Localization of mitotic factors on metaphase chromosomes

Abstract

The objective of this study was to determine whether the mitotic factors of HeLa cells, which induce meiotic maturation, i.e. germinal vesicle breakdown (GVBD) and chromosome condensation, when injected into fully grown Xenopus laevis oocytes, were localized in the cytoplasm or associated with the metaphase chromosomes. Cytoplasmic extracts Were prepared by lysing mitotic HeLa cells in low-salt hypotonic buffer and separating the chromosomes by centrifugation. The mitotic factors bound to chromosomes were extracted with high-salt (0·2 M-NaCl) buffer. Both the cytoplasmic and chromosomal protein fractions were evaluated for their maturation-promoting activity (MPA) in the Xenopus oocytes. The results of this study indicate that both the cytoplasmic and chromosomal fractions are identical in many respects, including their ability to induce GVBD, but the specific activity of the chromosomal fraction was at least threefold greater than that of the cytoplasmic fraction. These data suggest that a major portion of the mitotic factors is localized on the metaphase chromosomes. This association does not appear to be due to adventitious binding of mitotic proteins to chromosomes during the extraction procedures. Furthermore, when extracts were prepared in a similar way from early- and mid-G₂-phase HeLa cells, only the nuclear extracts had MPA and no activity was found in the cytoplasmic fraction. Both the cytoplasmic and nuclear extracts of late-G₂ cells exhibited MPA. These data support the conclusion that the mitotic factors become preferentially bound to chromatin as soon as they are synthesized, and as the cell synthesizes more of these factors in preparation for mitosis, increasing amounts of them are retained in the cytoplasm.