Cell cycle synchronization
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The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.
- MeSH
- algoritmy MeSH
- buněčný cyklus genetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory genetika metabolismus patologie MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transkriptom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- validační studie MeSH
A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.
- MeSH
- barvení a značení metody MeSH
- buněčné dělení MeSH
- buněčné kultury metody MeSH
- buněčný cyklus MeSH
- Chlamydomonas reinhardtii cytologie genetika růst a vývoj MeSH
- Chlorophyta cytologie genetika růst a vývoj MeSH
- chloroplasty genetika MeSH
- DNA rostlinná genetika MeSH
- fotoperioda * MeSH
- frakcionace buněk metody MeSH
- replikace DNA MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
- MeSH
- buněčný cyklus fyziologie MeSH
- časové faktory MeSH
- chromozomy rostlin * metabolismus MeSH
- genom rostlinný genetika MeSH
- genomika metody MeSH
- hydroxymočovina MeSH
- metafáze MeSH
- mitotický index MeSH
- nitrobenzeny MeSH
- organothiofosforové sloučeniny MeSH
- průtoková cytometrie metody MeSH
- pufry MeSH
- Saccharum cytologie genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Green algae dividing by multiple fission comprise unrelated genera but are connected by one common feature: under optimal growth conditions, they can divide into more than two daughter cells. The number of daughter cells, also known as the division number, is relatively stable for most species and usually ranges from 4 to 16. The number of daughter cells is dictated by growth rate and is modulated by light and temperature. Green algae dividing by multiple fission can thus be used to study coordination of growth and progression of the cell cycle. Algal cultures can be synchronized naturally by alternating light/dark periods so that growth occurs in the light and DNA replication(s) and nuclear and cellular division(s) occur in the dark; synchrony in such cultures is almost 100% and can be maintained indefinitely. Moreover, the pattern of cell-cycle progression can be easily altered by differing growth conditions, allowing for detailed studies of coordination between individual cell-cycle events. Since the 1950s, green algae dividing by multiple fission have been studied as a unique model for cell-cycle regulation. Future sequencing of algal genomes will provide additional, high precision tools for physiological, taxonomic, structural, and molecular studies in these organisms.
Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks-deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.
- MeSH
- buněčné dělení * MeSH
- buněčný cyklus * MeSH
- DNA antagonisté a inhibitory biosyntéza MeSH
- lidé MeSH
- replikace DNA * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.
- MeSH
- bílkoviny řas metabolismus MeSH
- buněčné kultury metody MeSH
- Chlamydomonas reinhardtii cytologie fyziologie MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- down regulace MeSH
- fyziologický stres MeSH
- kontrolní body buněčného cyklu * MeSH
- regulace genové exprese u rostlin MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
