Mouse Odf2 localizes to centrosomes and basal bodies in adult tissues and to the photoreceptor primary cilium

Odf2 (outer dense fiber 2) is the major protein of the cytoskeleton of the sperm tail. In somatic cells, it is a component of the centrosome in which it is located in the appendages of the mother centriole. Additionally, as shown previously by forced expression in cultured cells, Odf2 localizes to centrioles, basal bodies, and primary cilia, which are all structurally and functionally interconnected. The importance of Odf2 has become obvious by the absence of primary cilia in Odf2-deficient cells and by the embryonic lethality of the Odf2 gene trap insertional mouse. However, nothing is known about the endogenous localization of Odf2 in the tissues of adult mice. We show here that Odf2 protein localizes to centrosomes, to photoreceptor primary cilia, and to basal bodies of ciliated cells of the respiratory epithelium and of the kidney. Our results thus suggest that Odf2 contributes to assorted ciliopathies

Chronic exposure to cannabinoids during adolescence causes long-lasting behavioral deficits in adult mice

Regular use of marijuana during adolescence enhances the risk of long-lasting neurobiological changes in adulthood. The present study was aimed at assessing the effect of long-term administration of the synthetic cannabinoid WIN55212.2 during adolescence in young adult mice. Adolescent mice aged 5 weeks were subjected daily to the pharmacological action of WIN55212.2 for 3 weeks and were then left undisturbed in their home cage for a 5-week period and finally evaluated by behavioral testing. Mice that received the drug during adolescence showed memory impairment in the Morris water maze, as well as a dose-dependent memory impairment in fear conditioning. In addition, the administration of 3 mg/kg WIN55212.2 in adolescence increased adult hippocampal AEA levels and promoted DNA hypermethylation at the intragenic region of the intracellular signaling modulator Rgs7, which was accompanied by a lower rate of mRNA transcription of this gene, suggesting a potential causal relation. Although the concrete mechanisms underlying the behavioral observations remain to be elucidated, we demonstrate that long-term administration of 3 mg/kg of WIN during adolescence leads to increased endocannabinoid levels and altered Rgs7 expression in adulthood and establish a potential link to epigenetic changes.Beca Ramón y Caja

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

<p>Abstract</p> <p>Background</p> <p>Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation.</p> <p>Results</p> <p>We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed <it>in vitro </it>by GST pull-down assays and <it>in vivo </it>by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments.</p> <p>Conclusion</p> <p>We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.</p

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals

Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly

Cep120, a protein involved in maintenance of neural progenitor cells, is required for centriole duplication in cycling cells and for centriole amplification in tracheal epithelial cells

X Chromosome Inactivation and Differentiation Occur Readily in ES Cells Doubly-Deficient for MacroH2A1 and MacroH2A2

Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content

Meiotic Pairing and Segregation of Achiasmate Sex Chromosomes in Eutherian Mammals: The Role of SYCP3 Protein

In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (Meriones unguiculatus). We have analyzed the location of synaptonemal complex (SC) proteins SYCP1 and SYCP3, as well as three proteins involved in the process of meiotic recombination (RAD51, MLH1, and γ-H2AX). Our results show that although X and Y chromosomes are associated at pachytene and form a sex body, their axial elements (AEs) do not contact, and they never assemble a SC central element. Furthermore, MLH1 is not detected on the AEs of the sex chromosomes, indicating the absence of reciprocal recombination. At diplotene the organization of sex chromosomes changes strikingly, their AEs associate end to end, and SYCP3 forms an intricate network that occupies the Y chromosome and the distal region of the X chromosome long arm. Both the association of sex chromosomes and the SYCP3 structure are maintained until metaphase I. In anaphase I sex chromosomes migrate to opposite poles, but SYCP3 filaments connecting both chromosomes are observed. Hence, one can assume that SYCP3 modifications detected from diplotene onwards are correlated with the maintenance of sex chromosome association. These results demonstrate that some components of the SC may participate in the segregation of achiasmate sex chromosomes in eutherian mammals

A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations

Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse