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

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.

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome.

PURPOSE: Ciliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS. METHODS: Exome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants. RESULTS: We identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants. CONCLUSION: IFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

Comprehensive Parameter Space Mapping of Cell Cycle Dynamics under Network Perturbations.

Studies of quantitative systems and synthetic biology have extensively utilized models to interpret data, make predictions, and guide experimental designs. However, models often simplify complex biological systems and lack experimentally validated parameters, making their reliability in perturbed systems unclear. Here, we developed a droplet-based synthetic cell system to continuously tune parameters at the single-cell level in multiple dimensions with full dynamic ranges, providing an experimental framework for global parameter space scans. We systematically perturbed a cell-cycle oscillator centered on cyclin-dependent kinase (Cdk1), enabling comprehensive mapping of period landscapes in response to network perturbations. The data allowed us to challenge existing models and refine a new model that matches the observed response. Our analysis demonstrated that Cdk1 positive feedback inhibition restricts the cell cycle frequency range, confirming model predictions; furthermore, it revealed new cellular responses to the inhibition of the Cdk1-counteracting phosphatase PP2A: monomodal or bimodal distributions across varying inhibition levels, underscoring the complex nature of cell cycle regulation that can be explained by our model. This comprehensive perturbation platform may be generalizable to exploring other complex dynamic systems.

Mitotic waves in frog egg extracts: Transition from phase waves to trigger waves.

Cyclin-dependent kinase 1 (Cdk1) activity rises and falls throughout the cell cycle, a cell-autonomous process known as mitotic oscillations. These oscillators can synchronize when spatially coupled, providing a crucial foundation for rapid synchronous divisions in large early embryos like Drosophila (~ 0.5 mm) and Xenopus (~ 1.2 mm). While diffusion alone cannot achieve such long-range coordination, recent studies have proposed two types of mitotic waves, phase and trigger waves, to explain the phenomena. How the waves establish over time for efficient spatial coordination remains unclear. Using Xenopus laevis egg extracts and a Cdk1 FRET sensor, we observe a transition from phase waves to a trigger wave regime in an initially homogeneous cytosol. Adding nuclei accelerates such transition. Moreover, the system transitions almost immediately to this regime when externally driven by metaphase-arrested extracts from the boundary. Employing computational modeling, we pinpoint how wave nature, including speed-period relation, depends on transient dynamics and oscillator properties, suggesting that phase waves appear transiently due to the time required for trigger waves to entrain the system and that spatial heterogeneity promotes entrainment. Therefore, we show that both waves belong to a single biological process capable of coordinating the cell cycle over long distances.

Cilia regulate meiotic recombination in zebrafish.

Meiosis is essential for evolution and genetic diversity in almost all sexual eukaryotic organisms. The mechanisms of meiotic recombination, such as synapsis, have been extensively investigated. However, it is still unclear whether signals from the cytoplasm or even from outside of the cell can regulate the meiosis process. Cilia are microtubule-based structures that protrude from the cell surface and function as signaling hubs to sense extracellular signals. Here, we reported an unexpected and critical role of cilia during meiotic recombination. During gametogenesis of zebrafish, cilia were specifically present in the prophase stages of both primary spermatocytes and primary oocytes. By developing a germ cell-specific CRISPR/Cas9 system, we demonstrated that germ cell-specific depletion of ciliary genes resulted in compromised double-strand break repair, reduced crossover formation, and increased germ cell apoptosis. Our study reveals a previously undiscovered role for cilia during meiosis and suggests that extracellular signals may regulate meiotic recombination via this particular organelle.

Fault Detection Based on Near-Infrared Spectra for the Oil Desalting Process.

The fault detection problem of the oil desalting process is investigated in this paper. Different from the traditional fault detection approaches based on measurable process variables, near-infrared (NIR) spectroscopy is applied to acquire the process fault information from the molecular vibrational signal. With the molecular spectra data, principal component analysis was explored to calculate the Hotelling T2 and squared prediction error, which act as fault indicators. Compared with the traditional fault detection approach based on measurable process variables, NIR spectra-based fault detection illustrates more sensitivity to early failure because of the fact that the changes in the molecular level can be identified earlier than the physical appearances on the process. The application results show that the detection time of the proposed method is earlier than the traditional method by about 200 min.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

A robust and tunable mitotic oscillator in artificial cells.

Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions.