Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity—by degrading CDK activators or inhibitors—and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

https://science.sciencemag.org/content/sci/274/5293/1652.full.pdf

How proteolysis drives the cell cycle

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

https://science.sciencemag.org/content/sci/278/5346/2130.full.pdf

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts

A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B

Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.

/pdf/differences-in-the-localization-and-morphology-of-3vk26hynhc.pdf

Differences in the localization and morphology of chromosomes in the human nucleus

https://www.cell.com/cell/pdf/0092-8674(91)90042-W.pdf

Isolation of three novel human cyclins by rescue of G1 cyclin (cln) function in yeast

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.

/pdf/the-timing-of-the-formation-and-usage-of-replicase-clusters-1e6nbi7b4f.pdf

The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts.

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.

/pdf/cyclins-a-and-b-associate-with-chromatin-and-the-polar-3ixb7lf9il.pdf

Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos.

Cells of Drosophila embryos that are subjected to a 37 degrees C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphase-like state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.

/pdf/heat-shock-results-in-cell-cycle-delay-and-synchronisation-1k5imjpm5x.pdf

G. Maldonado-Codina

Papers

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts.

Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos.

Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos.

Cyclical changes in the subcellular distribution of proteins essential for mitosis during embryogenesis in Drosophila.