RESUMO
Polo-like-kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa), zebrafish embryos, and phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation by maintaining PCM cohesion in a PLK1-dependent manner. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to sequester PLK1 that limits PCM substrate phosphorylation events required for PCM cohesion.
Assuntos
Proteínas de Ciclo Celular , Centrossomo , Proteínas de Choque Térmico , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células HeLa , Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Quinase 1 Polo-LikeRESUMO
During the earliest division stages, zebrafish embryos have large cells that divide rapidly and synchronously to create a cellular layer on top of the yolk. Here, we describe a protocol for monitoring spindle dynamics during these early embryonic divisions. We outline techniques for injecting zebrafish embryos with small-molecule inhibitors toward polo-like kinases, preparing and mounting embryos for three-dimensional imaging using confocal microscopy. These techniques are used to understand how the early zebrafish embryo's centrosome constructs the mitotic spindle. For complete details on the use and execution of this protocol, please refer to Rathbun et al. (2020).
