MCB 252 Topic 30 Cell Cycle Embryonic Cycle and BiochemistryProf David Rivier MCB 252 Spring 2015 MCB 252 Cell Biology Topic 14 Cell Cycle Cell Cycle Embryonic Cycle and Biochemistry Reading Lodish Classic Experiment 19 1 pp 923 924 and sections 19 0 19 3 MCB 252 Cell Cycle I Embryonic Cell Cycle Biochemical Analysis Basic Features Cell fusion experiments Embryonic cell cycles Xenopus biochemical analysis Identification of MPF Identification of Cyclins The Eukaryotic Cell Cycle Flow cytometry to analyze DNA content Fluorescence activated cell sorter FACS Lodish et al Fig 9 2 Label cells with a DNA dye Alberts et al Fig 17 13 Cell fusion experiments Fuse cells together monitor effects S G1 G1 nucleus enters S S G2 G2 nucleus does not enter S S phase cells have factor that can induce DNA replication in G1 cells G2 cells do not respond to this factor Block to rereplication replicate once and only once Fuse Mitotic Cells w Interphase Cells Pollard and Earnshaw Fig 46 4 In all cases the nuclear envelope breaks down and chromosomes condense Advantages of studying embryonic cell cycle In embryos cycles are synchronized Early cycles are rapid Xenopus 1st cycle 75 next 11 are 30 each Provide good source of materials for biochemistry Xenopus egg 1 mm diameter 100 000x cytoplasm of human somatic cell Alberts et al Fig 17 8 Embryonic cell cycles Developing Xenopus embryos T14M1 Movie 16 lotscleavmov2 avi Xenopus laevis Alberts et al Figs 17 9 Maturation of Xenopus oocytes arrest 8 months grow synthesize materials needed for rapid embryonic development Progesterone secreted by follicle cells surrounding egg triggers maturation Maturation promoting factor MPF Yoshio Masui Mitotic and meiotic cells possess an activity that can drive interphase cells into mitosis 2 Transfer of cytoplasm from MII arrested eggs drives interphase oocytes into meiosis as if they had been treated with progesterone Lodish et al Sixth Edition Fig 20 6 Injections to assay MPF activity Xenopus oocytes eggs early embryos Lodish et al Sixth Edition Fig 20 7 MPF activity highly conserved cytoplasm from mitotically arrested mammalian cells also contains MPF activity Things to Remember We ll Return 1 MPF Identification of Cyclin Experiment to study translation in sea urchin embryos fertilized eggs take samples every 10 minutes add 35 S Met cell cleavage Tim Hunt 2001 Nobel Prize SDS PAGE autoradiography other proteins Cyclin protein levels alternate as cells progress through cell cycle Lodish et al Sixth Edition Fig 20 8 Xenopus egg extract that cycles in vitro Pollard and Earnshaw Fig 43 10 Role of Cyclin B Use egg extracts deplete and replenish CyclinB Experiment 1 Treat extracts w low doses RNAse to destroy mRNAs 2 Inactivate RNAse 3 Add back sea urchin cyclinB mRNA transcribed in vitro Untreated extract Cyclin protein levels correlate with MPF activity Lodish et al Fig 19 13 RNAse treated extract Role of Cyclin B continued RNAse treated extract cyclin B mRNA New Cyclin protein synthesis is sufficient to drive extracts into mitosis Lodish et al Fig 19 13 Cyclin degradation is required for exit from mitosis Add RNA encoding mutant form of Cyclin B 90 N terminal amino acids Induces mitosis but cannot be degraded Cyclin levels really do this Lodish et al Fig 19 13 What we have learned so far Mitotic and meiotic cells possess an activity that can drive interphase cells into mitosis This activity MPF is highly conserved among eukaryotes Cyclin protein levels alternate as cells progress through cell cycle and correlate with MPF activity New Cyclin protein synthesis is sufficient to drive extracts into mitosis Cyclin protein degradation is required for exit from mitosis Things to Remember We ll Return 1 MPF 2 Cyclin
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