Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual‐color bioluminescence reporter

A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red‐emitting Phrixothrix hirtus luciferase (PxRe) and a blue‐emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non‐mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual‐color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes. Copyright © 2013 John Wiley & Sons, Ltd.     

Dynamin at the actin-membrane interface

Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.     

The manipulation of calcium oscillations by harnessing self-organisation

This paper investigates how self-organisation might be harnessed for the manipulation and control of calcium oscillations. Calcium signalling mechanisms are responsible for a number of important functions within biological systems, such as fertilization, secretion, contraction, neuronal signalling and learning. In this paper, calcium oscillations are investigated as a biological periodic process. Within biological systems such periodic behaviour is one of the outcomes from self-organisation. The understanding of periodic processes in living systems can enable more accurate diagnosis and physiologically suitable clinical therapies to be proposed, for diseases such as cancer, epilepsy, cardiac diseases and other dynamic diseases. In this paper these ideas are investigated by means of the calcium-induced calcium release (CICR) model and a number of representative simulations of intra and inter-cellular calcium oscillations are used to illustrate the manipulation and control of these oscillations in normal and pathological situations.     

Intracellular sterol dynamics

We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated.     

An Algorithm to Automate Yeast Segmentation and Tracking

Our understanding of dynamic cellular processes has been greatly enhanced by rapid advances in quantitative fluorescence microscopy. Imaging single cells has emphasized the prevalence of phenomena that can be difficult to infer from population measurements, such as all-or-none cellular decisions, cell-to-cell variability, and oscillations. Examination of these phenomena requires segmenting and tracking individual cells over long periods of time. However, accurate segmentation and tracking of cells is difficult and is often the rate-limiting step in an experimental pipeline. Here, we present an algorithm that accomplishes fully automated segmentation and tracking of budding yeast cells within growing colonies. The algorithm incorporates prior information of yeast-specific traits, such as immobility and growth rate, to segment an image using a set of threshold values rather than one specific optimized threshold. Results from the entire set of thresholds are then used to perform a robust final segmentation.     

Selective regulation of cellular processes via protein cascades acting as band-pass filters for time-limited oscillations

We show by mathematical modelling that a two-level protein cascade can act as a band-pass filter for time-limited oscillations. The band-pass filters are then combined into a network of three-level signalling cascades that by filtering the frequency of time-limited oscillations selectively switches cellular processes on and off. The physiological relevance for the selective regulation of cellular processes is demonstrated for the case of regulation by time-limited calcium oscillations.     

Computational theories on the function of theta oscillations

Neural rhythms can be studied in terms of conditions for their generation, or in terms of their functional significance. The theta oscillation is a particularly prominent rhythm, reported to be present in many brain areas, and related to many important cognitive processes. The generating mechanisms of theta have extensively been studied and reviewed elsewhere; here we discuss ideas that have accumulated over the past decades on the computational roles it may subserve. Theories propose different aspects of theta oscillations as being relevant for their cognitive functions: limit cycle oscillations in neuronal firing rates, subthreshold membrane potential oscillations, periodic modulation of synaptic transmission and plasticity, and phase precession of hippocampal place cells. The relevant experimental data is briefly summarized in the light of these theories. Specific models proposing a function for theta in pattern recognition, memory, sequence learning and navigation are reviewed critically. Difficulties with testing and comparing alternative models are discussed, along with potentially important future research directions in the field.     

Conserved microtubule-actin interactions in cell movement and morphogenesis

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.     

Hypervariable genes--experimental error or hidden dynamics

In a homogeneous group of samples, not all genes of high variability stem from experimental errors in microarray experiments. These expression variations can be attributed to many factors including natural biological oscillations or metabolic processes. The behavior of these genes can tease out important clues about naturally occurring dynamic processes in the organism or experimental system under study. We developed a statistical procedure for the selection of genes with high variability denoted hypervariable (HV) genes. After the exclusion of low expressed genes and a stabilizing log-transformation, the majority of genes have comparable residual variability. Based on an F-test, HV genes are selected as having a statistically significant difference from the majority of variability stabilized genes measured by the 'reference group'. A novel F-test clustering technique, further noted as 'F-means clustering', groups HV genes with similar variability patterns, presumably from their participation in a common dynamic biological process. F-means clustering establishes, for the first time, groups of co-expressed HV genes and is illustrated with microarray data from patients with juvenile rheumatoid arthritis and healthy controls.     

Perisomatic feedback inhibition underlies cholinergically induced fast network oscillations in the rat hippocampus in vitro

Gamma frequency network oscillations are assumed to be important in cognitive processes, including hippocampal memory operations, but the precise functions of these oscillations remain unknown. Here, we examine the cellular and network mechanisms underlying carbachol-induced fast network oscillations in the hippocampus in vitro, which closely resemble hippocampal gamma oscillations in the behaving rat. Using a combination of planar multielectrode array recordings, imaging with voltage-sensitive dyes, and recordings from single hippocampal neurons within the CA3 gamma generator, active current sinks and sources were localized to the stratum pyramidale. These proximal currents were driven by phase-locked rhythmic inhibitory inputs to pyramidal cells from identified perisomatic-targeting interneurons. AMPA receptor-mediated recurrent excitation was necessary for the synchronization of interneuronal discharge, which strongly supports a synaptic feedback model for the generation of hippocampal gamma oscillations.     

Control Analysis for Autonomously Oscillating Biochemical Networks

It has hitherto not been possible to analyze the control of oscillatory dynamic cellular processes in other than qualitative ways. The control coefficients, used in metabolic control analyses of steady states, cannot be applied directly to dynamic systems. We here illustrate a way out of this limitation that uses Fourier transforms to convert the time domain into the stationary frequency domain, and then analyses the control of limit cycle oscillations. In addition to the already known summation theorems for frequency and amplitude, we reveal summation theorems that apply to the control of average value, waveform, and phase differences of the oscillations. The approach is made fully operational in an analysis of yeast glycolytic oscillations. It follows an experimental approach, sampling from the model output and using discrete Fourier transforms of this data set. It quantifies the control of various aspects of the oscillations by the external glucose concentration and by various internal molecular processes. We show that the control of various oscillatory properties is distributed over the system enzymes in ways that differ among those properties. The models that are described in this paper can be accessed on http://jjj.biochem.sun.ac.za.     

Mass spectrometry informs the structure and dynamics of membrane proteins involved in lipid and drug transport

Membrane proteins are important macromolecules that play crucial roles in many cellular and physiological processes. Over the past two decades, the use of mass spectrometry as a biophysical tool to characterise membrane proteins has grown steadily. By capturing these dynamic complexes in the gas phase, many unknown small molecule interactions have been revealed. One particular application of this research has been the focus on antibiotic resistance with considerable efforts being made to understand underlying mechanisms. Here we review recent advances in the application of mass spectrometry that have yielded both structural and dynamic information on the interactions of antibiotics with proteins involved in bacterial cell envelope biogenesis and drug efflux.     

Calcium imaging of cortical networks dynamics

Studies relating spontaneous network activities to cognitive processes and/or brain disorders constitute a recently expanding field of investigation. They are mostly based either on cellular recordings--usually performed in pharmacologically induced oscillations in brain slices--or on multi-cellular recordings using tetrodes or multiple electrodes. However, these research strategies cannot link the electrical recordings with morphological characterization of the neurons. The progress made in imaging techniques allows for the first time to have simultaneously a dynamic and global characterization of network activity and to determine the single-cell properties of the unitary microcircuits involved in this activity.     

Spatio-temporal modelling explains the effect of reduced plasma membrane Ca2+ efflux on intracellular Ca2+ oscillations in hepatocytes

In many non-excitable eukaryotic cells, including hepatocytes, Ca²⁺ oscillations play a key role in intra- and intercellular signalling, thus regulating many cellular processes from fertilisation to death. Therefore, understanding the mechanisms underlying these oscillations, and consequently understanding how they may be regulated, is of great interest. In this paper, we study the influence of reduced Ca²⁺ plasma membrane efflux on Ca²⁺ oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane Ca²⁺ efflux increases the frequency of Ca²⁺ oscillations, but does not affect the duration of individual transients. This phenomenon can be best explained by taking into account not only the temporal, but also the spatial dynamics underlying the generation of Ca²⁺ oscillations in the cell. Here we divide the cell into a grid of elements and treat the Ca²⁺ dynamics as a spatio-temporal phenomenon. By converting an existing temporal model into a spatio-temporal one, we obtain theoretical predictions that are in much better agreement with the experimental observations.     

Is a constant low-entropy process at the root of glycolytic oscillations?

We measured temporal oscillations in thermodynamic variables such as temperature, heat flux, and cellular volume in suspensions of non-dividing yeast cells which exhibit temporal glycolytic oscillations. Oscillations in these variables have the same frequency as oscillations in the activity of intracellular metabolites, suggesting strong coupling between them. These results can be interpreted in light of a recently proposed theoretical formalism in which isentropic thermodynamic systems can display coupled oscillations in all extensive and intensive variables, reminiscent of adiabatic waves. This interpretation suggests that oscillations may be a consequence of the requirement of living cells for a constant low-entropy state while simultaneously performing biochemical transformations, i.e., remaining metabolically active. This hypothesis, which is in line with the view of the cellular interior as a highly structured and near equilibrium system where energy inputs can be low and sustain regular oscillatory regimes, calls into question the notion that metabolic processes are essentially dissipative.     

Dynamic Circadian Protein–Protein Interaction Networks Predict Temporal Organization of Cellular Functions

Essentially all biological processes depend on protein–protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (∼24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner.