RESUMEN
Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in the transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we showed that ODF2 controls CP110 levels. The overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but the overexpression of NEURL4 or HYLS1 is. The co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation.
Asunto(s)
Cuerpos Basales , Centriolos , Cilios , Dimerización , SirolimusRESUMEN
Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.
Asunto(s)
Cilios , Proteínas de Choque Térmico , Cilios/genética , Cilios/metabolismo , Proteínas de Choque Térmico/metabolismo , Centriolos/metabolismo , Cuerpos Basales/metabolismo , Regulación de la Expresión GénicaRESUMEN
ODF1 is a major protein of the accessory fibres of the mammalian sperm tail. In addition, ODF1 is found in the connecting piece, a complex structure located at the posterior end of the nucleus that connects the sperm head and tail. The tight coupling of the sperm head and tail is critical for the progressive motility of the sperm to reach the oocyte for fertilisation. The depletion of ODF1 by homologous recombination in mice led to male infertility. Although sperm tails were present in the epididymis, no intact spermatozoa were found. Instead, the depletion of ODF1 resulted in sperm decapitation, suggesting that ODF1 is essential for the formation of the coupling apparatus and the tight linkage of the sperm head and tail. However, the development of the linkage complex in the absence of ODF1 has never been investigated. Here, I analysed the fine structure of the developing connecting piece by transmission electron microscopy. I show that the connecting piece develops as in wild-type spermatids. Structural abnormalities were not observed when ODF1 was absent. Thus, ODF1 is dispensable for the development of the connecting piece. However, the decapitation of ODF1-deficient spermatozoa indicates that the heads and tails of the spermatozoa are not linked, so that they separate when force is applied.
Asunto(s)
Decapitación , Espermátides , Animales , Decapitación/metabolismo , Masculino , Mamíferos , Ratones , Semen , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatozoides/metabolismoRESUMEN
The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium.
Asunto(s)
Fibroblastos , Miofibroblastos , Animales , Embrión de Mamíferos , Matriz Extracelular , Fibroblastos/metabolismo , Ratones , Células 3T3 NIHRESUMEN
The dynamics of neuronal microtubules are essential for brain plasticity. Vesicular transport and synaptic transmission, additionally, requires acetylation of α-tubulin, and aberrant tubulin acetylation and neurobiological deficits are associated. Prolonged exposure to a stressor or consumption of drugs of abuse, like marihuana, lead to neurological changes and psychotic disorders. Here, we studied the effect of psychosocial stress and the administration of cannabinoid receptor type 1 drugs on α-tubulin acetylation in different brain regions of mice. We found significantly decreased tubulin acetylation in the prefrontal cortex in stressed mice. The impact of cannabinoid drugs on stress-induced microtubule disturbance was investigated by administration of the cannabinoid receptor agonist WIN55,212-2 and/or antagonist rimonabant. In both, control and stressed mice, the administration of WIN55,212-2 slightly increased the tubulin acetylation in the prefrontal cortex whereas administration of rimonabant acted antagonistically indicating a cannabinoid receptor type 1 mediated effect. The analysis of gene expression in the prefrontal cortex showed a consistent expression of ApoE attributable to either psychosocial stress or administration of the cannabinoid agonist. Additionally, ApoE expression inversely correlated with acetylated tubulin levels when comparing controls and stressed mice treated with WIN55,212-2 whereas rimonabant treatment showed the opposite.
Asunto(s)
Cannabinoides , Tubulina (Proteína) , Acetilación , Animales , Apolipoproteínas E/genética , Agonistas de Receptores de Cannabinoides/metabolismo , Cannabinoides/metabolismo , Cannabinoides/farmacología , Expresión Génica , Ratones , Microtúbulos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Corteza Prefrontal/metabolismo , Receptores de Cannabinoides/metabolismo , Rimonabant/farmacología , Estrés Psicológico , Tubulina (Proteína)/metabolismoRESUMEN
The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell's environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders.
Asunto(s)
Cuerpos Basales/patología , Centrosoma/patología , Células Germinativas/patología , Infertilidad Masculina/patología , Humanos , Infertilidad Masculina/etiología , Masculino , EspermatogénesisRESUMEN
The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.
Asunto(s)
Acetiltransferasas/metabolismo , Competencia Celular/fisiología , Tubulina (Proteína)/metabolismo , Acetilación , Acetiltransferasas/genética , Animales , Cilios/metabolismo , Fibroblastos/metabolismo , Ratones , Células 3T3 NIH , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
BACKGROUND: The Drosophila melanogaster mutant white-mottled is a well-established model for position-effect variegation (PEV). Transposition of the euchromatic white gene into the vicinity of the pericentric heterochromatin caused variegated expression of white due to heterochromatin spreading. The establishment of the euchromatin-heterochromatin boundary and spreading of silencing is regulated by mutually exclusive histone modifications, i.e. the methylations of histone H3 at lysine 9 and lysine 4. Demethylation of H3K4, catalysed by lysine-specific demethylase LSD1, is required for subsequent methylation of H3K9 to establish heterochromatin. LSD1 is therefore essential for heterochromatin formation and spreading. We asked whether drug-mediated inhibition of LSD affects the expression of white and if this induced change can be transmitted to those generations that have never been exposed to the triggering signal, i.e. transgenerational epigenetic inheritance. RESULTS: We used the lysine-specific demethylase 1 (LSD1)-inhibitor Tranylcypromine to investigate its effect on eye colour expression in consecutive generations by feeding the parental and F1 generations of the Drosophila melanogaster mutant white-mottled. Quantitative Western blotting revealed that Tranylcypromine inhibits H3K4-demethylation both in vitro in S2 cells as well as in embryos when used as feeding additive. Eye colour expression in male flies was determined by optical measurement of pigment extracts and qRT-PCR of white gene expression. Flies raised in the presence of Tranylcypromine and its solvent DMSO showed increased eye pigment expression. Beyond that, eye pigment expression was also affected in consecutive generations including F3, which is the first generation without contact with the inhibitor. CONCLUSIONS: Our results show that feeding of Tranylcypromine and DMSO caused desilencing of white in treated flies of generation F1. Consecutive generations, raised on standard food without further supplements, are also affected by the drug-induced alteration of histone modifications. Although eye pigment expression eventually returned to the basal state, the observed long-lasting effect points to a memory capacity of previous epigenomes. Furthermore, our results indicate that food compounds potentially affect chromatin modification and hence gene expression and that the alteration is putatively inherited not only parentally but transgenerationally.
Asunto(s)
Drosophila , Preparaciones Farmacéuticas , Animales , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Silenciador del Gen , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Oxidorreductasas N-DesmetilantesRESUMEN
Development of spermatozoa requires remodelling and formation of particular structures. In elongating spermatids, the transient microtubular manchette contributes to the formation of the head-tail coupling apparatus (HTCA) and the sperm tail. The HTCA derives from the centrosome in that the proximal centriole inserts into the nuclear indentation and the distal centriole gives rise to the sperm flagellum. Although impairments in the formation of HTCA and sperm tail cause male infertility their molecular constituents are only partially known. The WD40-protein CFAP52 is implicated in motile cilia, but its relevance for male germ cell differentiation is not known. Here we show that CFAP52 is widespread expressed and localizes to a subset of microtubular structures. In male germ cells, CFAP52 is a component of the transient manchette and the sperm tail. However, expression of Cfap52 is not restricted to motile cilia-bearing cells. In NIH3T3 cells, CFAP52 localizes to the centrosome, the basal body, and the mitotic spindle poles, but not to the primary cilium. Our results demonstrate that CFAP52 is not restricted to motile cilia but instead most likely functions in constituting the centrosome/basal body matrix and the sperm tail.
Asunto(s)
Proteínas Portadoras/metabolismo , Cilios/metabolismo , Flagelos/metabolismo , Espermátides/metabolismo , Animales , Cuerpos Basales/metabolismo , Proteínas Portadoras/genética , Centriolos/metabolismo , Centrosoma/metabolismo , Cilios/genética , Citoesqueleto/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Células 3T3 NIH , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatogénesis/fisiologíaRESUMEN
Terminal differentiation of male germ cells into functional spermatozoa requires shaping and condensation of the nucleus as well as the formation of sperm-specific structures. A transient microtubular structure, the manchette, is mandatory for sperm head shaping and the development of the connecting piece and the sperm tail. The connecting piece or head-to-tail coupling apparatus (HTCA) mediates the tight linkage of sperm head and tail causing decapitation and infertility when faulty. Using mice as the experimental model, several proteins have already been identified affecting the linkage complex, manchette or tail formation when missing. However, our current knowledge is far too rudimentary to even draft an interacting protein network. Depletion of the major outer dense fiber protein 1 (ODF1) mainly caused decapitation and male infertility but validated binding partners collaborating in the formation of sperm-specific structures are largely unknown. Amongst all candidate proteins affecting the HTCA when missing, the structural protein CCDC42 attracted our attention. The coiled-coil domain containing 42 (CCDC42) is important for HTCA and sperm tail formation but is otherwise largely uncharacterized. We show here that CCDC42 is expressed in spermatids and localizes to the manchette, the connecting piece and the tail. Beyond that, we show that CCDC42 is not restricted to male germ cells but is also expressed in somatic cells in which it localizes to the centrosome. Although centrosomal and sperm tail location seems to be irrespective of ODF1 we asked whether both proteins may form an interacting network in the male germ cell. We additionally considered ODF2, a prevalent protein involved in the formation of spermatid-specific cytoskeletal structures, as a putative binding partner. Our data depict for the first time the subcellular location of CCDC42 in spermatids and deepen our knowledge about the composition of the spermatid/sperm-specific structures. The presence of CCDC42 in the centrosome of somatic cells together with the obvious restricted male-specific phenotype when missing strongly argues for a compensatory function by other still unknown proteins most likely of the same family.
RESUMEN
The centrosome, as the main microtubule-organizing center, safeguards chromosome segregation by supporting the bipolar spindle. Centrosome aberrations are causally related to chromosome segregation disorders, both characterizing cancer cells. Thus, a restriction to only having one centrosome per cell and cell cycle-dependent duplication of the centrosome is mandatory. Duplicated centrosomes remain physically connected, in order to function as a single entity, until onset of mitosis when centrosome disjunction is licensed by disassembly of linker proteins and accumulation of ß-catenin. The crucial role ß-catenin plays in centrosome disjunction inevitably demands for restricting its premature accumulation. ODF2 (also known as cenexin) is an essential centrosomal component, but its relevance for the interphase centrosome has not been elucidated. We show here that ODF2 plays a central role in centrosome cohesion. Depletion of ODF2 induces premature centrosome splitting and formation of tripolar spindles that are likely caused by the observed accumulation of centrosomal ß-catenin. Our data collectively indicate that ODF2 restricts ß-catenin accumulation at the centrosome, thus preventing premature centrosome disjunction.
Asunto(s)
Centrosoma/metabolismo , Proteínas de Choque Térmico/genética , beta Catenina/metabolismo , Proteínas de Choque Térmico/metabolismo , HumanosRESUMEN
Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.
Asunto(s)
Proteínas Nucleares/deficiencia , Cabeza del Espermatozoide/química , Cabeza del Espermatozoide/ultraestructura , Cola del Espermatozoide/química , Cola del Espermatozoide/ultraestructura , Animales , Masculino , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismoRESUMEN
INTRODUCTION: Vulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident-intruder confrontations to study the brain corticostriatal-function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB1 receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures. AIMS AND METHODS: The investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid-like molecules and changes in the transcriptome. RESULTS: Stressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N-arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB1 activation, N-oleoylethanolamide and N-palmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co-administration with both cannabinoids induced an up-regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2-arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co-administration tended to produce a null effect under stress. CONCLUSIONS: The endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha-6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid-like mediators might be a valuable option for treating stress-related neuropsychiatric symptoms.
Asunto(s)
Cannabinoides/toxicidad , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Vías Nerviosas/efectos de los fármacos , Estrés Psicológico/patología , Animales , Peso Corporal/efectos de los fármacos , Antagonistas de Receptores de Cannabinoides , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Rimonabant/farmacología , Estrés Psicológico/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Tirapazamina/farmacologíaRESUMEN
Cortical glutamatergic neurons are generated by radial glial cells (RGCs), specified by the expression of transcription factor (TF) Pax6, in the germinative zones of the dorsal telencephalon. Here, we demonstrate that Pax6 regulates the structural assembly of the interphase centrosomes. In the cortex of the Pax6-deficient Small eye (Sey/Sey) mutant, we find a defect of the appendages of the mother centrioles, indicating incomplete centrosome maturation. Consequently, RGCs fail to generate primary cilia, and instead of staying in the germinative zone for renewal, RGCs detach from the ventricular surface thus affecting the interkinetic nuclear migration and they exit prematurely from mitosis. Mechanistically, we show that TF Pax6 directly regulates the activity of the Odf2 gene encoding for the appendage-specific protein Odf2 with a role for the assembly of mother centriole. Our findings demonstrate a molecular mechanism that explains important characteristics of the centrosome disassembly and malfunctioning in developing cortex lacking Pax6.
Asunto(s)
Centriolos/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/embriología , Proteínas del Ojo/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Centriolos/ultraestructura , Proteínas del Ojo/análisis , Proteínas del Ojo/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/genética , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células 3T3 NIH , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/análisis , Factores de Transcripción Paired Box/genética , Regiones Promotoras Genéticas , Proteínas Represoras/análisis , Proteínas Represoras/genéticaRESUMEN
The small heat shock protein ODF1/HSPB10 is essential for male fertility in mice. Targeted deletion of Odf1 resulted in acephalic sperm in homozygous mice of mixed background (C57BL/6J//129/Sv), whereas heterozygous animals are fully fertile. To further elucidate the function of ODF1, we generated incipient congenic mice with targeted deletion of Odf1 by successive backcrossing on the 129/Sv background. We observed that fecundity of heterozygous Odf1(+/-) male mice was severely reduced over backcross generations. However, neither aberrant sperm parameters nor sperm anomalies could be observed. Ultra-structural analyses of sperm from incipient congenic heterozygous Odf1(+/-) males of backcross generation N7 revealed no obvious pathological findings. However, we observed an enlargement of the distance between nuclear membrane and capitulum, indicating a weakening of the sperm head-to-tail coupling. Severe male subfertility provoked by haplo-deficiency of ODF1 is therefore most probably caused by impaired head-to-tail coupling that eventually might induce sperm decapitation on the specific conditions of in vivo fertilisation. As subfertility in haplo-deficient ODF1 male mice could not be diagnosed by semen analysis, it seems to be a paradigm for unexplained infertility that is a frequent diagnosis for male fertility impairment in humans.
Asunto(s)
Fertilidad , Haploinsuficiencia , Proteínas de Choque Térmico/metabolismo , Infertilidad Masculina/metabolismo , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Animales , Genotipo , Proteínas de Choque Térmico/genética , Heterocigoto , Homocigoto , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Masculino , Ratones de la Cepa 129 , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Conducta Sexual Animal , Cabeza del Espermatozoide/ultraestructura , Motilidad Espermática , Cola del Espermatozoide/ultraestructuraRESUMEN
The centrosome/basal body protein ODF2/Cenexin is necessary for the formation of the primary cilium. Primary cilia are essential organelles that sense and transduce environmental signals. Primary cilia are therefore critical for embryonic and postnatal development as well as for tissue homeostasis in adulthood. Impaired function of primary cilia causes severe human diseases. ODF2 deficiency prevents formation of the primary cilium and is embryonically lethal. To explore the regulation of primary cilia formation we analyzed the promoter region of Odf2 and its transcriptional activity. In cycling cells, Odf2 transcription is depressed but becomes up-regulated in quiescent cells. Low transcriptional activity is mediated by sequences located upstream from the basal promoter, and neither transcription factors with predicted binding sites in the Odf2 promoter nor Rfx3 or Foxj, which are known to control ciliary gene expression, could activate Odf2 transcription. However, co-expression of either C/EBPα, c-Jun or c-Jun and its regulator MEKK1 enhances Odf2 transcription in cycling cells. Our results provide the first analysis of transcriptional regulation of a ciliary gene. Furthermore, we suggest that transcription of even more ciliary genes is largely inhibited in cycling cells but could be activated by cell cycle arrest and by the stress signaling JNK pathway.
Asunto(s)
Puntos de Control del Ciclo Celular , Regulación de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Sistema de Señalización de MAP Quinasas , Activación Transcripcional , Células Cultivadas , Cilios/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Técnicas para Inmunoenzimas , Fosforilación , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatozoides/citología , Animales , Línea Celular , Fertilidad , Eliminación de Gen , Proteínas de Choque Térmico/genética , Masculino , Ratones , Mutación , Motilidad Espermática , Espermatogénesis , Espermatozoides/metabolismo , Espermatozoides/ultraestructuraRESUMEN
SUN domain proteins are integral proteins of the inner nuclear membrane and functions in linkage of the nuclear lamina to the cytoskeleton. Moreover, SUN domain proteins seem to mediate the tethering of the centrosome to the nuclear membrane, and they are involved in telomere attachment to the nuclear envelope in meiotic cells, and in germ cell development in invertebrates. In contrast to the widely expressed SUN domain proteins in mammals, SUN1 and SUN2, which have been analysed in great detail, there is virtually nothing known about testicular SUN domain proteins. Since a hallmark of male germ cell development is the profound remodelling of the nuclear envelope, emphasized, for example, by the reshaping of the nucleus during spermiogenesis, and the biogenesis of its tightly associated acrosome, SUN domain proteins might be engaged in these processes. We have isolated a novel SUN domain protein, SPAG4L-2, that differs from SPAG4L by an N-terminal insertion of 25 amino acids. Spag4l and Spag4l-2 are exclusively expressed in testis at about equimolar amounts, and show elevated transcription during ongoing spermiogenesis coincident with the appearance of round spermatids. Molecular dissection of the protein followed by cytological and biochemical investigations revealed that SPAG4L-2 and SPAG4L are transmembrane proteins that localize to the nuclear envelope. SPAG4L/4L-2 are restricted to the apical nuclear region of round spermatids that face the acrosomic vesicle, and thus are most probably involved in linkage of the acrosomic vesicle to the spermatid nucleus, and in acrosome biogenesis.
Asunto(s)
Acrosoma/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Espermátides/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
CHARGE syndrome is an autosomal dominant disorder caused in about two-third of cases by mutations in the CHD7 gene. For other genetic diseases e.g. hereditary spastic paraplegia, it was shown that interacting partners are involved in the underlying cause of the disease. These data encouraged us to search for CHD7 binding partners by a yeast two-hybrid library screen and CHD8 was identified as an interacting partner. The result was confirmed by a direct yeast two-hybrid analysis, co-immunoprecipitation studies and by a bimolecular fluorescence complementation assay. To investigate the function of CHD7 missense mutations in the CHD7-CHD8 interacting area on the binding capacity of both proteins, we included three known missense mutations (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one newly identified missense mutation (p.Trp2091Arg) in the CHD7 gene and performed both direct yeast two-hybrid and co-immunoprecipitation studies. In the direct yeast two-hybrid system, the CHD7-CHD8 interaction was disrupted by the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg, whereas in the co-immunoprecipitation studies disruption of the CHD7-CHD8 interaction by the mutations could not be observed. The results lead to the hypothesis that CHD7 and CHD8 proteins are interacting directly and indirectly via additional linker proteins. Disruption of the direct CHD7-CHD8 interaction might change the conformation of a putative large CHD7-CHD8 complex and could be a disease mechanism in CHARGE syndrome.
Asunto(s)
Anomalías Múltiples/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Anomalías Múltiples/metabolismo , Atresia de las Coanas/complicaciones , Atresia de las Coanas/genética , Atresia de las Coanas/metabolismo , Coloboma/complicaciones , Coloboma/genética , Coloboma/metabolismo , Sordera/complicaciones , Sordera/congénito , Sordera/genética , Sordera/metabolismo , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Oído/anomalías , Células HeLa , Cardiopatías Congénitas/complicaciones , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Humanos , Mutación/fisiología , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Infantilismo Sexual/complicaciones , Infantilismo Sexual/genética , Infantilismo Sexual/metabolismo , Síndrome , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: 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. RESULTS: 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 in vitro by GST pull-down assays and in vivo 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. CONCLUSION: 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.