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1.
Proc Natl Acad Sci U S A ; 119(12): e2115883119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35302885

RESUMO

SignificanceEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process are the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study provides observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.


Assuntos
Cromossomos , Meiose , Cromossomos/genética , Prófase
2.
Plant Cell ; 34(6): 2404-2423, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35294035

RESUMO

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of posttranscriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, for example, during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored nonfully spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.


Assuntos
Larix , Animais , Corpos Enovelados/genética , Corpos Enovelados/metabolismo , Larix/genética , Larix/metabolismo , Meiose , Prófase , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
J Microbiol ; 60(2): 177-186, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35102525

RESUMO

Genetic variation in eukaryotes is mediated during meiosis by the exchange of genetic material between homologous chromosomes to produce recombinant chromosomes. Cohesin is essential to promote proper chromosome segregation, chromosome morphogenesis, and recombination in meiotic cells. Cohesin consists of three main subunits-Smc1, Smc3, and the kleisin subunit Mcd1/Scc1 (Rec8 in meiosis)-and cohesin accessory factors. In Saccharomyces cerevisiae, the cohesin regulatory subunit Pds5 plays a role in homolog pairing, meiotic axis formation, and interhomolog recombination. In this study, we examine the prophase functions of Pds5 by performing physical analysis of recombination and three-dimensional high-resolution microscopy analysis to identify its roles in meiosis-specific recombination and chromosome morphogenesis. To investigate whether Pds5 plays a role in mitotic-like recombination, we inhibited Mek1 kinase activity, which resulted in switching to sister template bias by Rad51-dependent recombination. Reductions in double-strand breaks and crossover products and defective interhomolog recombination occurred in the absence of Pds5. Furthermore, recombination intermediates, including single-end invasion and double-Holliday junction, were reduced in the absence of Pds5 with Mek1 kinase inactivation compared to Mek1 kinase inactivation cells. Interestingly, the absence of Pds5 resulted in increasing numbers of chromosomes with hypercompaction of the chromosome axis. Thus, we suggest that Pds5 plays an essential role in recombination by suppressing the pairing of sister chromatids and abnormal compaction of the chromosome axis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , MAP Quinase Quinase 1/metabolismo , Meiose , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Cromossomos Fúngicos , DNA Fúngico , Recombinação Homóloga , Prófase
4.
J Cell Sci ; 135(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35112707

RESUMO

Formation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders and infertility. We found that oocytes contain prominent nuclear F-actin structures that are correlated with meiotic developmental capacity. We demonstrate that nuclear F-actin is a conserved feature of healthy mammalian oocytes and declines significantly with female reproductive ageing. Actin monomers used for nuclear F-actin assembly are sourced from an excess pool in the oocyte cytoplasm. Increasing monomeric G-actin transfer from the cytoplasm to the nucleus or directly enriching the nucleus with monomers led to assembly of stable nuclear F-actin bundles that significantly restrict chromatin mobility. By contrast, reducing G-actin monomer transfer by blocking nuclear import triggered assembly of a dense cytoplasmic F-actin network that is incompatible with healthy oocyte development. Overall, our data suggest that the large oocyte nucleus helps to maintain cytoplasmic F-actin organisation and that defects in this function are linked with reproductive age-related female infertility. This article has an associated First Person interview with Federica Giannini, joint first author of the paper.


Assuntos
Actinas , Meiose , Animais , Feminino , Humanos , Mamíferos , Oócitos , Oogênese , Prófase
5.
Mol Cell ; 82(3): 696-708.e4, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35090599

RESUMO

We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Cromossomos , DNA/metabolismo , Linfoma de Células B/metabolismo , Proteínas Nucleares/metabolismo , Prófase , Proteoma , Proteômica , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Galinhas , Cromatina/genética , DNA/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas Nucleares/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fatores de Tempo
6.
Cell Res ; 32(3): 254-268, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34980897

RESUMO

In response to DNA double-strand breaks (DSBs), DNA damage repair factors are recruited to DNA lesions and form nuclear foci. However, the underlying molecular mechanism remains largely elusive. Here, by analyzing the localization of DSB repair factors in the XY body and DSB foci, we demonstrate that pre-ribosomal RNA (pre-rRNA) mediates the recruitment of DSB repair factors around DNA lesions. Pre-rRNA exists in the XY body, a DSB repair hub, during meiotic prophase, and colocalizes with DSB repair factors, such as MDC1, BRCA1 and TopBP1. Moreover, pre-rRNA-associated proteins and RNAs, such as ribosomal protein subunits, RNase MRP and snoRNAs, also localize in the XY body. Similar to those in the XY body, pre-rRNA and ribosomal proteins also localize at DSB foci and associate with DSB repair factors. RNA polymerase I inhibitor treatment that transiently suppresses transcription of rDNA but does not affect global protein translation abolishes foci formation of DSB repair factors as well as DSB repair. The FHA domain and PST repeats of MDC1 recognize pre-rRNA and mediate phase separation of DSB repair factors, which may be the molecular basis for the foci formation of DSB repair factors during DSB response.


Assuntos
Meiose , RNA Ribossômico , Proteínas de Ciclo Celular/metabolismo , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Prófase , Precursores de RNA
7.
Ann Palliat Med ; 10(8): 9174-9183, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488403

RESUMO

BACKGROUND: Cytokines play critical roles in the inflammatory processes underlying liver failure. The relevance of interleukin-35 (IL-35), an anti-inflammatory cytokine, in liver failure remains uncharacterized. This study was conducted to investigate whether the IL-35 level in patients with prophase of liver failure (PLF) is associated with prognosis and the possible mechanism of immune regulation for IL-35. METHODS: This retrospective study enrolled 42 patients with PLF at the Department of Infection, First Affiliated Hospital of Soochow University between January 2016 and December 2018. Thirty patients with hepatitis and 30 healthy controls were also enrolled. We divide patients with prophase of liver failure into improvement group who recovered quickly (n=33) and deteriorate group who deteriorated to overt liver failure (n=9). Serum IL-35 level was measured by enzyme-linked immunosorbent assay (ELISA). The ratio of regulatory T cells to T-helper type-17 cells (Treg/Th17) in peripheral blood was determined by flow cytometry. RESULTS: Serum IL-35 level was higher in patients with PLF who showed subsequent improvement than in patients with PLF who showed deterioration to overt liver failure. The Treg/Th17 ratio was higher in patients with PLF who showed improvement than in patients with PLF who developed overt liver failure. The serum IL-35 level and Treg/Th17 ratio were positively correlated in patients with PLF. CONCLUSIONS: High serum IL-35 level is associated with better prognosis in patients with PLF.


Assuntos
Interleucina-17 , Falência Hepática , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prófase , Estudos Retrospectivos , Células Th17
8.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445207

RESUMO

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.


Assuntos
Núcleo Celular/metabolismo , Larix/metabolismo , Pólen/metabolismo , Prófase , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Núcleo Celular/genética , Larix/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética
9.
Cell Death Dis ; 12(8): 780, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373449

RESUMO

FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci-/- mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia.


Assuntos
Anemia de Fanconi/metabolismo , Histonas/metabolismo , Meiose , Espermatogênese , Animais , Apoptose , Diferenciação Celular , Epigênese Genética , Deleção de Genes , Infertilidade Masculina/genética , Lisina/metabolismo , Masculino , Metilação , Camundongos , Especificidade de Órgãos/genética , Prófase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Cromossomos Sexuais/metabolismo , Espermatogônias/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo
10.
PLoS Genet ; 17(5): e1009247, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34014920

RESUMO

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)-the epigenetic determinant of centromere identity-is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of 'mitotic drive' that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.


Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Epigênese Genética , Células Germinativas/metabolismo , Células-Tronco/metabolismo , Animais , Autorrenovação Celular , Senescência Celular , Proteínas Cromossômicas não Histona/deficiência , Proteínas de Drosophila/deficiência , Drosophila melanogaster/metabolismo , Feminino , Fase G2 , Masculino , Prófase , Fase S
11.
Nat Plants ; 7(6): 739-747, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031540

RESUMO

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dinitrobenzenos , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Filogenia , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Prófase , Domínios Proteicos , Sulfanilamidas , Tabaco/genética , Proteínas de Transporte Vesicular/metabolismo
12.
Chromosoma ; 130(2-3): 149-162, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33825974

RESUMO

The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective rec12- cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Prófase , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Complexo Sinaptonêmico/metabolismo
13.
Mol Cell ; 81(10): 2231-2245.e11, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33826921

RESUMO

Long undecoded transcript isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed important aspects of LUTI-based repression, how these features affect gene regulation on a global scale is unknown. Using transcript leader and direct RNA sequencing, here, we identify 74 LUTI candidates that are specifically induced in meiotic prophase. Translational repression of these candidates appears to be ubiquitous and is dependent on upstream open reading frames. However, LUTI-based transcriptional repression is variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.


Assuntos
Regulação Fúngica da Expressão Gênica , Genômica , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Genes Reporter , Meiose/genética , Sequenciamento por Nanoporos , Nucleossomos/metabolismo , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Prófase/genética , Biossíntese de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
14.
Plant Cell ; 33(1): 27-43, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33751090

RESUMO

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.


Assuntos
Arabidopsis/metabolismo , Microtúbulos/metabolismo , /metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Prófase/fisiologia
15.
Elife ; 102021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33502312

RESUMO

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here, we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism.


Most mammalian, yeast and other eukaryote cells have two sets of chromosomes, one from each parent, which contain all the cell's DNA. Sex cells ­ like the sperm and egg ­ however, have half the number of chromosomes and are formed by a specialized type of cell division known as meiosis. At the start of meiosis, each cell replicates its chromosomes so that it has twice the amount of DNA. The cell then undergoes two rounds of division to form sex cells which each contain only one set of chromosomes. Before the cell divides, the two duplicated sets of chromosomes pair up and swap sections of their DNA. This exchange allows each new sex cell to have a unique combination of DNA, resulting in offspring that are genetically distinct from their parents. This complex series of events is tightly regulated, in part, by a protein called the 'small ubiquitin-like modifier' (or SUMO for short), which attaches itself to other proteins and modifies their behavior. This process, known as SUMOylation, can affect a protein's stability, where it is located in the cell and how it interacts with other proteins. However, despite SUMO being known as a key regulator of meiosis, only a handful of its protein targets have been identified. To gain a better understanding of what SUMO does during meiosis, Bhagwat et al. set out to find which proteins are targeted by SUMO in budding yeast and to map the specific sites of modification. The experiments identified 2,747 different sites on 775 different proteins, suggesting that SUMO regulates all aspects of meiosis. Consistently, inactivating SUMOylation at different times revealed SUMO plays a role at every stage of meiosis, including the replication of DNA and the exchanges between chromosomes. In depth analysis of the targeted proteins also revealed that SUMOylation targets different groups of proteins at different stages of meiosis and interacts with other protein modifications, including the ubiquitin system which tags proteins for destruction. The data gathered by Bhagwat et al. provide a starting point for future research into precisely how SUMO proteins control meiosis in yeast and other organisms. In humans, errors in meiosis are the leading cause of pregnancy loss and congenital diseases. Most of the proteins identified as SUMO targets in budding yeast are also present in humans. So, this research could provide a platform for medical advances in the future. The next step is to study mammalian models, such as mice, to confirm that the regulation of meiosis by SUMO is the same in mammals as in yeast.


Assuntos
Meiose , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Sumoilação , Pareamento Cromossômico , Prófase , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Dev Biol ; 470: 147-153, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33278404

RESUMO

The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 â€‹°C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.


Assuntos
Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular , Ciclo Celular , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/citologia , Mitose , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Ciclina B/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/embriologia , Embrião não Mamífero/enzimologia , Ativação Enzimática , Metáfase , Prometáfase , Prófase , Proteína Fosfatase 1/metabolismo , Temperatura
17.
J Biol Chem ; 296: 100164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33293370

RESUMO

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.


Assuntos
Aneuploidia , Aurora Quinase B/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Processamento de Proteína Pós-Traducional , Treonina/metabolismo , Alanina/metabolismo , Substituição de Aminoácidos , Anáfase , Ácido Aspártico/metabolismo , Aurora Quinase B/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Segregação de Cromossomos , Edição de Genes , Humanos , Metáfase , Modelos Biológicos , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Prófase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Transgenes
18.
Proc Natl Acad Sci U S A ; 117(49): 31301-31308, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229589

RESUMO

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.


Assuntos
Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Laminopatias/patologia , Mecanotransdução Celular , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Interfase , Longevidade , Meiose , Modelos Biológicos , Mutação/genética , Poro Nuclear/metabolismo , Prófase
19.
PLoS Genet ; 16(11): e1008968, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33175901

RESUMO

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved "closure motif" region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico , Segregação de Cromossomos , Cromossomos , Meiose/fisiologia , Fosforilação , Prófase/fisiologia , Domínios Proteicos
20.
Mol Biol Cell ; 31(25): 2841-2862, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026960

RESUMO

Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulate specific meiotic stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the Sertoli-cell androgen receptor knockout (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild-type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and scRNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.


Assuntos
Androgênios/metabolismo , Meiose/fisiologia , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Androgênios/fisiologia , Animais , Masculino , Prófase Meiótica I , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prófase , Receptores Androgênicos/fisiologia , Análise de Sequência de RNA/métodos , Células de Sertoli/fisiologia , Transdução de Sinais , Análise de Célula Única/métodos , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo
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