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.     

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.     

Stathmin Is Dispensable for Tumor Onset in Mice

The microtubule-destabilizing protein stathmin is highly expressed in several types of tumor, thus deserving the name of oncoprotein 18. High levels of stathmin expression and/or activity favor the metastatic spreading and mark the most aggressive tumors, thus representing a realistic marker of poor prognosis. Stathmin is a downstream target of many signaling pathways, including Ras-MAPK, PI3K and p53, involved in both tumor onset and progression. We thus hypothesized that stathmin could also play a role during the early stages of tumorigenesis, an issue completely unexplored. In order to establish whether stathmin expression is necessary for tumor initiation, we challenged wild type (WT), stathmin heterozygous and stathmin knock-out (KO) mice with different carcinogens. Using well-defined mouse models of carcinogenesis of skin, bladder and muscle by the means of 7,12-dimethylbenz[α]antracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and 3-methylcholanthrylene (3MC) treatments, respectively, we demonstrated that knock-out of stathmin has no impact on the onset of cancer in mice. No significant difference was noticed either when the Ras oncogene was mutated (skin carcinogenesis model) or when the p53 pathway was inactivated (bladder carcinomas and fibrosarcomas). Finally, we concomitantly impinged on p53 and Ras pathways, by generating WT and stathmin KO mouse embryo fibroblasts transformed with papilloma virus large T antigen (LgTAg) plus the K-Ras^G12V oncogene. In vivo growth of xenografts from these transformed fibroblasts did not highlight any significant difference depending on the presence or absence of stathmin. Overall, our work demonstrates that stathmin expression is dispensable for tumor onset, at least in mice, thus making stathmin a virtually exclusive marker of aggressive disease and a promising therapeutic target for advanced cancers.     

Ezrin Is Highly Expressed in Early Thymocytes,but Dispensable for T Cell Development in Mice

Background

Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.

Methodology/Principal Findings

We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin^−/− mice likely arise as a consequence of nutritional stress.

Conclusions/Significance

We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.     

Piwi Genes Are Dispensable for Normal Hematopoiesis in Mice

Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized “piwi” refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal lineage reconstitution after competitive transplantation. We further show that the three piwi genes are dispensable during hematopoietic recovery after myeloablative stress by 5-FU. Collectively, our data suggest that the function of the piwi gene subfamily is not required for normal adult hematopoiesis.     

The Hedgehog Receptor Patched1 in T Cells Is Dispensable for Adaptive Immunity in Mice

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and T_reg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.     

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.     

Transcription Factor GATA1 Is Dispensable for Mast Cell Differentiation in Adult Mice

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1^−/y), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1^−/y bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5′ end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit⁺ FcεRIα⁺ mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.     

mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility

The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.     

Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.     

Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.     

DNA Methyltransferase 3b Is Dispensable for Mouse Neural Crest Development

The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.     

The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAV_UK. Various EIAV_UK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAV_UK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.