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Free keywords:
Animal; *Cell Cycle/ph [Physiology]; Cell Differentiation/de [Drug Effects]; Cell Line, Transformed; Cells, Cultured; *Cyclic AMP/me [Metabolism]; *Cyclic AMP/pd [Pharmacology]; Cysteine Endopeptidases/an [Analysis]; *Cysteine Endopeptidases/me [Metabolism]; Cysteine Endopeptidases/ul [Ultrastructure]; DNA, Viral/ge [Genetics]; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Antibody Technique; *Forskolin/pd [Pharmacology]; *Genes, ras; *Granulosa Cells/cy [Cytology]; Granulosa Cells/de [Drug Effects]; *Granulosa Cells/en [Enzymology]; Microscopy, Electron; *Mitosis/ph [Physiology]; Multienzyme Complexes/an [Analysis]; *Multienzyme Complexes/me [Metabolism]; Multienzyme Complexes/ul [Ultrastructure]; Simian virus 40/ge [Genetics]; Transfection
Abstract:
We describe the isolation and characterization of proteasomes from recently established immortalized ovarian granulosa cell lines and their intracellular distribution during mitosis and during cAMP-induced differentiation, as revealed by immunofluorescence microscopy. In interphase, proteasomes were localized in small clusters throughout the cytoplasm and the nuclear matrix. In prophase, a substantial increase in proteasomal staining was observed in the perichromosomal area. A dramatic increase occurred in metaphase and in early anaphase; the chromosomes remained unstained. In late anaphase, intensive staining remained associated mainly with the spindle fibers. In telophase and early interphase of the daughter cells, intensive staining of proteasomes persisted in the nuclei. In contrast, in cells stimulated to differentiate by forskolin, which substantially elevates intracellular cAMP in these cell lines, only a weak staining of proteasomes was revealed in both the nucleus and the cytoplasm. Double staining of nondividing cells with antibodies to proteasomes and to tubulin did not show colocalization of proteasomes and microtubules. In contrast, dividing cells show a preferential concentration of proteasomes around spindle microtubules during metaphase and anaphase. The observed spatial and temporal distribution pattern of proteasomes during mitosis is highly reminiscent of the behavior of cyclins [Pines, J. & Hunter, T. (1991) J. Cell Biol. 115, 1-17]. Since proteasome accumulation appears to coincide with disappearance of cyclins A and B1 from the spindle apparatus, it is suggested that proteasomes may play a role in termination of mitosis by degrading the cyclins, which act as regulatory elements.
