RESUMO
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Assuntos
Divisão Celular , Polaridade Celular , Drosophila melanogaster , Células Epiteliais , Metáfase , Fuso Acromático , Estresse Mecânico , Animais , Metáfase/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fuso Acromático/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/citologia , Polaridade Celular/fisiologia , Padronização Corporal , Miosina Tipo II/metabolismo , Embrião não Mamífero/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Gastrulação/fisiologiaRESUMO
Chromosome mis-segregation during mitosis leads to aneuploidy, which is a hallmark of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although their mechanistic origins and likelihood of correction are incompletely understood. Here, we combine lattice light-sheet microscopy, endogenous protein labeling, and computational analysis to define the life history of >104 kinetochores. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable risk of mis-segregation. We show that the majority of lazy kinetochores are corrected rapidly in anaphase by Aurora B; if uncorrected, they result in a higher rate of micronuclei formation. Quantitative analyses of the kinetochore life histories reveal a dynamic signature of metaphase kinetochore oscillations that forecasts their anaphase fate. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with an additional layer of anaphase error correction required for stable karyotype propagation.
Assuntos
Anáfase/fisiologia , Aurora Quinase B/metabolismo , Cinetocoros/metabolismo , Segregação de Cromossomos/fisiologia , Humanos , Metáfase/fisiologia , Microtúbulos/metabolismo , Mitose/fisiologia , Fuso Acromático/metabolismoRESUMO
Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mitocôndrias/fisiologia , Mitofagia/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Cromossomos/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Metáfase/genética , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Mitofagia/genética , Oócitos/crescimento & desenvolvimento , RNA Mensageiro/genéticaRESUMO
Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.
Assuntos
Cromossomos/metabolismo , Meiose/fisiologia , Metáfase/fisiologia , Oócitos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Camundongos , Securina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.
Assuntos
Segregação de Cromossomos/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Metáfase/fisiologia , Cromátides , Células HeLa , Humanos , Cinetocoros/fisiologia , Microscopia de Fluorescência/métodos , Microtúbulos/fisiologia , Mitose/fisiologia , Modelos Teóricos , Fuso AcromáticoRESUMO
The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.
Assuntos
Metáfase/fisiologia , Fuso Acromático/fisiologia , Animais , Fenômenos Biomecânicos , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Elasticidade , Cristais Líquidos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Mitose , Fuso Acromático/química , Fuso Acromático/efeitos dos fármacos , Tensão Superficial , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis-Menten constant (Km ) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus-oocyte complexes.
Assuntos
Células do Cúmulo/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Oogênese/fisiologia , Serina/metabolismo , Animais , Comunicação Celular/fisiologia , Junções Comunicantes/metabolismo , Metáfase/fisiologia , CamundongosRESUMO
The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.
Assuntos
Blastômeros/fisiologia , Blastômeros/efeitos da radiação , Lasers , Tetraploidia , Animais , Blastômeros/citologia , Divisão Celular/efeitos da radiação , Fusão Celular/métodos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos da radiação , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Metáfase/fisiologia , Metáfase/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , GravidezRESUMO
The mechanisms of endometriosis-related infertility remain still unknown. Endometriosis and clinical markers of oocyte quality are a very important problem of reproduction. The purpose of the study is to assess the quality of oocytes in women with infertility associated with endometriosis. The study included infertile reproductive aged women, between 29 and 40 years who underwent IVF and ICSI procedures. The patients were divided into three groups: group I involved 50 (n = 50) patients with recurrent unilateral endometriomas, group II included 50 patients (n = 50) unilateral endometriomas after surgical treatment and control group with 30 (n = 30) patients with tubal factor infertility. Clinical and morphological assessment of oocyte quality was performed in all IVF/ICSI cycles. The results of the study demonstrate a statistically significant increase in the number of immature oocytes of metaphase MI and immature oocytes at the GV germinal vesicle stage in patients with infertility associated with endometriosis, compared with the control group (p<.005). There is deterioration in the quality of the obtained oocytes in patients with the presence of endometrioma more than 3 cm in diameter. The results of this study allow to conclude that endometriomas negatively affect quality of oocyte and ovarian reserve, whereas endometriomas after cystectomy, have a deleterious and sustained effect on ovarian reserve.
Assuntos
Endometriose/patologia , Infertilidade Feminina/patologia , Oócitos/patologia , Doenças Ovarianas/patologia , Adulto , Estudos de Casos e Controles , Células Cultivadas , Endometriose/complicações , Feminino , Fertilização in vitro , Humanos , Técnicas de Maturação in Vitro de Oócitos , Infertilidade Feminina/etiologia , Metáfase/fisiologia , Recuperação de Oócitos , Doenças Ovarianas/complicações , Reserva Ovariana/fisiologia , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Injeções de Esperma IntracitoplásmicasRESUMO
End binding protein 1 (EB1) is a key regulator of microtubule dynamics that orchestrates hierarchical interaction networks at microtubule plus ends to control proper cell division. EB1 activity is known to be regulated by serine/threonine phosphorylation; however, how tyrosine phosphorylation affects EB1 activity remains poorly understood. In this study, we mapped the tyrosine phosphorylation pattern of EB1 in synchronized cells and identified two tyrosine phosphorylation sites (Y217 and Y247) in mitotic cells. Using phospho-deficient (Y/F) and phospho-mimic (Y/D) mutants, we revealed that Y247, but not Y217, is critical for astral microtubule stability. The Y247D mutant contributed to increased spindle angle, indicative of defects in spindle orientation. Time-lapse microscopy revealed that the Y247D mutant significantly delayed mitotic progression by increasing the duration times of prometaphase and metaphase. Structural analysis suggests that Y247 mutants lead to instability of the hydrophobic cavity in the EB homology (EBH) domain, thereby affecting its interactions with p150glued, a protein essential for Gαi/LGN/NuMA complex capture. These findings uncover a crucial role for EB1 phosphorylation in the regulation of mitotic spindle orientation and cell division.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Fosforilação/fisiologia , Linhagem Celular Tumoral , Complexo Dinactina/metabolismo , Células HeLa , Humanos , Metáfase/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Ligação Proteica/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans, an important pathogen of humans, is essential for chromosome segregation. However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Esp1p-Interacting Protein1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APCCdc20-dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.
Assuntos
Segregação de Cromossomos/fisiologia , Securina/metabolismo , Separase/metabolismo , Candida albicans/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Endopeptidases/metabolismo , Metáfase/fisiologia , Mitose/fisiologia , Ligação Proteica , Securina/fisiologia , Separase/fisiologia , CoesinasRESUMO
Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mitofagia/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Metáfase/genética , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Modelos Biológicos , Oócitos/crescimento & desenvolvimento , Quinazolinonas/farmacologia , Interferência de RNA , RNA-Seq , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , TranscriptomaRESUMO
The microtubule-based spindle is subjected to various mechanical forces during cell division. How the structure generates and responds to forces while maintaining overall integrity is unknown because we have a poor understanding of the relationship between filament architecture and mechanics. Here, to fill this gap, we combine microneedle-based quantitative micromanipulation with high-resolution imaging, simultaneously analyzing forces and local filament motility in the Xenopus meiotic spindle. We find that microtubules exhibit a compliant, fluid-like mechanical response at the middle of the spindle half, being distinct from those near the pole and the equator. A force altering spindle length induces filament sliding at this compliant array, where parallel microtubules predominate, without influencing equatorial antiparallel filament dynamics. Molecular perturbations suggest that kinesin-5 and dynein contribute to the spindle's local mechanical difference. Together, our data establish a link between spindle architecture and mechanics and uncover the mechanical design of this essential cytoskeletal assembly.
Assuntos
Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Fenômenos Biomecânicos/fisiologia , Divisão Celular , Dineínas/metabolismo , Feminino , Cinesinas/metabolismo , Masculino , Metáfase/fisiologia , Microtúbulos/fisiologia , Fuso Acromático/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere-associated protein E (CENP-E), a plus-end-directed kinesin motor, is required for congression of pole-proximal chromosomes in metaphase. CENP-E accumulates at the outer plate of kinetochores and mediates the kinetochore-microtubule capture. CENP-E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP-E interacts with Bub1-related kinase, Aurora B and core kinetochore components during kinetochore-microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin-7 CENP-E. We highlight the complicated interactions between CENP-E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP-E in mitosis and meiosis, including the kinetochore-microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP-E in tumourigenesis and CENP-E's specific inhibitors.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cinesinas/metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Microtúbulos/metabolismo , Animais , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HeLa , Humanos , Cinesinas/química , Camundongos , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.
Assuntos
Divisão Celular/fisiologia , Fuso Acromático/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Dineínas/metabolismo , Cinesinas/metabolismo , Metáfase/fisiologia , Micromanipulação/métodos , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Xenopus laevis/fisiologiaRESUMO
Separation of sister chromatids is a drastic and irreversible step in the cell cycle. The key biochemistry behind this event is the proteolysis mediated by the ubiquitin ligase called the anaphase promoting complex, or APC/C. Securin and cyclin B1 are the two established substrates for APC/C whose degradation releases separase and inactivates cyclin B1-dependent kinase 1 (cdk1), respectively, at the metaphase-to-anaphase transition. In this study, we have combined biochemical quantifications with mathematical simulations to characterize the kinetic regulation of securin and cyclin B1, in the cytoplasmic and chromosomal compartments, and found that they are differentially distributed and degraded with different rates. Modeling their interaction with separase predicted that activation timing of separase well coincides with the decline of securin-separase concentration in the cytoplasm. Notably, it also coincides with the peak of cyclin B1-separase level on chromosomes, which appeared crucial to coordinate the timing for separase activation and cdk1 inhibition. We have also conducted phosphoproteomic analysis and identified Ki67 as a chromosomal cdk1 substrate whose dephosphorylation is facilitated by cyclin B1-separase interaction in anaphase.
Assuntos
Anáfase/fisiologia , Ciclina B1/metabolismo , Metáfase/fisiologia , Securina/metabolismo , Algoritmos , Citocinese , Células HeLa , Humanos , Mitose/fisiologia , Modelos Biológicos , Fosforilação , Transporte Proteico , Separase/metabolismoRESUMO
Higher plants possess a large number of kinesins, but lack the minus-end directed dynein motors. However, the kinesin class XIV has strongly expanded, and minus-end directed motors from this class may have taken over functions of cytoplasmic dyneins. In this study, we address the functional aspects of a novel rice homologue of the Arabidopsis class-XIV kinesins ATK1 and ATK5. Since a loss-of-function rice mutant of this kinesin is not viable, the function was studied in tobacco BY-2 as heterologous system. OsDLK-GFP stably expressed in BY-2 cells decorates cortical microtubules, but also can shift into the nucleus of interphase cells. Because of this peculiar localisation, we coined the name Dual Localisation Kinesin (DLK). The nuclear import of this protein is strongly and reversibly promoted in response to cold. During mitosis, OsDLK is repartitioned between spindle and phragmoplast. Motility assays in vitro using show that OsDLK can convey mutual sliding of microtubules and moves at a velocity comparable to other class-XIV kinesins. When tobacco cells overexpressing OsDLK are synchronised, they exhibit a delayed entry into metaphase, while the later phases of mitosis are accelerated. The data are discussed in relation to additional functions of this kinesin type, beyond their transport along microtubules.
Assuntos
Núcleo Celular/metabolismo , Temperatura Baixa , Cinesinas/metabolismo , Proteínas Motores Moleculares/metabolismo , Oryza/genética , Oryza/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas de Arabidopsis/química , Movimento Celular , Dineínas/metabolismo , Ácidos Graxos Insaturados/farmacologia , Genoma de Planta , Proteínas de Homeodomínio/química , Cinesinas/química , Metáfase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Plasmídeos , Homologia de Sequência , Nicotiana/citologia , Transativadores/química , Transformação GenéticaRESUMO
In mammals, journey from metaphase-I (M-I) to metaphase-II (M-II) is important since oocyte extrude first polar body (PB-I) and gets converted into haploid gamete. The molecular and cellular changes associated with meiotic cell cycle progression from M-I to M-II stage and extrusion of PB-I remain ill understood. Several factors drive oocyte meiosis from M-I to M-II stage. The mitogen-activated protein kinase3/1 (MAPK3/1), signal molecules and Rho family GTPases act through various pathways to drive cell cycle progression from M-I to M-II stage. The down regulation of MOS/MEK/MAPK3/1 pathway results in the activation of anaphase-promoting complex/cyclosome (APC/C). The active APC/C destabilizes maturation promoting factor (MPF) and induces meiotic resumption. Several signal molecules such as, c-Jun N-terminal kinase (JNK2), SENP3, mitotic kinesin-like protein 2 (MKlp2), regulator of G-protein signaling (RGS2), Epsin2, polo-like kinase 1 (Plk1) are directly or indirectly involved in chromosomal segregation. Rho family GTPase is another enzyme that along with cell division cycle (Cdc42) to form actomyosin contractile ring required for chromosomal segregation. In the presence of origin recognition complex (ORC4), eccentrically localized haploid set of chromosomes trigger cortex differentiation and determine the division site for polar body formation. The actomyosin contractile activity at the site of division plane helps to form cytokinetic furrow that results in the formation and extrusion of PB-I. Indeed, oocyte journey from M-I to M-II stage is coordinated by several factors and pathways that enable oocyte to extrude PB-I. Quality of oocyte directly impact fertilization rate, early embryonic development, and reproductive outcome in mammals.
Assuntos
Mamíferos/fisiologia , Metáfase/fisiologia , Oócitos/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Mamíferos/metabolismo , Fator Promotor de Maturação/metabolismo , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Quinase 1 Polo-LikeRESUMO
Anaphase in epithelia typically does not ensue until after spindles have achieved a characteristic position and orientation, but how or even if cells link spindle position to anaphase onset is unknown. Here, we show that myosin-10 (Myo10), a motor protein involved in epithelial spindle dynamics, binds to Wee1, a conserved regulator of cyclin-dependent kinase 1 (Cdk1). Wee1 inhibition accelerates progression through metaphase and disrupts normal spindle dynamics, whereas perturbing Myo10 function delays anaphase onset in a Wee1-dependent manner. Moreover, Myo10 perturbation increases Wee1-mediated inhibitory phosphorylation on Cdk1, which, unexpectedly, concentrates at cell-cell junctions. Based on these and other results, we propose a model in which the Myo10-Wee1 interaction coordinates attainment of spindle position and orientation with anaphase onset.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Metáfase/fisiologia , Modelos Biológicos , Miosinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Epitélio/metabolismo , Miosinas/genética , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Fuso Acromático/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.