RESUMO
In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Metáfase/fisiologia , Ouriços-do-Mar/citologia , Ouriços-do-Mar/fisiologia , Fuso Acromático/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Fracionamento Celular , Centrossomo/química , Chaperonina com TCP-1 , Chaperoninas/metabolismo , Técnicas de Cultura , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Técnica Direta de Fluorescência para Anticorpo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/metabolismo , Fuso Acromático/química , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividus is shown by means of in situ immunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 molecules observed in nontreated eggs. That the constitutive hsp70 plays a role in sea urchin mitosis is indicated.