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.

Protein homeostasis from diffusion-dependent control of protein synthesis and degradation

“…By following microtubule polymerization and depolymerization, cell cycles were detected down to dilutions of 0.2x, and the oscillations persisted for at least 14 hours with more than 10 complete periods (Figure 1C, D, Movies S1-S3). In agreement with a previous study (Jin et al, 2022), the diluted extracts did not show signs of significant cell cycle defects except that the duration of interphase and the cell cycle period increased with increasing dilution.…”

Scaling of biosynthesis and metabolism with cell size

“…Furthermore, when cells reach sizes beyond their physiological range, overall proteome content no longer scales with size and the cytoplasm becomes diluted, presumably due to defective coordination of cell volume growth and biosynthesis. The consequences of cytoplasm dilution are an active area of research, with recent evidence demonstrating effects on reaction rates ( Jin et al., 2022 ; Molines et al., 2022 ) and phase separation ( Delarue et al., 2018 ) that could negatively impact cell function. New techniques enabling cellular density measurement with unprecedented precision ( Miettinen et al., 2022 ; Oh et al., 2022 ), as well as theoretical approaches applying principles of colloidal physics to study cytoplasmic crowding ( Maheshwari et al., 2019 ), are likely to provide exciting new insights.…”

A nested bistable module within a negative feedback loop ensures different types of oscillations in signaling systems

“…The 2014 work by Tsai et al on a cell cycle system shows the importance of analyzing different oscillatory types in a computational model, since it explains experimental findings 14 . And a recent work on the cell cycle model investigates the oscillatory robustness as a function of the cytoplasmic density, where they find limit cycles forming a hysteresis loop 53 . Simulations explain this through subcritical Hopf bifurcations, showing the significance of dynamical studies on experimental examples.…”