RESUMO
Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.
Assuntos
Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , 4-Nitroquinolina-1-Óxido/farmacologia , Proteínas de Ciclo Celular/metabolismo , Centrossomo/efeitos dos fármacos , Centrossomo/ultraestrutura , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Retroalimentação Fisiológica , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Quinolonas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The mitotic spindle, due to its striking form, has been imaged for well over 100 years. Composed largely of microtubules and chromosomes, the spindle also contains numerous proteins whose roles include biochemical and biophysical regulation of mitosis. Given the transient, dynamic nature of the spindle, the light microscope continues to be the main tool employed to unlock its mysteries. In this chapter, we will discuss modern light microscopy techniques commonly used for imaging this intricate cellular machine as well as provide examples and protocols. We will also describe some biological preparations and experimental regimes for investigation of the mitotic spindle.
Assuntos
Rastreamento de Células/métodos , Fuso Acromático/metabolismo , Animais , Caenorhabditis elegans/citologia , Divisão Celular/genética , Linhagem Celular , Recuperação de Fluorescência Após Fotodegradação , Humanos , Mamíferos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microscopia de Contraste de Fase/métodos , Microscopia de Polarização/métodos , Microscopia de Vídeo/métodos , Microtúbulos/genética , Mitose/genética , Saccharomyces cerevisiae/citologia , Fuso Acromático/genéticaRESUMO
Polo-like kinases (PLKs) are evolutionarily conserved kinases essential for cell cycle regulation. These kinases are characterized by the presence of a C-terminal phosphopeptide-interaction domain, the polo-box domain (PBD). How the functional domains of PLKs work together to promote cell division is not understood. To address this, we performed a genetic screen to identify mutations that independently modulate the kinase and PBD activities of yeast PLK/Cdc5. This screen identified a mutagenic hotspot in the F-helix region of Cdc5 kinase domain that allows one to control kinase activity in vivo. These mutations can be systematically engineered into other major eukaryotic cell cycle kinases to similarly regulate their activity in live cells. Here, using this approach, we show that the kinase activity of Cdc5 can promote the execution of several stages of mitosis independently of PBD activity. In particular, we observe that the activation of Cdc14 and execution of mitotic exit are uniquely sensitive to the modulation of Cdc5 kinase activity. In contrast, PBD-defective mutants are capable of completing mitosis but are unable to maintain spindle pole body integrity. Consistent with this defect, PBD-deficient cells progressively double the size of their genome and ultimately lose genome integrity. Collectively, these results highlight the specific contributions of Cdc5 functional domains to cell division and reveal unexpected mechanisms controlling spindle pole body behavior and genome stability.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica/fisiologia , Mitose/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Proteínas de Ciclo Celular/isolamento & purificação , Eletroforese , Citometria de Fluxo , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Mitose/genética , Mutação/genética , Fosforilação , Proteínas Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
A defining feature of mitosis is the reorganization of chromosomes into highly condensed structures capable of withstanding separation and large-scale intracellular movements. This reorganization is promoted by condensin, an evolutionarily conserved multisubunit ATPase. Here we show, using budding yeast, that condensin is regulated by phosphorylation specifically in anaphase. This phosphorylation depends on several mitotic regulators, and the ultimate effector is the Polo kinase Cdc5. We demonstrate that Cdc5 directly phosphorylates all three regulatory subunits of the condensin complex in vivo and that this causes a hyperactivation of condensin DNA supercoiling activity. Strikingly, abrogation of condensin phosphorylation is incompatible with viability, and cells expressing condensin mutants that have a reduced ability to be phosphorylated in vivo are defective in anaphase-specific chromosome condensation. Our results reveal the existence of a regulatory mechanism essential for the promotion of genome integrity through the stimulation of chromosome condensation in late mitosis.