Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.     

Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+, Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 and pmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.     

The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation

SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.     

The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca²⁺-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca²⁺-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown.

Objective

Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca²⁺-mobilization and cytoskeletal rearrangements.

Methods and Results

We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2^-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca²⁺-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2^-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation.

Conclusion

These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss.     

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.     

Lysine Methylation of the Valosin-Containing Protein (VCP) Is Dispensable for Development and Survival of Mice

Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1–4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt ^-/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt ^-/- mouse organs or three human Vcpkmt ^-/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt ^-/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.     

PXA Domain-Containing Protein Pxa1 Is Required for Normal Vacuole Function and Morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

Replication Protein A (RPA1a) Is Required for Meiotic and Somatic DNA Repair But Is Dispensable for DNA Replication and Homologous Recombination in Rice

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.In a population of organisms, it is crucial to maintain the integrity of genome among individuals as well as shuffle genetic information at the population level. To maintain such genetic integrity, cells have evolved elaborate mechanisms such as base excision repair (BER; Hegde et al., 2008), nucleotide excision repair (NER; Shuck et al., 2008), homologous recombination (HR; Li and Heyer, 2008) repair, and nonhomologous end joining (Weterings and Chen, 2008) pathways to repair diverse types of DNA damage. To allow for variation, however, organisms utilize meiosis to shuffle genetic material so as to increase genetic diversity in populations and in the species.DNA double-strand break (DSB) repair is particularly important in maintaining the integrity of genome among individuals and shuffling genetic information among population, because DSBs are generated not only in meiotic cells but also from the action of certain endogenous or exogenous DNA-damaging agents and during repair of other kinds of DNA lesions by NER or BER (West et al., 2004; Bleuyard et al., 2006). The past decade has witnessed an explosion in understanding of this complex process by using yeast (Saccharomyces cerevisiae) as a model organism (Aylon and Kupiec, 2004). Cells can repair DSBs by the relatively inaccurate process of rejoining the two broken ends directly (i.e. nonhomologous end joining) or much more accurately by HR (Bleuyard et al., 2006; Wyman and Kanaar, 2006). These two pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type (Shrivastav et al., 2008). According to the current general model for meiotic DSB repair (Bishop and Zickler, 2004; Ma, 2006; San Filippo et al., 2008), when DSBs occur the MRN complex (composed of Mre11, Rad50, and NBS1) resects the DSBs to generate 5′→3′ single-stranded DNA (ssDNA) ends. Subsequently, the replication protein A (RPA) protein complex binds to the ssDNA ends to protect them from attack by endogenous exonucleases; then, in concert with catalysis by Rad52, Rad55, and Rad57, the recombinase Rad51 displaces RPA, resulting in the generation of a Rad51 nucleoprotein filament that in turn catalyzes the search and invasion into the recombination partner with the help of proteins belonging to the RAD52 epistasis group to form a D loop that accompanies DNA synthesis. Thereafter, at least two competing mechanisms may come into play. One is the DSB repair pathway, in which the capture of the second DSB end and additional DNA synthesis result in an intermediate that harbors two Holliday junctions. The subsequent resolution of Holliday junctions results in the formation of crossovers. Alternatively, in the synthesis-dependent strand annealing pathway, the D loop dissociates and the invading single strand with newly synthesized DNA reanneals with the other DSB end, followed by gap-filling DNA synthesis and ligation, forming only noncrossover products (Ma, 2006; San Filippo et al., 2008).RPA is comprised of three subunits of RPA1, 2, and 3, alternatively termed as RPA70, 32, and 14, respectively, according to their apparent M_rs (Wold, 1997; Iftode et al., 1999). RPA is an essential protein in various DNA metabolism pathways such as DNA replication, repair, and HR (Wold, 1997; Iftode et al., 1999). In these pathways, the most basic function of RPA is binding to ssDNA to protect it from exonucleases, and its general roles in DNA metabolism depend on its interactions with other proteins in various pathways (Wold, 1997; Iftode et al., 1999). For example, in human NER pathway, RPA binds to damaged DNA and interacts with xeroderma pigmentosum damage-recognition protein, XPA, in the damage recognition step, and then the endonucleases XPG and ERCC1/XPF are recruited to the RPA-XPA-damaged DNA complex in the excision step (He et al., 1995). Interactions of RPA with those proteins are critical in this process (Wold, 1997; Iftode et al., 1999). A great deal of protein dynamics research has indicated that the interactions between RPA and other DNA-metabolism proteins are choreographed on the ssDNA to recruit the required protein present at the proper time (Fanning et al., 2006).Human, animals, and fungi have single copy for each subunit of RPA (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, have multiple genes for most RPA subunits (Ishibashi et al., 2006; Shultz et al., 2007). Most of them have not unveiled exact function up to now. To elucidate the molecular basis of meiosis in rice, we performed a large-scale screen for sterile mutants using our T-DNA insertion mutant library (Wu et al., 2003). Previously, we reported the cloning of OsPAIR3, a novel gene required for homologous chromosome pairing and synapsis in rice (Yuan et al., 2009). Here we report the characterization of another sterile mutant with a T-DNA insertion in OsRPA1a. Our results indicate that OsRPA1a is essential for DNA repair but may play redundant roles in DNA replication and recombination in rice.     

The Septin CDCrel-1 Is Dispensable for Normal Development and Neurotransmitter Release

Septins are GTPases required for the completion of cytokinesis in a variety of organisms, yet their role in this process is not known. Septins may have additional functions since the mammalian septin CDCrel-1 is predominantly expressed in the nervous system, a largely postmitotic tissue. While relatively little is known about the function of this protein, we have previously shown that it is involved in regulated secretion. In addition, the gene encoding this protein maps to a locus often deleted in velo-cardiofacial and DiGeorge syndromes, and CDCrel-1 has recently been shown to be a direct target of the E3 ubiquitin ligase activity of Parkin, a causative agent in autosomal recessive forms of Parkinson's disease. Here we show that CDCrel-1 expression rises at the time of synaptic maturation and that CDCrel-1 is present in a complex that includes the septins Nedd5 and CDC10. To investigate its function in the nervous system, we generated homozygotic CDCrel-1 null mice and showed that these mice appear normal with respect to synaptic properties and hippocampal neuron growth in vitro. Moreover, we found that while the expression of a number of synaptic proteins is not affected in the CDCrel-1 mutant mice, the expression of other septins is altered. Together, these data suggest that CDCrel-1 is not essential for neuronal development or function, and that changes in expression of other septins may account for its functional redundancy.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.