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1.
Nucleic Acids Res ; 50(21): 12400-12424, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-35947650

RESUMO

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.


Assuntos
Metiltransferases , Neurônios Motores , RNA Nuclear Pequeno , Animais , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HeLa , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Fenótipo , RNA Nuclear Pequeno/metabolismo , Metiltransferases/metabolismo
2.
PLoS Genet ; 16(5): e1008815, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453722

RESUMO

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.


Assuntos
Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN/genética , Animais , Regulação para Baixo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Letais , Tamanho do Órgão , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/metabolismo
3.
J Cell Sci ; 133(2)2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31907206

RESUMO

Morgana (Mora, also known as CHORD in flies) and its mammalian homologue, called CHORDC1 or CHP1, is a highly conserved cysteine and histidine-rich domain (CHORD)-containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of mora causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Mora localises to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT supercomplex and with additional well-known Hsp90 co-chaperones. Acute inhibition of Mora function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds to microtubules directly and promotes microtubule polymerisation in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Fuso Acromático/metabolismo , Animais , Humanos , Polimerização
4.
J Med Genet ; 58(4): 254-263, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32527956

RESUMO

BACKGROUND: Mutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. METHODS: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy. RESULTS: We show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs. CONCLUSIONS: Our results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.


Assuntos
Proteínas de Transporte/genética , Infertilidade Masculina/genética , Microtúbulos/genética , Serina Endopeptidases/genética , Animais , Segregação de Cromossomos/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Predisposição Genética para Doença , Humanos , Infertilidade Masculina/patologia , Masculino , Meiose/genética , Mutação/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/patologia , Fuso Acromático/genética , Fuso Acromático/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologia
5.
PLoS Genet ; 15(9): e1008371, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527906

RESUMO

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.


Assuntos
Duplicação Cromossômica/genética , Segregação de Cromossomos/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Elementos Reguladores de Transcrição/genética , Fuso Acromático/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/genética
6.
PLoS Genet ; 13(5): e1006784, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28505193

RESUMO

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.


Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Linhagem Celular , Drosophila/genética , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Mitose , Ubiquitinação
7.
Chromosoma ; 127(4): 489-504, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30120539

RESUMO

Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.


Assuntos
Cromossomos de Insetos/genética , Proteínas de Drosophila/genética , Drosophila/citologia , Meiose , Espermatócitos/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Quebra Cromossômica , Segregação de Cromossomos , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Larva/citologia , Masculino , Mutação , Fenótipo , Espermatócitos/citologia
8.
Nucleic Acids Res ; 45(6): 3068-3085, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27940556

RESUMO

Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.


Assuntos
Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Proteínas Cromossômicas não Histona/fisiologia , Reparo do DNA , DNA de Cadeia Simples/ultraestrutura , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Proteínas de Drosophila/ultraestrutura , Microscopia de Força Atômica , Domínios Proteicos , Multimerização Proteica , Proteína de Replicação A/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/ultraestrutura
9.
BMC Biol ; 16(1): 68, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907103

RESUMO

BACKGROUND: S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). RESULTS: We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. CONCLUSIONS: We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.


Assuntos
Linhagem Celular/ultraestrutura , Drosophila melanogaster/citologia , Membranas Intracelulares/ultraestrutura , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Mitose , Animais , Microscopia Eletrônica de Transmissão/métodos
10.
PLoS Genet ; 11(6): e1005260, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26110638

RESUMO

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Telômero/genética , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Antígeno Nuclear de Célula em Proliferação/metabolismo , Telômero/metabolismo , Cromossomo Y/genética , Cromossomo Y/metabolismo
11.
PLoS Genet ; 11(6): e1005167, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26110528

RESUMO

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas/metabolismo , Telômero/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Ciclo Celular/genética , Células Cultivadas , Dano ao DNA/genética , Replicação do DNA , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Fibroblastos/fisiologia , Genes p53 , Humanos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas/genética , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
12.
Genes Dev ; 24(15): 1596-601, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20679394

RESUMO

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. Here we show that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; we speculate that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Regulação da Expressão Gênica , Modelos Moleculares , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/isolamento & purificação
13.
PLoS Genet ; 10(3): e1004199, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24651653

RESUMO

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, has been implicated in preventing human pathologies, such as diabetes and cancer. However, the mechanisms underlying the beneficial effects of PLP are still unclear. Using Drosophila as a model system, we show that PLP deficiency, caused either by mutations in the pyridoxal kinase-coding gene (dPdxk) or by vitamin B6 antagonists, results in chromosome aberrations (CABs). The CAB frequency in PLP-depleted cells was strongly enhanced by sucrose, glucose or fructose treatments, and dPdxk mutant cells consistently displayed higher glucose contents than their wild type counterparts, an effect that is at least in part a consequence of an acquired insulin resistance. Together, our results indicate that a high intracellular level of glucose has a dramatic clastogenic effect if combined with PLP deficiency. This is likely due to an elevated level of Advanced Glycation End-products (AGE) formation. Treatment of dPdxk mutant cells with α-lipoic acid (ALA) lowered both AGE formation and CAB frequency, suggesting a possible AGE-CAB cause-effect relationship. The clastogenic effect of glucose in PLP-depleted cells is evolutionarily conserved. RNAi-mediated silencing of PDXK in human cells or treatments with PLP inhibitors resulted in chromosome breakage, which was potentiated by glucose and reduced by ALA. These results suggest that patients with concomitant hyperglycemia and vitamin B6 deficiency may suffer chromosome damage. This might impact cancer risk, as CABs are a well-known tumorigenic factor.


Assuntos
Instabilidade Cromossômica/genética , Glucose/metabolismo , Piridoxal Quinase/genética , Deficiência de Vitamina B 6/genética , Animais , Aberrações Cromossômicas , Drosophila , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Modelos Animais , Mutação , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/administração & dosagem , Deficiência de Vitamina B 6/patologia
14.
PLoS Genet ; 10(10): e1004739, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340516

RESUMO

Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.


Assuntos
Estruturas Cromossômicas/genética , DNA Topoisomerases Tipo II/genética , Heterocromatina/genética , Mitose/genética , Alelos , Animais , Cromatina/genética , Drosophila melanogaster , Regulação da Expressão Gênica , Masculino , Mutação , Fenótipo , Espermatócitos , Cromossomo X/genética , Cromossomo Y/genética
15.
Cell Biol Int ; 40(9): 984-90, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27317357

RESUMO

The spindle microtubule (MT) flux is the continuous translocation of MTs toward the spindle poles caused by MT polymerization at plus ends coupled to depolymerization at minus ends. Poleward flux is observed in both mitotic and meiotic spindles; it is evolutionarily conserved and contributes to the regulation of spindle length and anaphase chromosome movement. MT photobleaching is a tool frequently used to measure poleward flux. Spindles containing fluorescently tagged tubulin are photobleached to generate a non-fluorescent stripe, which moves toward the spindle poles allowing a measure of the flux. However, this method only permits rapid measurements of the flux, because the fluorescence of the bleached stripe recovers rapidly due to the spindle MT turnover. Here, we describe a modification of the current photobleaching-based method for flux measurement. We photobleached two large areas at the opposite sides of the metaphase plate in spindles of Drosophila S2 cells expressing Cherry-tagged tubulin, leaving unbleached only the area near the chromosomes. We then measured the speed with which the fluorescent MTs move toward the poles. We found that this method allows a measure of the flux over a two- to threefold longer time than the "single stripe" method, providing a reliable evaluation of the flux rate.


Assuntos
Análise do Fluxo Metabólico/métodos , Microtúbulos/metabolismo , Polos do Fuso/metabolismo , Anáfase/fisiologia , Animais , Segregação de Cromossomos , Drosophila , Cinesinas/metabolismo , Cinetocoros/metabolismo , Mitose/genética , Mitose/fisiologia , Fuso Acromático/genética , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo
16.
J Cell Sci ; 125(Pt 3): 584-8, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22389398

RESUMO

The spindle is a highly dynamic molecular machine that mediates precise chromosome segregation during cell division. Spindle size can vary dramatically, not only between species but also between different cells of the same organism. However, the reasons for spindle size variability are largely unknown. Here we show that variations in spindle size can be linked to a precise developmental requirement. Drosophila species have dramatically different sperm flagella that range in length from 0.3 mm in D. persimilis to 58.3 mm in D. bifurca. We found that males of different species exhibit striking variations in meiotic spindle size, which positively correlate with sperm length, with D. bifurca showing 30-fold larger spindles than D. persimilis. This suggests that primary spermatocytes of Drosophila species manufacture and store amounts of tubulin that are proportional to the axoneme length and use these tubulin pools for spindle assembly. These findings highlight an unsuspected plasticity of the meiotic spindle in response to the selective forces controlling sperm length.


Assuntos
Drosophila/ultraestrutura , Cauda do Espermatozoide/ultraestrutura , Animais , Evolução Biológica , Segregação de Cromossomos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Masculino , Meiose , Microscopia de Fluorescência , Especificidade da Espécie , Cauda do Espermatozoide/metabolismo , Tubulina (Proteína)/metabolismo
17.
J Cell Sci ; 125(Pt 17): 4014-25, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22685323

RESUMO

The Zw10 protein, in the context of the conserved Rod-Zwilch-Zw10 (RZZ) complex, is a kinetochore component required for proper activity of the spindle assembly checkpoint in both Drosophila and mammals. In mammalian and yeast cells, the Zw10 homologues, together with the conserved RINT1/Tip20p and NAG/Sec39p proteins, form a second complex involved in vesicle transport between Golgi and ER. However, it is currently unknown whether Zw10 and the NAG family member Rod are also involved in Drosophila membrane trafficking. Here we show that Zw10 is enriched at both the Golgi stacks and the ER of Drosophila spermatocytes. Rod is concentrated at the Golgi but not at the ER, whereas Zwilch does not accumulate in any membrane compartment. Mutations in zw10 and RNAi against the Drosophila homologue of RINT1 (rint1) cause strong defects in Golgi morphology and reduce the number of Golgi stacks. Mutations in rod also affect Golgi morphology, whereas zwilch mutants do not exhibit gross Golgi defects. Loss of either Zw10 or Rint1 results in frequent failures of spermatocyte cytokinesis, whereas Rod or Zwilch are not required for this process. Spermatocytes lacking zw10 or rint1 function assemble regular central spindles and acto-myosin rings, but furrow ingression halts prematurely due to defective plasma membrane addition. Collectively, our results suggest that Zw10 and Rint1 cooperate in the ER-Golgi trafficking and in plasma membrane formation during spermatocyte cytokinesis. Our findings further suggest that Rod plays a Golgi-related function that is not required for spermatocyte cytokinesis.


Assuntos
Membrana Celular/metabolismo , Citocinese , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Células Cultivadas , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Cinetocoros/metabolismo , Masculino , Mutação/genética , Transporte Proteico , Interferência de RNA , Homologia de Sequência de Aminoácidos , Espermatócitos/citologia , Espermatócitos/metabolismo , Frações Subcelulares/metabolismo
18.
J Cell Sci ; 124(Pt 5): 706-17, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21285248

RESUMO

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. In Drosophila, mutants without centrosomes can form functional anastral spindles and survive to adulthood. Here we show that mutations in the Drosophila misato (mst) gene inhibit kinetochore-driven MT growth, lead to the formation of monopolar spindles and cause larval lethality. In most prophase cells of mst mutant brains, asters are well separated, but collapse with progression of mitosis, suggesting that k-fibers are essential for maintenance of aster separation and spindle bipolarity. Analysis of mst; Sas-4 double mutants showed that mitotic cells lacking both the centrosomes and the mst function form polarized MT arrays that resemble monopolar spindles. MT regrowth experiments after cold exposure revealed that in mst; Sas-4 metaphase cells MTs regrow from several sites, which eventually coalesce to form a single polarized MT array. By contrast, in Sas-4 single mutants, chromosome-driven MT regrowth mostly produced robust bipolar spindles. Collectively, these results indicate that kinetochore-driven MT formation is an essential process for proper spindle assembly in Drosophila somatic cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Fenótipo , Fuso Acromático/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Cinetocoros/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitose , Modelos Biológicos , Mutação
19.
Exp Cell Res ; 318(12): 1375-80, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22580224

RESUMO

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. Here we review the studies on spindle formation in Drosophila centrosome-containing cells. Mutants with impaired centrosome function assemble functional anastral spindles in somatic tissues and survive to adulthood. In contrast, mutants defective in chromosome-driven MT formation form highly aberrant mitotic spindles and die at larval stages. The requirements for spindle assembly in Drosophila male meiotic cells are diametrically opposed to those of somatic cells. Spermatocytes assemble morphologically normal spindles in the complete absence of chromosome-induced MTs, but are unable to organize a functional spindle in the absence of centrosomal MTs. Male meiotic spindles are much larger than mitotic spindles as they contain most of the tubulin needed for sperm tail formation. We suggest that the centrosome-based mechanism of spindle assembly in spermatocytes reflects their need for rapid and efficient polymerization of a particularly large amount of tubulin.


Assuntos
Centrossomo/metabolismo , Drosophila , Cinetocoros/metabolismo , Microtúbulos/fisiologia , Fuso Acromático/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Fuso Acromático/genética
20.
Cells ; 12(6)2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36980263

RESUMO

The Drosophila abnormal spindle (asp) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM (Abnormal Spindle-like Microcephaly-associated; or MCPH5) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse (Aspm) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp/Aspm/ASPM genes play the same conserved functions in Drosophila, mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly.


Assuntos
Microcefalia , Animais , Humanos , Camundongos , DNA , Drosophila/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Mitose , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo
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