Monitoring of IP3 dynamics during Ca2+ oscillations in HSY human parotid cell line with FRET-based IP3 biosensors

Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger that elicits a wide range of spatial and temporal Ca2+ signals, and this signaling versatility is exploited to regulate diverse cellular responses. In the present study, we have developed a series of IP3 biosensors that exhibit strong pH stability and varying affinities for IP3, as well as a method for the quantitative measurement of cytosolic concentrations of IP3 ([IP3]i) in single living cells. We applied this method to elucidate IP3 dynamics during agonist-induced Ca2+ oscillations, and demonstrated cell type-dependent differences in IP3 dynamics ; a non-fluctuating rise in [IP3]i and repetitive IP3 spikes during Ca2+ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP3 spikes in HSY-EA1 cells varied from 10 to 100 nM, and the [IP3]i spike peak was preceded by a Ca2+ spike peak. These results suggest that repetitive IP3 spikes in HSY-EA1 cells are passive reflections of Ca2+ oscillations, and are unlikely to be essential for driving Ca2+ oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP3 and Ca2+

A biophysical model explains the spontaneous bursting behavior in the developing retina

During early development, waves of activity propagate across the retina and play a key role in the proper wiring of the early visual system. During the stage II these waves are triggered by a transient network of neurons, called Starburst Amacrine Cells (SACs), showing a bursting activity which disappears upon further maturation. While several models have attempted to reproduce retinal waves, none of them is able to mimic the rhythmic autonomous bursting of individual SACs and reveal how these cells change their intrinsic properties during development. Here, we introduce a mathematical model, grounded on biophysics, which enables us to reproduce the bursting activity of SACs and to propose a plausible, generic and robust, mechanism that generates it. The core parameters controlling repetitive firing are fast depolarizing $V$-gated calcium channels and hyperpolarizing $V$-gated potassium channels. The quiescent phase of bursting is controlled by a slow after hyperpolarization (sAHP), mediated by calcium-dependent potassium channels. Based on a bifurcation analysis we show how biophysical parameters, regulating calcium and potassium activity, control the spontaneously occurring fast oscillatory activity followed by long refractory periods in individual SACs. We make a testable experimental prediction on the role of voltage-dependent potassium channels on the excitability properties of SACs and on the evolution of this excitability along development. We also propose an explanation on how SACs can exhibit a large variability in their bursting periods, as observed experimentally within a SACs network as well as across different species, yet based on a simple, unique, mechanism. As we discuss, these observations at the cellular level have a deep impact on the retinal waves description.Comment: 25 pages, 13 figures, submitte

A Ca2+^{2+} puff model based on integrodifferential equations

The calcium (Ca$^{2+}$) signalling system is important for many cellular processes within the human body. Signals are transmitted within the cell by releasing Ca$^{2+}$ from the endoplasmic reticulum (ER) into the cytosol via clusters of Ca$^{2+}$ channels. Mathematical models of Ca$^{2+}$ release via inositol 1,4,5-trisphosphate receptors (IP$_{3}$R) help with understanding underlying Ca$^{2+}$ dynamics but data-driven modelling of stochastic Ca$^{2+}$ release events, known as Ca$^{2+}$ puffs, is a difficult challenge. Parameterising Markov models for representing the IP$_{3}$R with steady-state single channel data obtained at fixed combinations of the ligands Ca$^{2+}$ and inositol-trisphosphate (IP$_{3}$) has previously been demonstrated to be insufficient. However, by extending an IP$_{3}$R model based on steady-state data with an integral term that incorporates the delayed response of the channel to varying Ca$^{2+}$ concentrations we succeed in generating realistic Ca$^{2+}$ puffs. By interpreting the integral term as a weighted average of Ca$^{2+}$ concentrations that extend over a time interval of length $\tau$ into the past we conclude that the IP$_{3}$R requires a certain amount of memory of past ligand concentrations.Comment: 31 pages, 8 figures, 1 tabl

The Cerebellar Nodulus/Uvula Integrates Otolith Signals for the Translational Vestibulo-Ocular Reflex

Background: The otolith-driven translational vestibulo-ocular reflex (tVOR) generates compensatory eye movements to linear head accelerations. Studies in humans indicate that the cerebellum plays a critical role in the neural control of the tVOR, but little is known about mechanisms of this control or the functions of specific cerebellar structures. Here, we chose to investigate the contribution of the nodulus and uvula, which have been shown by prior studies to be involved in the processing of otolith signals in other contexts. Methodology/Principal Findings: We recorded eye movements in two rhesus monkeys during steps of linear motion along the interaural axis before and after surgical lesions of the cerebellar uvula and nodulus. The lesions strikingly reduced eye velocity during constant-velocity motion but had only a small effect on the response to initial head acceleration. We fit eye velocity to a linear combination of head acceleration and velocity and to a dynamic mathematical model of the tVOR that incorporated a specific integrator of head acceleration. Based on parameter optimization, the lesion decreased the gain of the pathway containing this new integrator by 62%. The component of eye velocity that depended directly on head acceleration changed little (gain decrease of 13%). In a final set of simulations, we compared our data to the predictions o

The effect of gap junctional coupling on the spatiotemporal patterns of Ca2+ signals and the harmonization of Ca2+-related cellular responses

The calcium ion (Ca²⁺), a universal signaling molecule, is widely recognized to play a fundamental role in the regulation of various biological processes. Agonist–evoked Ca²⁺ signals often manifest as rhythmic changes in the cytosolic free Ca²⁺ concentration (ccyt) called Ca²⁺ oscillations. Stimuli intensity was found to be proportional to the oscillation frequency and the evoked down-steam cellular response. Stochastic receptor expression in individual cells in a cell population inevitably leads to individually different oscillation frequencies and individually different Ca²⁺-related cellular responses. However, in many organs, the neighboring cells have to overcome their individually different sensitivity and produce a synchronized response. Gap junctions are integral membrane structures that enable the direct cytoplasmic exchange of Ca²⁺ ions and InsP₃ molecules between neighboring cells. By simulations, we were able to demonstrate how the strength of intercellular gap junctional coupling in relation to stimulus intensity can modify the spatiotemporal patterns of Ca²⁺ signals and harmonize the Ca²⁺-related cellular responses via synchronization of oscillation frequency. We demonstrate that the most sensitive cells are the wave initiator cells and that a highly sensitive region plays an important role in the determination of the Ca²⁺ phase wave direction. This sensitive region will then also progressively determine the global behavior of the entire system

Routes of Ca²⁺ shuttling during Ca²⁺ oscillations FOCUS ON THE ROLE OF MITOCHONDRIAL Ca²⁺ HANDLING AND CYTOSOLIC Ca²⁺ BUFFERS

In some cell types, Ca²⁺ oscillations are strictly dependent on Ca²⁺ influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca²⁺ influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca²⁺ influx played a pivotal role. However, when the Ca²⁺ transport across the plasma membrane by the “lanthanum insulation method” was blocked prior to the induction of the serum-induced Ca²⁺ oscillations, mitochondrial Ca²⁺ transport was found to be able to substitute for the plasmalemmal Ca²⁺ exchange function, thus rendering the oscillations independent of extracellular Ca²⁺. However, in a physiological situation, the Ca²⁺-buffering capacity of mitochondria was found not to be essential for Ca²⁺ oscillations. Moreover, brief spontaneous Ca²⁺ changes were observed in the mitochondrial Ca²⁺ concentration without apparent changes in the cytosolic Ca²⁺ concentration, indicating the presence of a mitochondrial autonomous Ca²⁺ signaling mechanism. In the presence of calretinin, a Ca²⁺-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca²⁺ ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca²⁺ shuttling pathways in primary mesothelial cells during Ca²⁺ oscillations: Ca²⁺ shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca²⁺ buffers

Kinetics of the thapsigargin-induced Ca2+ mobilisation: A quantitative analysis in the HEK-293 cell line

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump and, when applied acutely, it initiates a Ca2+ mobilisation that begins with the loss of Ca2+ from the endoplasmic reticulum (ER) and culminates with store-operated Ca2+ entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca2+ levels using FURA-2 and the FRET-based ER Ca2+ sensor D1ER, respectively. Our analysis predicts an ER Ca2+ leak of 5–6 µM⋅s−1 for the typical basal ER Ca2+ level of 335–407 µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6–1.0 µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28–30 nM⋅s−1 in 1 mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10–13 nM⋅s−1. Using puromycin (PURO), which enhances the ER Ca2+ leak, we show that TG-induced cytosolic Ca2+ transients are directly related to ER Ca2+ levels and to the ER Ca2+ leak. A one-compartment model incorporating ER Ca2+ leak and cytosolic Ca2+ clearance accounted satisfactorily for the basic features of TG-induced Ca2+ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca2+ transients most likely reflects an increase in ER Ca2+ leak

Water Quality Management in the Nitra River Basin

The Nitra River is a tributary of the Vih which enters the Danube downstream of Bratislava. The watershed area is slightly larger than 5000 km2, and more than 650000 inhabitants live there. The quality of the river is one of the poorest in Slovakia due to numerous municipal and industrial discharges and the low level of wastewater treatment. The ongoing economic transition and shortage of financial resources for environmental management call for the development of regional short-run, least-cost policies. The development of such policies was the main objective of this joint study with the participation of IIASA, the Water Research Institute (VUVH, Bratislava) and the Vih River Basin Authority. The present state of emissions and water quality was evaluated on the basis of available, routine types of information (including observations from the basin-wide water quality monitoring network) and additional data collection. It was found that industrial discharges form problems which can be handled mostly locally with a straight-forward strategy. In contrast, the management of municipal discharges -- representing about 70% of the total BOD5 emission in the catchment -- is a more complex issue requiring the development of a regional policy. The definition of ambient water quality criteria (or the usage of a combination of ambient and effluent criteria) reflecting water use is a pre-requisite of the establishment of a least-cost policy. Thus, the application of water quality models is necessary to relate emissions to receiving water quality (as well as their changes). Due to the nature of the problem, a number of oxygen and nutrient balance models were used, ranging from the traditional Streeter-Phelps model to the latest version of U.S. EPA's QUAL model family. The models were calibrated and validated on the basis of two comprehensive longitudinal water quality profile observations. These observations were gathered under low-flow conditions to correspond with the design requirements of the strategy development. Due to the presence of uncertainties of different origins, the methodology of Hornberger, Spear, and Young (based on the so-called "behavior definition") was applied for parameter estimation of simpler models which then were directly incorporated into an optimization model. This optimization model was based on dynamic programming, utilizing structural features of river basin water pollution problems. Elements of the water quality control policy model or decision support system (including the linked hydraulic and water quality model(s), the parameter estimation and uncertainty analysis routines, the dynamic programming, the database, the graphical user-interface, etc.) were developed in a rather generic fashion to allow a transfer from one watershed to another. This philosophy corresponds to the broader goals of IIASA's Water Resources Project dealing with issues of the management of degraded river basins in Central and Eastern Europe and the development of associated methodologies for which the Nitra River served as a case study. Starting from the existing municipal wastewater treatment facilities, a number of alternatives were developed for each site on the basis of various combinations of well-proven physical, biological, and chemical processes to which different effluent quality (BOD-5, TP, NH4-N, NO3-N, etc.) as well as investment, operation, maintenance, and repair costs belong. The technological alternatives (and their major parameters) serve as input to the management optimization model. A special focus was devoted to phased plant development and innovative, cost-effective upgrading of highly overloaded plants by adding chemicals in low dosage. The issue of upgrading was also experimentally analyzed by jar tests at different treatment plants. The objectives of the policy model were formulated in terms of minimizing the total annual cost or the investment cost. Constraints might incorporate ambient water quality (characterized by DO, BOD-5 and NH4-N), effluent criteria, and/or minimum level of treatment. The derived least-cost policies were compared to policies based strictly on effluent criteria and to those based on the application of "best available technology." The effluent criteria based policy stems from the new Slovakian legislation if its ambient criteria element was excluded (the legislation defines the simultaneous usage of effluent and ambient criteria and an eleven-year long transition period after which more stringent standards should be met). The role of industrial emissions was demonstrated in a sensitivity fashion, while the influence of parameter uncertainty on the developed policies was analyzed by an a posteriori Monte Carlo simulation and a multi-objective assessment. The study shows that significant cost savings are possible in comparison to uniform, effluent standard policies. They also suggest that a long-term strategy should be realized on the basis of a sequence of properly phased least-cost policies corresponding to ambient (or regionally variable) standards to be tightened gradually as financial resources become available

Assessment of Ca2+ Dynamics in Human Retinal Pigment Epithelial Cell Cultures

Retinal pigment epithelium is a monolayer of cells located beneath photoreceptors of the retina maintaining their functionality. Malfunction of RPE leads to retinal degenerative diseases, such as age-related macular degeneration and Stargardt disease. Ca2+ is a ubiquitous ion that takes part in regulation of vital cellular processes. The knowledge of Ca2+ dynamics is essential for understanding RPE physiology. This is especially important for functionality assessment of cells intended for transplantation and for drug testing. The aim of this thesis was to study spontaneous and mechanically induced Ca2+ activity in human RPE and to assess the effect of cellular maturation and wounding on the [Ca2+]i dynamics. For this, various methods, such as fluorescent Ca2+ imaging, immunofluorescence staining, PCR, and mathematical modeling were applied. In addition, novel methods were developed to analyze large amounts of Ca2+ imaging data. ARPE-19 and human embryonic stem cell-derived RPE cells (hESC-RPE) were used as RPE cell models. In this thesis, it was shown that both ARPE-19 and hESC-RPE exhibit intercellular Ca2+ waves upon mechanical stimulation. With live-cell Ca2+ imaging and mathematical modeling, it was demonstrated that in ARPE-19 cells, the mechanically induced Ca2+ waves propagate intracellularly through gap junctions and extracellularly involving diffusion of a paracrine factor. By applying in-house image analysis tools for the experimental fluorescence time-series, it was found that in hESC-RPE cells, spontaneous [Ca2+]i transients and the ability to propagate intercellular Ca2+ waves upon mechanical stimulation strongly depend on the maturation status of the cells. Finally, it was demonstrated that wounding affects spontaneous Ca2+ activity close to the wound edges, and cells within the healed areas resemble Ca2+ dynamics of immature hESC-RPE. To conclude, this thesis has provided important insights into human RPE Ca2+ dynamics, as well as into the events of single cell mechanical stimulation and large scale monolayer wounding. In addition, it was demonstrated that maturation drastically affects RPE Ca2+ dynamics. This knowledge and the developed image analysis algorithms contribute to understanding RPE physiology and can facilitate establishment of novel tools for assessment of RPE functionality prior to transplantation and in drug testing assays

Uncertainty in Modeling Regional Environmental Systems: The Generalization of a Watershed Acidification Model for Predicting Broad Scale Effects

Policies aimed at the alleviation of negative environmental impacts have increasingly been based on predictions made with models of the environmental systems involved. The usefulness of these models is limited by many uncertainties, e.g., is the complexity of a system properly reflected in the model structure and chosen aggregation-level? Is the data used in the model representative of the system? Is the temporal and spatial scale used in the model appropriate for understanding the system's behavior? The combination of these uncertainties may lead to unexpected results. This study identifies key factors determining watershed responses to acid deposition in different regions, using the RAINS Lake Acidification Model. The study aims at: -- Providing a method for defining a region such that environmental policy directed at alleviating watershed acidification will become more suitable for the large scale management of surface water quality. -- Depicting regional characteristics that allow for the usage of a less detailed and thus more aggregated model. -- Providing an operational concept of critical loads for policy insight into watershed quality as a function of the spatial zoning of watersheds. The results of the study are: -- The 5-step method of flexible zoning introduced in this study, allows for a probabilistic investigation of the compatibility between models and available spatial data. -- The calibration of models to previously defined regions may be of limited use for policy purposes because predictions of environmental effects (i.e., watershed acidification) as a result of changing deposition patterns over large regions may be error prone. -- Cumulative distributions of model predictions about the acidification of watersheds should be used to assess critical and target loads for broad regions