By Levi Clancy for Student Reader on
Cycles in M-Phase Promoting Factor (MPF) activity control mitosis.
As a protein kinase, MPF likely acts via phosphorylation of the major histone protein H1 and the major nuclear envelope protein lamin. This leads to the degradation of the nuclear envelope and condensation of chromatin into chromosomes in anticipation of mitosis. Found in all organisms, MPF is a heterodimer composed of cyclin-dependent kinase (cdk1) and cyclin B.
Cycles of MPF activity are based on synthesis and degradation of cyclin B.
During the M phase (mitosis) the heterodimer is functional and drives cells into metaphase. Cyclin B is degraded, inactivating MPF once cells go into mitosis. During the S phase, newly synthesized Cyclin B from maternal mRNA leads to formation of a new functional MPF heterodimer.
Cytoplasm from mammalian cells arrested in metaphase of mitosis by treatment with drugs that inhibit the polymerization of microtubules (e.g. colchicine or nocodazole) had high oocyte maturation promoting factor (MPF).
When the ctoplasm from Xenopus eggs was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down. Also, when the cytoplasm from mammalian cells arrested in mitotic metaphase by treatment with micrtotubule inhibitors was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down.
This is like the mitosis promoting activity first observed in the cell fusion experiments. So, Xenopus ooctye MATURATION PROMOTING FACTOR = mammalian MITOSIS PROMOTING FACTOR. Fortunately, MPF is the abbreviation for both Maturation Promoting Factor (revealed by injection into Xenopus oocytes) and Mitosis Promoting Factor (revealed by fusing an M-phase cell to a cell in G1).
Oocytes remain arrested in G2 until they mature and addition of a sperm nucleus (via fertilization when in vivo). This is a great model for figuring out what the egg must have synthesized beforehand for mitosis to occur.
|Preparation||G2-arrested frog oocytes are arrested and suspended in buffer. They are passed through an electric field, which stimulates the oocytes to mature into eegs. The buffer is removed and the eggs are powerfully centrifuged, tearing them apart into three different layers resembling a parfait: lipid at top; cytoplasm in the middle; and yolk at the bottom. The layer of cytoplasm is then extracted from this egg parfait.|
|Sperm Nuclei||When sperm chromatin is added to an egg extract, a nuclear envelope forms around the sperm chromatin and the chromosomes decondense (30 min), then the chromosomes condense and the nuclear envelope breaks down (60 min), then the chromosomes decondense, a nuclear envelope forms around the chromosomes and the DNA is replicated (80 min).|
|Untreated||When sperm nuclei were added to untreated egg extract, mitotic cycles ensued as expected.|
|RNase||When egg extract was first treated with RNase and sperm nuclei were added, chromosomes did not condense and no new proteins were synthesized. RNase degrades mRNA but leaves tRNA and rRNA intact (needed for transcription and translation).|
|RNase + cyclin B mRNA||When cyclin B mRNA was added to egg extracted treated with RNase, then mitosis ensued as expectd. Thus, cyclin B is the only protein that must be synthesized in the egg extract for cycles of mitosis to ensue after fertlization.|
|RNase + nondegradable cyclin B mRNA||(nondegradable cyclin B mRNA refers to mRNA encoding a cyclin B mutant that can't be degraded). These results demonstrate that cyclin B must be degraded for cells to exit mitosis: chromosome decondensation and formation of a nuclear envelope.|
|Results||A cyclin is periodically synthesized and degraded in the egg extract. When MPF activity is assayed (here by the simple histone H1 kinase assay), MPF activity peaks when the cyclin concentration peaks. Nuclear envelope breakdown and chromosome condensation occur when MPF activity peaks.|