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In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density.

ABSTRACT: Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes.

SUBMITTER: Jin M

PROVIDER: S-EPMC8832984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote.ResultsA synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression.ConclusionThe analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.

Project description:During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.

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In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density.

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