Related Experiment Videos
Nucleolin, defective for MPF phosphorylation, localizes normally during mitosis and nucleologenesis
1Department of Biological Sciences, Louisiana State University, Baton Rouge 70803-1806, USA.
Abstract:
To determine what effect maturation promoting factor (MPF, p34(cdc2) kinase/cyclin B) phosphorylation has on nucleolin's distribution during mitotic nucleolar disassembly and reassembly, we altered Chinese hamster ovary (CHO) nucleolin (the N protein) such that it cannot be phosphorylated by p34(cdc2). As expected, the transiently expressed epitope-tagged N protein showed no apparent defect in nucleolar localization in interphase CHO cells, even after hypotonic shock and recovery to quickly disassemble and then reassemble interphase nucleoli. In mitotic CHO cells, the N protein localized to the perichromosomal sheath and the cytoplasm, as is typical for nucleolin. Similar to epitope-tagged wild-type nucleolin, the N protein also maintained its association with persistent nucleoli characteristic of mitotic Chinese hamster lung (Dede) cells. In synchronized HeLa cells, the N protein again localized to the perichromosomal sheath and the cytoplasm as nucleoli disassembled during prophase. In HeLa cell telophase, the N protein localized normally to nucleolus-derived foci within the cytoplasm and prenucleolar bodies within reforming nuclei. The observations indicate that MPF phosphorylation is not essential for nucleolin's localizations to the perichromosomal sheath and the cytoplasm during prophase and metaphase, and that functional MPF phosphorylation sites are not essential for nucleolin's localizations during nucleologenesis.
Insights
Maturation promoting factor (MPF) phosphorylation does not affect nucleolin distribution during cell division. This finding clarifies the role of MPF in nucleolin localization during mitosis and nucleologenesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Nucleolin is a key protein involved in nucleolar structure and function.
- Maturation promoting factor (MPF) is a critical regulator of cell cycle progression, including mitosis.
- MPF phosphorylates numerous substrates, but its specific role in nucleolin localization during mitosis is not fully understood.
Purpose of the Study:
- To investigate the effect of MPF phosphorylation on nucleolin's distribution during mitotic nucleolar disassembly and reassembly.
- To determine if MPF phosphorylation sites are essential for nucleolin localization in different cell cycle stages.
Main Methods:
- Chinese hamster ovary (CHO) nucleolin was altered to prevent phosphorylation by p34(cdc2) (MPF kinase).
- Epitope-tagged wild-type and mutant nucleolin were transiently expressed in CHO and HeLa cells.
- Cellular localization of nucleolin was analyzed during interphase, mitosis (prophase, metaphase), and telophase using microscopy.
Main Results:
- Mutant nucleolin, unable to be phosphorylated by MPF, localized normally to interphase nucleoli.
- During mitosis, mutant nucleolin localized to the perichromosomal sheath and cytoplasm, similar to wild-type nucleolin.
- MPF phosphorylation was not essential for nucleolin's localization to the perichromosomal sheath, cytoplasm, or prenucleolar bodies during nucleologenesis.
Conclusions:
- MPF phosphorylation is not required for nucleolin's proper localization during mitotic nucleolar disassembly and reassembly.
- Nucleolin's association with persistent nucleoli and its distribution in the cytoplasm and prenucleolar bodies are independent of MPF phosphorylation.
- These findings highlight the robustness of nucleolin localization mechanisms during the cell cycle.