shared 74% similarity with Apis melifera innexin3. The expression profile of the BmINX3 mRNA is similar to that of previously described BmINX2, expressed in ovary and testis after 5th instar larvae and in fat body after gut purge. However, during embryogenesis, the expression of BmINX3 mRNA is restricted to the blastokinesis stage. Microscopic observation of the BmInx2 and BmInx3 fused to fluorescent proteins showed an overlapping cytoplasmic expression, whereas the BmInx4 is accumulated in the cytoplasmic surface at which two cells have physical contact. This finding of innexins distribution in silkworm would provide an essential basis for future studies of the functions and interactions of innexins.     

<Emphasis Type="Italic">qRfg3</Emphasis>, a novel quantitative resistance locus against <Emphasis Type="Italic">Gibberella</Emphasis> stalk rot in maize

Key message

A quantitative trait locus  qRfg3 imparts recessive resistance to maize Gibberella stalk rot. qRfg3 has been mapped into a 350-kb interval and could reduce the disease severity index by ~26.6%.

Abstract

Gibberella stalk rot, caused by the fungal pathogen Fusarium graminearum, severely affects maize yield and grain quality worldwide. To identify more resistance quantitative trait loci (QTLs) against this disease, we analyzed a recombinant inbred line (RIL) population derived from a cross between resistant H127R and susceptible C7-2 inbred lines. Within this population, maize resistance to Gibberella stalk rot had high broad-sense heritability. A major QTL, qRfg3, on chromosome 3 was consistently detected across three field trials, accounting for 10.7–19.4% of the total phenotypic variation. Using a progeny-based sequential fine-mapping strategy, we narrowed qRfg3 down to an interval of ~350 kb. We further demonstrated that qRfg3 is a recessive resistance locus to Gibberella stalk rot that reduced the disease severity index by ~26.6%. Both the gene location and recessive genetic mode distinguish qRfg3 from other stalk rot resistance loci. Hence, qRfg3 is valuable as a complement to existing resistance QTLs to improve maize resistance to Gibberella stalk rot.

     

Gap junctions in Drosophila: developmental expression of the entire innexin gene family

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.     

Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

Background

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated; one reason is that there is no follicle culture system that can reproduce theca cell layer formation in vitro. Therefore, a three-dimensional follicle culture system that can reproduce theca cell layer formation is required.

Methods

A collagen gel was used in the follicle culture system. To determine the optimum conditions for follicle culture that can reproduce theca cell layer formation, the effects of hormonal treatment and cell types co-cultured with follicles were examined. In addition, immunohistochemistry was used to examine the properties of the cell layers formed in the outermost part of follicles.

Results

Follicles maintained a three-dimensional shape and grew in collagen gel. By adding follicle-stimulating hormone (FSH) and co-culturing with interstitial cells, the follicles grew well, and cell layers were formed in the outermost part of follicles. Immunohistochemistry confirmed that the cells forming the outermost layers of the follicles were theca cells.

Conclusion

In this study, follicle culture system that can reproduce theca cell layer formation in vitro was established. In our opinion, this system is suitable for the analysis of theca cell layer formation and contributes to our understanding of the mechanisms of folliculogenesis.     

Exome sequencing identifies novel and recurrent mutations in <Emphasis Type="Italic">GJA8</Emphasis> and <Emphasis Type="Italic">CRYGD</Emphasis>associated with inherited cataract

Background

Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families.

Results

In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with `coralliform' lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function.

Conclusions

Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families.

     

Evaluation of <Emphasis Type="Italic">ZmCCT</Emphasis> haplotypes for genetic improvement of maize hybrids

Key message

The elite ZmCCT haplotypes which have no transposable element in the promoter could enhance maize resistance to Gibberella stalk rot and improve yield-related traits, while having no or mild impact on flowering time. Therefore, they are expected to have great value in future maize breeding programs.

Abstract

A CCT domain-containing gene, ZmCCT, is involved in both photoperiod response and stalk rot resistance in maize. At least 15 haplotypes are present at the ZmCCT locus in maize germplasm, whereas only three of them are found in Chinese commercial maize hybrids. Here, we evaluated ZmCCT haplotypes for their potential application in corn breeding. Nine resistant ZmCCT haplotypes that have no CACTA-like transposable element in the promoter were introduced into seven elite maize inbred lines by marker-assisted backcrossing. The resultant 63 converted lines had 0.7-5.1 Mb of resistant ZmCCT donor segments with over 90% recovery rates. All converted lines tested exhibited enhanced resistance to maize stalk rot but varied in photoperiod sensitivity. There was a close correlation between the hybrids and their parental lines with respect to both resistance performance and photoperiod sensitivity. Furthermore, in a given hybrid A5302/83B28, resistant ZmCCT haplotype could largely improve yield-related traits, such as ear length and 100-kernel weight, resulting in enhanced grain yield. Of nine resistant ZmCCT haplotypes, haplotype H5 exhibited excellent performance for both flowering time and stalk rot resistance and is thus expected to have potential value in future maize breeding programs.

     

Epithelial-specific histone modification of the <Emphasis Type="Italic">miR-96/182</Emphasis> locus targeting <Emphasis Type="Italic">AMAP1</Emphasis> mRNA predisposes p53 to suppress cell invasion in epithelial cells

Background

TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1.

Methods

Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells.

Results

We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3′-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells.

Conclusion

Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

     

Regulation of Epithelial Stem Cell Replacement and Follicle Formation in the Drosophila Ovary

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. We show that one daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when ∼16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis.THE Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell (reviewed in Blanpain et al. 2007; Kirilly and Xie 2007). New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (reviewed in Spradling 1993). Follicle formation begins in the germarium (Figure 1A), a structure at the tip of each ovariole that houses 2–3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches (reviewed in Morrison and Spradling 2008). Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte (reviewed in de Cuevas et al. 1997). At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a “lens shape” characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Figure 1B).Open in a separate windowFigure 1.—Prefollicle cells associate with cysts in an ordered fashion downstream from the FSCs. (A) A diagram of the Drosophila germarium showing the four subregions: 1, 2a, 2b, and 3. Two GSCs (orange) reside in region 1 and produce cysts (yellow ovals). Two FSCs reside at the border of regions 2a and 2b and produce follicle cells that encapsulate region 2b and region 3 cysts. (B) A diagram of two follicles that have budded from the germarium showing their pairs of anterior and posterior polar cells as well as the interconnecting 4–6 stalk cells. (C) Germaria stained with anti-traffic jam (green) to mark somatic cells, anti-vasa (red) to mark germ cells, and DAPI (blue). The numbers of somatic cells associated with each cyst (indicated) were reconstructed from three-dimensional image stacks. (D–F) Small transient clones stained with anti-LacZ (green, the clonal marker), anti-FasIII (red), and DAPI (blue). Regions 2b and 3 cysts are outlined in white. Pink dots indicate labeled FSC daughters; however, not all labeled cells are marked because some are not visible in the presented plane of focus. (D) A 4-cell clone associated with the first cyst in region 2b. (E) An 8-cell clone associated with the second region 2b cyst. (F) A 15-cell clone associated with the region 3 cyst. (G) Model of follicle layer acquisition. One FSC daughter, the cmc (light green, left) contacts the anterior face of the incoming cyst (2a/b, orange) and founds mostly anterior follicle cells (light green). Another FSC daughter, the pmc (dark green, left) contacts the posterior cyst face and founds mostly posterior follicle cells (dark green). Bar, 10 μm; anterior is to the left.A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning (reviewed in Huynh and St. Johnston 2004). However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs (Margolis and Spradling 1995; Nystul and Spradling 2007), low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Zhang and Kalderon 2001; Vied and Kalderon 2009).The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production (Ruohola et al. 1991; Larkin et al. 1996; Grammont and Irvine 2001), and depends on Notch signals received from the germline (Lopez-Schier and St. Johnston 2001). Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells (McGregor et al. 2002; Torres et al. 2003; Assa-Kunik et al. 2007). While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b (Margolis and Spradling 1995). In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2 (Torres et al. 2003). Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5 (Besse and Pret 2003). Moreover, polar and stalk are believed to arise exclusively from “polar/stalk” precursors that separate from the rest of the FSC lineage (Larkin et al. 1996; Tworoger et al. 1999; Besse and Pret 2003) and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Torres et al. 2003; Assa-Kunik et al. 2007).Here we have analyzed the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and we show that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. Our results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution.     

Integrative analysis of the Pekin duck (<Emphasis Type="Italic">Anas anas</Emphasis>) MicroRNAome during feather follicle development

Background

The quality and yield of duck feathers are very important economic traits that might be controlled by miRNA regulation. The aim of the present study was to investigate the mechanism underlying the crosstalk between individual miRNAs and the activity of signaling pathways that control the growth of duck feathers during different periods. We therefore conducted a comprehensive investigation using Solexa sequencing technology on the Pekin duck microRNAome over six stages of feather development at days 11, 15, and 20 of embryonic development (during the hatching period), and at 1 day and 4 and 10 weeks posthatch.

Results

There were a total of 354 known miRNAs and 129 novel candidate miRNAs found based on comparisons with known miRNAs in the Gallus gallus miRBase. The series of miRNAs related to feather follicle formation as summarized in the present study showed two expression patterns, with primary follicle developed during embryonic stage and secondary follicle developed mainly at early post hatch stage. Analysis of miRNA expression profiles identified 18 highly expressed miRNAs, which might be directly responsible for regulation of feather development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that in addition to Wnt and transforming growth factor (TGFβ) signaling pathways, which were widely reported in response to follicle formation, another group of signaling pathways that regulate lipid synthesis and metabolism, such as the phosphatidylinositol signaling system and glycerolipid metabolism and signaling, are also responsible for follicle formation.

Conclusion

The highly expressed miRNAs provide a valuable reference for further investigation into the functional miRNAs important for feather development. Lipid synthesis and metabolism related signaling pathways might be responsible for lipid formation on the surface of feather, and should be paid much more attention for their relation to feather quality.

     

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V₁ and membrane V₀ sectors inactivates MgATPase activity of the V₁-ATPase. This inactivation requires the V₁ H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761–21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V₁ and V₀ subunits were identified by two-hybrid assay. The B subunit of the V₁ catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V₁ subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V₀ subunit Vph1p. V₁-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V₁ when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V₁ lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V₁ was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V₁ complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V₁-ATPase activity and precludes V₀ interactions.V-ATPases are ubiquitous proton pumps responsible for compartment acidification in all eukaryotic cells (1, 2). These pumps couple hydrolysis of cytosolic ATP to proton transport into the lysosome/vacuole, endosomes, Golgi apparatus, clathrin-coated vesicles, and synaptic vesicles. Through their role in organelle acidification, V-ATPases are linked to cellular functions as diverse as protein sorting and targeting, zymogen activation, cytosolic pH homeostasis, and resistance to multiple types of stress (3). They are also recruited to the plasma membrane of certain cells, where they catalyze proton export (4, 5).V-ATPases are evolutionarily related to ATP synthases of bacteria and mitochondria and consist of two multisubunit complexes, V₁ and V₀, which contain the sites for ATP hydrolysis and proton transport, respectively. Like the ATP synthase (F-ATPase), V-ATPases utilize a rotational catalytic mechanism. ATP binding and hydrolysis in the three catalytic subunits of the V₁ sector generate sequential conformational changes that drive rotation of a central stalk (6–8). The central stalk subunits are connected to a ring of proteolipid subunits in the V₀ sector that bind protons to be transported. The actual transport is believed to occur at the interface of the proteolipids and V₀ subunit a. Rotational catalysis will be productive in proton transport only if V₀ subunit a is held stationary, whereas the proteolipid ring rotates (8). This “stator function” resides in a single peripheral stalk in F-ATPases (9, 10), but is distributed among up to three peripheral stalks in V-ATPases (11–13). The peripheral stator stalks link V₀ subunit a to the catalytic headgroup and ensures that there is rotation of the central stalk complex relative to the V₀ a subunit and catalytic headgroup.Eukaryotic V-ATPases are highly conserved in both their overall structure and the sequences of individual subunits. Although homologs of most subunits of eukaryotic V-ATPases are present in archaebacterial V-ATPases (also known as A-ATPases), the C and H subunits are unique to eukaryotes. Both subunits have been localized at the interface of the V₁ and V₀ sectors, suggesting that they are positioned to play a critical role in structural and functional interaction between the two sectors (14–16). The yeast C and H subunits are the only eukaryotic V-ATPase subunits for which x-ray crystal structures are available (17, 18). The structure of the C subunit revealed an elongated “dumbbell-shaped” molecule, with foot, head, and neck domains (18). The structure of the H subunit indicated two domains. The N-terminal 348 amino acids fold into a series of HEAT repeats and are connected by a 4-amino acid linker to a C-terminal domain containing amino acids 352–478 (17). These two domains have partially separable functions in the context of the assembled V-ATPase (19). Complexes containing only the N-terminal domain of the H subunit (H-NT)² supported some ATP hydrolysis but little or no proton pumping in isolated vacuolar vesicles (19, 20). The C-terminal domain (H-CT) assembled with the rest of the V-ATPase in the absence of intact subunit H, but supported neither ATPase nor proton pumping activity (19). However, co-expression of the H-NT and H-CT domains results in assembly of both sectors with the V-ATPase and allows increased ATP-driven proton pumping in isolated vacuolar vesicles. These results suggest that the H-NT and H-CT domains play distinct and complementary roles even when the two domains are not covalently attached.In addition to their role as dedicated proton pumps, eukaryotic V-ATPases are also distinguished from F-ATPases and archaeal V-ATPases in their regulation. Eukaryotic V-ATPases are regulated in part by reversible disassembly of the V₁ complex from the V₀ complex (1, 21, 22). In yeast, disassembly of previously assembled complexes occurs in response to glucose deprivation, and reassembly is rapidly induced by glucose readdition to glucose-deprived cells. Disassembly down-regulates pump activity, and both the disassembled sectors are inactivated. Inhibition of ATP hydrolysis in free V₁ sectors is particularly critical, because release of an active ATPase into the cytosol could deplete cytosolic ATP stores. This inhibition is dependent in part on the H subunit. V₁ complexes isolated from vma13Δ mutants, which lack the H subunit gene (V₁(-H) complexes) have MgATPase activity. Consistent with a physiological role for H subunit inhibition of V₁, heterozygous diploids containing elevated levels of free V₁ complexes without subunit H have severe growth defects (23). V₁ complexes containing subunit H have no MgATPase activity, but retain some CaATPase activity, suggesting a role for nucleotides in inhibition (24, 25). Consistent with such a role, both the CaATPase activity of native V₁ and the MgATPase activity of V₁(-H) complexes are lost within a few minutes of nucleotide addition (24).A number of points of interaction between the H subunit and the V₁ and V₀ complexes have been identified through two-hybrid assays, binding of expressed proteins, and cross-linking experiments. These experiments have indicated that the H subunit binds to V₁ subunits E and G of the V-ATPase peripheral stalks (26, 27), the catalytic subunit (V₁ subunit A) (28), regulatory V₁ subunit B (15), and the N-terminal domain of subunit a (28). Recently, Jeffries and Forgac (29) have found that cysteines introduced into the C-terminal domain of subunit H can be cross-linked to subunit F in isolated V₁ sectors via a 10-Å cross-linking reagent.In this work, we examine both the subunit-subunit interactions and functional roles of the H-NT and H-CT domains in inhibition of V₁-ATPase activity. When expressed in yeast cells lacking subunit H, H-NT can be isolated with cytosolic V₁ complexes, but H-CT cannot. We find that both of these domains contribute to inhibition of ATPase activity, but that stable binding to V₁ and full inhibition of activity requires both domains. We also find that the H-CT can bind to the cytosolic N-terminal domain of V₀ subunit Vph1p (Vph1-NT) in isolation, but does not support tight binding of Vph1-NT to isolated V₁ complexes.     

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673–685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an α-helical coiled-coil, ∼80 residues in length, whereas the C-terminal residues interact to form a globular domain containingα- and β-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit (Lokanath, N. K., Matsuura, Y., Kuroishi, C., Takahashi, N., and Kunishima, N. (2007) J. Mol. Biol. 366, 933–944). However, upon the addition of a peptide comprising the C-terminal 21 residues of the H subunit (or full-length H subunit), dimeric E subunit C-terminal domain dissociates to form a 1:1 heterodimer. NMR spectroscopy was used to show that H subunit C-terminal peptide binds to E subunit C-terminal domain via the terminal α-helices, with little involvement of the β-sheet region. Based on these data, we propose a structural model of the A-ATPase peripheral stalk.The archaeal ATP synthase (A₁A₀-ATPase),² along with the related F₁F₀- and V₁V₀-ATPases (proton pumping vacuolar ATPases), is a rotary molecular motor (1–4). The rotary ATPases are bilobular in overall architecture, with one lobe comprising the water-soluble A₁, F₁, or V₁ and the other comprising the membrane-bound A₀, F₀, or V₀ domain, respectively. The subunit composition of the A-ATPase is A₃B₃DE₂FH₂ for the A₁ and CIK_x for the A₀. In the A₁ domain, the three A and B subunits come together in an alternating fashion to form a hexamer with a hydrophobic inner cavity into which part of the D subunit is inserted. Subunits D and F comprise the central stalk connection to A₀, whereas two heterodimeric EH complexes are thought to form the peripheral stalk attachment to A₀ seen in electron microscopy reconstructions (5, 6). In the A₀ domain (subunits CIK_x), the K subunits (proteolipids) form a ring that is linked to the central stalk by the C subunit, whereas the cytoplasmic N-terminal domain of the I subunit probably mediates the binding of the EH peripheral stalks to A₀, as suggested for the bacterial A/V-type enzyme (7). Although closer in structure to the proton-pumping V-ATPase, the A-ATPase functions in vivo as an ATP synthase, coupling ion motive force to ATP synthesis, most likely via a similar rotary mechanism as demonstrated for the bacterial A/V- and the vacuolar type enzymes (8, 9). During catalysis, substrate binding occurs sequentially on the three catalytic sites, which are formed predominantly by the A subunits. This is accompanied by conformation changes in the A₃B₃ hexamer that are linked to the rotation of the embedded D subunit together with the rotor subunits F, C, and the proteolipid ring. Each copy of K contains a lipid-exposed carboxyl residue (Asp or Glu), which is transiently interfaced with the membrane-bound domain of I during rotation, thereby catalyzing ion translocation. The EH peripheral stalks function to stabilize the A₃B₃ hexamer against the torque generated during rotation of the central stalk. Much work has been accomplished to elucidate the architectural features of the rotational and catalytic domains, especially in the related F- and V-type enzymes. However, the peripheral stalk complexes in the A- and V-type enzymes remain an area open to question. Although the stoichiometry of the peripheral stalks in the A/V-type and the vacuolar type ATPases have recently been resolved to two and three, respectively (6, 10), the overall structure of the peripheral stalk, including the nature of attachment to the A₃B₃ hexamer and I subunit (called subunit a in the F- and V-ATPase), is not well understood. Some structural information exists in the form of the A-ATPase E subunit C-terminal domain (11), although isolation from its binding partner H may have influenced its conformation.Previously, our lab has characterized the Thermoplasma acidophilum A-ATPase E and H subunits individually and in complex (12). We found that despite their tendency to oligomerize when isolated separately, upon mixing, E and H form a tight heterodimer that was monodisperse and elongated in solution, which is consistent with its role as the peripheral stalk element in the A-ATPase. Here, we have expanded our study of the A-ATPase EH complex through the production of various N- and C-terminal truncation mutants of both binding partners. The data show that the EH complex is comprised of two distinct domains, one that contains both N termini interacting via a coiled-coil and a second that contains both C termini folded in a globular structure containing mixed secondary structure. Consistent with recent crystallographic data for the related A-ATPase from Pyrococcus horikoshii (11), we found that the isolated C-terminal domain of the E subunit exists as a stable homodimer in solution. However, the addition of subunit H or a peptide consisting of the 21 C-terminal residues of the subunit to the dimeric C-terminal domain of subunit E resulted in dissociation of the homodimer with concomitant formation of a 1:1 heterodimer containing the C termini of both polypeptides. This study delineates and characterizes the two domains of the EH complex and will aid in the further exploration of the nature of peripheral stalk attachment and function in the intact A₁A₀-ATPase.     

Rab11 is required for epithelial cell viability, terminal differentiation, and suppression of tumor-like growth in the Drosophila egg chamber

Background

The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary.

Methodology/Principal Findings

Here we investigate the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells. We show that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. We also show that rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but that none of the cells complete their differentiation programs and that the epithelial cells undergo premature programmed cell death. Finally we show, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity.

Conclusions/Significance

While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, our study is the first to identify such activity for regulators of endocytic recycling. Our studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues.     

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> DU202

Background

Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis.

Methods

Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed.

Results

OMV secretion was increased >?twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which β-lactamase OXA-23, various proteases, outer membrane proteins, β-barrel assembly machine proteins, peptidyl-prolyl cis–trans isomerases and inherent prophage head subunit proteins were significantly upregulated.

Conclusion

In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

     

HspX protein as a candidate vaccine against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: an overview

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.