Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism. © 2012 Selivanov et al

Design and implementation of a mammalian synthetic gene oscillator

The core goal of synthetic biology as a discipline is to design, develop and characterize biological parts in order to precisely control cellular behaviour. Much of the research in this field has been focused on the development of gene regulatory networks, namely switches and oscillators. The study of synthetic gene oscillators has attracted significant attention in the past decade due to their intriguing dynamics and relevance in controlling inflammatory, metabolic and circadian signalling pathways. Additionally, the precise expression dynamics and molecular mechanisms that underlie the mammalian circadian clock structure are not fully understood. The work presented herein regards the design and implementation of a tuneable mammalian synthetic gene oscillator with a novel biological structure. To this end, an approach based on a combination of in silico design and in vivo part validation, in conjunction with a comparative analysis of previously implemented synthetic gene oscillators, was taken when assembling the proposed system. The topology of the system relies on a delayed negative feedback loop, consisting of the coupled regulatory activities of the transcription regulators LacI, tTA, and Gal4. The numerical solution and stability analysis of an ODE-based model describing the dynamics of the system are indicative that the proposed system is capable of generating sustained oscillations across a wide range of parameter values. The biological parts that comprise the system have been monitored and validated in HEK293T cells through time-lapse fluorescence microscopy and image analysis. The in vivo performance of the proposed mammalian synthetic gene oscillator was also assessed in the HEK293T cell line, and monitored using time-lapse fluorescence microscopy. Damped fluorescence oscillations were observed: these could be tuned by a differential IPTG concentration gradient and abolished by doxycycline. The proposed mammalian synthetic gene oscillator provides valuable insight into the gene expression regulatory processes leading to oscillatory behaviour, and has the potential to foster progress in future synthetic biology-based therapies.Open Acces