RESUMO
Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.
Assuntos
Aurora Quinase B , Segregação de Cromossomos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Animais , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Masculino , Mamíferos/metabolismo , Meiose/genética , Camundongos , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Treonina/metabolismoRESUMO
PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.
RESUMO
Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo-like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo-like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.
Assuntos
Proteínas de Ciclo Celular , Centrossomo , Animais , Proteínas de Ciclo Celular/genética , Masculino , Meiose , Camundongos , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Fuso Acromático , Quinase 1 Polo-LikeAssuntos
Cromossomos/genética , Citogenética/história , Meiose , Animais , Gafanhotos , História do Século XX , História do Século XXIRESUMO
Cohesin cofactors regulate the loading, maintenance, and release of cohesin complexes from chromosomes during mitosis but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists as two paralogs in somatic and germline cells, PDS5A and PDS5B, with unclear functions. Here, we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous excision of Pds5A and Pds5B results in severe defects during early prophase I while their individual depletion does not, suggesting their functional redundancy. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after the strong depletion of PDS5A/B proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I.
Assuntos
Meiose , Telômero , Animais , Proteínas de Ciclo Celular/genética , Masculino , Meiose/genética , Camundongos , Mitose , Espermatócitos , Complexo Sinaptonêmico , Telômero/genéticaRESUMO
A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose/fisiologia , Oócitos/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteínas de Ciclo Celular/análise , Centrômero/ultraestrutura , Camundongos , Oócitos/metabolismo , Oócitos/ultraestrutura , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , CoesinasRESUMO
The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Complexo Sinaptonêmico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Espermatócitos/metabolismo , CoesinasRESUMO
Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Meiose , Membrana Nuclear/metabolismo , Telômero/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Prófase Meiótica I , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Estágio Paquíteno , Ligação Proteica , Espermatócitos/patologiaRESUMO
CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.
Assuntos
Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Nanismo/genética , Recombinação Homóloga/genética , Meiose/genética , Microcefalia/genética , Espermatócitos/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Dano ao DNA , Fácies , Imuno-Histoquímica , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Recombinação Genética/genética , Contagem de Espermatozoides , Espermatócitos/patologiaRESUMO
In most organisms, telomeres attach to the nuclear envelope at the onset of meiosis to promote the crucial processes of pairing, recombination and synapsis during prophase I. This attachment of meiotic telomeres is mediated by the specific distribution of several nuclear envelope components that interact with the attachment plates of the synaptonemal complex. We have determined by immunofluorescence and electron microscopy that the ablation of the kinase CDK2 alters the nuclear envelope in mouse spermatocytes, and that the proteins SUN1, KASH5 (also known as CCDC155) and lamin C2 show an abnormal cap-like distribution facing the centrosome. Strikingly, some telomeres are not attached to the nuclear envelope but remain at the nuclear interior where they are associated with SUN1 and with nuclear-envelope-detached vesicles. We also demonstrate that mouse testis CDK2 phosphorylates SUN1 in vitro. We propose that during mammalian prophase I the kinase CDK2 is a key factor governing the structure of the nuclear envelope and the telomere-led chromosome movements essential for homolog pairing.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Prófase Meiótica I/fisiologia , Membrana Nuclear/metabolismo , Espermatócitos/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/genética , Proteínas do Citoesqueleto , Laminina/genética , Laminina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Fosforilação/fisiologia , Espermatócitos/citologia , Telômero/genéticaRESUMO
Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mamíferos/metabolismo , Meiose , Anáfase/efeitos dos fármacos , Animais , Centrômero/efeitos dos fármacos , Cromátides/efeitos dos fármacos , Cromátides/metabolismo , Etoposídeo/farmacologia , Masculino , Meiose/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitose/efeitos dos fármacos , CoesinasRESUMO
Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos/genética , Meiose/fisiologia , Mitose/fisiologia , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Células Germinativas , Humanos , Mamíferos , CamundongosAssuntos
Caseína Quinase I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Proteína Fosfatase 2/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Segregação de Cromossomos , Modelos Biológicos , Mutação/genética , Fosforilação , Saccharomycetales/citologia , Saccharomycetales/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , CoesinasRESUMO
Cyclin-dependent kinase 2 (CDK2) was assumed to be essential in the mammalian cell cycle both at the G1-S transition and throughout the S phase. Interestingly, ablation of Cdk2 in mice does not have substantial consequences for embryonic or postnatal development, but both males and females are infertile. In the present study, we have analysed the meiotic alterations leading to infertility in Cdk2-/- male mice. We have studied the distribution and dynamics of several proteins related to meiosis progression, such as synaptonemal complex proteins, cohesin complexes, and centromere-, telomere- and recombination-related proteins. Cdk2-/- spermatocytes show an incomplete chromosome pairing, an extensive non-homologous synapsis and arrest at a pachytene-like stage with unrepaired programmed double-strand breaks. In these spermatocytes, some telomeres do not attach to the nuclear envelope, and sex chromosomes do not form a sex body. Our data demonstrate an unpredicted participation of CDK2 in the accurate pairing and recombination between homologues during mammalian meiosis.
Assuntos
Pareamento Cromossômico/genética , Quinase 2 Dependente de Ciclina/fisiologia , Meiose/genética , Recombinação Genética/genética , Espermatócitos/fisiologia , Animais , Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quebras de DNA de Cadeia Dupla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogênese/genéticaRESUMO
The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Proteínas Cromossômicas não Histona/metabolismo , Cinesinas/metabolismo , Meiose , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Centrômero/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Masculino , Prófase Meiótica I , Camundongos , Espermatócitos/ultraestruturaRESUMO
Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Meiose/fisiologia , Mitose/fisiologia , Anáfase/fisiologia , Aneuploidia , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Infertilidade Feminina/genética , Infertilidade Feminina/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Troca de Cromátide Irmã , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , CoesinasRESUMO
Chromosome shaping and individualization are necessary requisites to warrant the correct segregation of genomes in either mitotic or meiotic cell divisions. These processes are mainly prompted in vertebrates by three multiprotein complexes termed cohesin and condensin I and II. In the present study we have analyzed by immunostaining the appearance and subcellular distribution of condensin I in mouse mitotic and meiotic chromosomes. Our results demonstrate that in either mitotically or meiotically dividing cells, condensin I is loaded onto chromosomes by prometaphase. Condensin I is detectable as a fuzzy axial structure running inside chromatids of condensed chromosomes. The distribution of condensin I along the chromosome length is not uniform, since it preferentially accumulates close to the chromosome ends. Interestingly, these round accumulations found at the condensin I axes termini colocalized with telomere complexes. Additionally, we present the relative distribution of the condensin I and cohesin complexes in metaphase I bivalents. All these new data have allowed us to propose a comprehensive model for meiotic chromosome structure.
Assuntos
Adenosina Trifosfatases/metabolismo , Estruturas Cromossômicas , Cromossomos de Mamíferos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose/fisiologia , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Animais , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/ultraestrutura , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Complexos Multiproteicos/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espermatogônias/citologia , Espermatogônias/fisiologia , Telômero/metabolismoRESUMO
The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Gafanhotos/genética , Prófase Meiótica I , Proteínas Nucleares/metabolismo , Sistema A de Transporte de Aminoácidos/metabolismo , Animais , Células Cultivadas , Pareamento Cromossômico , Cromossomos/metabolismo , Drosophila , Gafanhotos/metabolismo , Masculino , Modelos Biológicos , Subunidades Proteicas/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/metabolismo , Distribuição Tecidual , CoesinasRESUMO
Shugoshin (SGO) is a family of proteins that protect centromeric cohesin complexes from release during mitotic prophase and from degradation during meiosis I. Two mammalian SGO paralogues - SGO1 and SGO2 - have been identified, but their distribution and function during mammalian meiosis have not been reported. Here, we analysed the expression of SGO2 during male mouse meiosis and mitosis. During meiosis I, SGO2 accumulates at centromeres during diplotene, and colocalizes differentially with the cohesin subunits RAD21 and REC8 at metaphase I centromeres. However, SGO2 and RAD21 change their relative distributions during telophase I when sister-kinetochore association is lost. During meiosis II, SGO2 shows a striking tension-dependent redistribution within centromeres throughout chromosome congression during prometaphase II, as it does during mitosis. We propose a model by which the redistribution of SGO2 would unmask cohesive centromere proteins, which would be then released or cleaved by separase, to trigger chromatid segregation to opposite poles.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Meiose/fisiologia , Espermatócitos/fisiologia , Animais , Proteínas de Ligação a DNA , Imunofluorescência , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismoRESUMO
Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a "cone"-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.