1. Ultrafast multicellular calcium imaging of calcium spikes in mouse beta cells in tissue slicesJurij Dolenšek, Viljem Pohorec, Maša Skelin, Marko Gosak, Andraž Stožer, 2025, izvirni znanstveni članek Opis: Background: The crucial steps in beta cell stimulus-secretion coupling upon stimulation with glucose are oscillatory changes in metabolism, membrane potential, intracellular calcium concentration, and exocytosis. The changes in membrane potential consist of bursts of spikes, with silent phases between them being dominated by membrane repolarization and absence of spikes. Assessing intra- and intercellular coupling at the multicellular level is possible with ever-increasing detail, but our current ability to simultaneously resolve spikes from many beta cells remains limited to double-impalement electrophysiological recordings. Methods: Since multicellular calcium imaging of spikes would enable a better understanding of coupling between changes in membrane potential and calcium concentration in beta cell collectives, we set out to design an appropriate methodological approach. Results: Combining the acute tissue slice method with ultrafast calcium imaging, we were able to resolve and quantify individual spikes within bursts at a temporal resolution of >150 Hz over prolonged periods, as well as describe their glucose-dependent properties. In addition, by simultaneous patch-clamp recordings we were able to show that calcium spikes closely follow membrane potential changes. Both bursts and spikes coordinate across islets in the form of intercellular waves, with bursts typically displaying global and spikes more local patterns. Conclusions: This method and the associated findings provide additional insight into the complex signaling within beta cell networks. Once extended to tissue from diabetic animals and human donors, this approach could help us better understand the mechanistic basis of diabetes and find new molecular targets.
Ključne besede: beta cell, calcium imaging, calcium oscillations, calcium spikes, physiology
Objavljeno v DKUM: 24.01.2025; Ogledov: 0; Prenosov: 7
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2. Assessing different temporal scales of calcium dynamics in networks of beta cell populationsJan Zmazek, Maša Skelin, Rene Markovič, Jurij Dolenšek, Marko Marhl, Andraž Stožer, Marko Gosak, 2021, izvirni znanstveni članek Opis: Beta cells within the pancreatic islets of Langerhans respond to stimulation with coherent oscillations of membrane potential and intracellular calcium concentration that presumably drive the pulsatile exocytosis of insulin. Their rhythmic activity is multimodal, resulting from networked feedback interactions of various oscillatory subsystems, such as the glycolytic, mitochondrial, and electrical/calcium components.How these oscillatory modules interact and affect the collective cellular activity, which is a prerequisite for proper hormone release, is incompletely understood. In the present work, we combined advanced confocal Ca2+ imaging in fresh mouse pancreas tissue
slices with time series analysis and network science approaches to unveil the glucosedependent characteristics of different oscillatory components on both the intra- and inter-cellular level. Our results reveal an interrelationship between the metabolically driven low-frequency component and the electrically driven high-frequency component, with the latter exhibiting the highest bursting rates around the peaks of the slow
component and the lowest around the nadirs. Moreover, the activity, as well as the average synchronicity of the fast component, considerably increased with increasing stimulatory glucose concentration, whereas the stimulation level did not affect any of these parameters in the slow component domain. Remarkably, in both dynamical components, the average correlation decreased similarly with intercellular distance, which implies that intercellular communication affects the synchronicity of both types of oscillations. To explore the intra-islet synchronization patterns in more detail, we constructed functional connectivity maps. The subsequent comparison of network characteristics of different oscillatory components showed more locally clustered and segregated networks of fast oscillatory activity, while the slow oscillations were more global, resulting in several long-range connections and a more cohesive structure. Besides the structural differences, we found a relatively weak relationship between the fast and slow network layer, which suggests that different synchronization mechanisms
shape the collective cellular activity in islets, a finding which has to be kept in mind in future studies employing different oscillations for constructing networks.
Ključne besede: islets of Langerhans, beta cell network, calcium oscillations, multimodal activity analysis, confocal imaging, functional connectivity, multiplex network
Objavljeno v DKUM: 06.06.2024; Ogledov: 171; Prenosov: 6
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3. Critical and supercritical spatiotemporal calcium dynamics in beta cellsMarko Gosak, Andraž Stožer, Rene Markovič, Jurij Dolenšek, Matjaž Perc, Marjan Rupnik, Marko Marhl, 2017, izvirni znanstveni članek Opis: A coordinated functioning of beta cells within pancreatic islets is mediated by oscillatory membrane depolarization and subsequent changes in cytoplasmic calcium concentration. While gap junctions allow for intraislet information exchange, beta cells within islets form complex syncytia that are intrinsically nonlinear and highly heterogeneous. To study spatiotemporal calcium dynamics within these syncytia, we make use of computational modeling and confocal high-speed functional multicellular imaging. We show that model predictions are in good agreement with experimental data, especially if a high degree of heterogeneity in the intercellular coupling term is assumed. In particular, during the first few minutes after stimulation, the probability distribution of calcium wave sizes is characterized by a power law, thus indicating critical behavior. After this period, the dynamics changes qualitatively such that the number of global intercellular calcium events increases to the point where the behavior becomes supercritical. To better mimic normal in vivo conditions, we compare the described behavior during supraphysiological non-oscillatory stimulation with the behavior during exposure to a slightly lower and oscillatory glucose challenge. In the case of this protocol, we observe only critical behavior in both experiment and model. Our results indicate that the loss of oscillatory changes, along with the rise in plasma glucose observed in diabetes, could be associated with a switch to supercritical calcium dynamics and loss of beta cell functionality.
Ključne besede: beta cells, islets of Langerhans, self-organized criticality, intercellular dynamics, calcium waves, glucose oscillations, computational model, confocal calcium imaging
Objavljeno v DKUM: 23.01.2018; Ogledov: 1752; Prenosov: 394
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4. Modeling of molecular and cellular mechanisms involved in [Ca sup 2+] signal encoding in airway myocytesMarko Marhl, Denis Noble, Etienne Roux, 2006, pregledni znanstveni članek Opis: In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca2+ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. Inparticular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. Themodel of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca2+ influx triggered by initialcytosolic Ca2+ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca2+ handling were developed to investigate the role of a mixed population of InsP3 receptor isoforms and the cellular environment in the occurrence of Ca2+ oscillations, and the respective role ofthe sarcoplasmic reticulum, mitochondria, and cytosolic Ca2+-binding proteins in cytosolic Ca2+ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.
Ključne besede: biophysics, mathematical modelling, modelling, calcium oscillations, contractions, airway smooth muscle cells, muscle cells, smooth muscles, encoding, decoding, bow-tie structures
Objavljeno v DKUM: 07.06.2012; Ogledov: 2034; Prenosov: 49
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5. Theoretical and experimental investigation of calcium-contraction coupling in airway smooth musclePrisca Mbikou, Aleš Fajmut, Milan Brumen, Etienne Roux, 2006, izvirni znanstveni članek Opis: We investigated theoretically and experimentally the ▫$Ca^{2+}$▫-contraction couplingin rat tracheal smooth muscle. ▫$[Ca^{2+}]_i$▫, isometric contraction and myosin light chain (MLC) phosphorylation were measured in response to 1 mM carbachol. Theoretical modeling consisted in coupling a model of ▫$Ca^{2+}-dependent$▫ MLC kinase (MLCK) activation with a four-state model of smooth muscle contractile apparatus. Stimulation resulted in a short-time contraction obtained within 1 min, followed by a long-time contraction up to the maximal force obtained in 30 min. ML-7 and Wortmannin (MLCK inhibitors) abolished the contraction. Chelerythrine (PKC inhibitor) did not change the short-time, but reduced the long-time contraction. ▫$[Ca^{2+}]_i$▫ responses of isolated myocytes recorded during the first 90 s consisted in a fast peak, followed by a plateau phase and, in 28 % of the cells, superimposed ▫$Ca^{2+}$▫ oscillations. MLC phosphorylation was maximal at 5 s and then decreased, whereas isometric contraction followed a Hill-shaped curve. The model properlypredicts the time course of MLC phosphorylation and force of the short-time response. With oscillating ▫$Ca^{2+}$▫ signal, the predicted force does not oscillate. According to the model, the amplitude of the plateau and the frequency of oscillations encode for the amplitude of force, whereas the peak encodes for force velocity. The long-time phase of the contraction, associated with a second increase in MLC phosphorylation, may be explained, at least partially, by MLC phosphatase (MLCP) inhibition, possibly via PKC inhibition.
Ključne besede: biophysics, mathematical modelling, modelling, calcium oscillations, contractions, force development, muscle cells, smooth muscles, myosin kinase
Objavljeno v DKUM: 07.06.2012; Ogledov: 2091; Prenosov: 102
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6. Selective regulation of protein activity by complex Ca[sup]2+ oscillations : a theoretical studyBeate Knoke, Marko Marhl, Stefan Schuster, 2007, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Opis: Calcium oscillations play an important role in intracellular signal transduction. As a second messenger, ▫$Ca^{2+}$▫ represents a link between several input signals and several target processes in the cell. Whereas the frequency of simple ▫$Ca^{2+}$▫ oscillations enables a selective activation of a specific protein and herewith a particular process, the question arises of how at the same time two or more classes of proteins can be specifically regulated. The question is general and concerns the problem of how one second messenger can transmit more than one signal simultaneously (bow-tie structure of signalling). To investigate whether a complex ▫$Ca^{2+}$▫ signal like bursting, a succession of low-peak and high-peak oscillatory phases, could selectively activate different proteins, several bursting patterns with simplified square pulses were applied in a theoretical model. The results indicate that bursting ▫$Ca^{2+}$▫ oscillations allow a differential regulation of two different calcium-binding proteins, and hence, perform the desired function.
Ključne besede: biophysics, calcium oscillations, cellular dynamics, mathematical models, signalling, bow-tie structures, bursting, decoding
Objavljeno v DKUM: 07.06.2012; Ogledov: 2022; Prenosov: 42
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7. Noise-induced spatial dynamics in the presence of memory lossMatjaž Perc, Marko Marhl, 2007, izvirni znanstveni članek Opis: We study the spatial dynamics of noise-induced waves in two-dimensional excitable media in dependence on the duration of the artificially imposed refractory time that is introduced to each constitutive system unit after an excitation. Due to the introduction of refractory times, a randomly induced spatial wave is temporarily unable to transmit information to the opposite site of its propagation direction. Thus, once the wave leaves the absorbing boundaries of the spatial grid the system has little or no recollection, depending on the duration of the refractory time, of its existence. We show that even in the presence of such memory loss, self-organization of excitatory events leads to noise-induced spatial periodicity in the media. We present a simple analytical treatment of a two-unit system to capture and explain the essence of the observed phenomenon. Since refractory times are widespread in biological systems, our results provide interesting insights into functioning of real-life organisms at the cellular as well as tissue level.
Ključne besede: noise, spatiotemporal noise, intensity, pattern formation, refractory time, calcium oscillations
Objavljeno v DKUM: 07.06.2012; Ogledov: 2229; Prenosov: 99
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8. Role of cascades in converting oscillatory signals into stationary step-like responsesMarko Marhl, Vladimir Grubelnik, 2007, izvirni znanstveni članek Opis: In biological signal transduction pathways intermediates are often oscillatory and need to be converted into smooth output signals at the end. We show by mathematical modelling that protein kinase cascades enable converting oscillatory signals into sharp stationary step-like outputs. The importance of this result is demonstrated for the switch-like protein activation by calcium oscillations, which is of biological importance for regulating different cellular processes. In addition, we found that protein kinase cascades cause memory effects in the protein activation, which might be of a physiological advantage since a smaller amount of calcium transported in the cell is required for an effective activation of cellular processes.
Ključne besede: physics, calcium oscillations, mathematical modelling, calcium, calcium oscillations, sygnalling cascade, protein kinase cascades, signal transduction, ultrasensitivity, biochemical switch, cellular dynamics
Objavljeno v DKUM: 07.06.2012; Ogledov: 2187; Prenosov: 94
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9. Spatio-temporal modelling explains the effect of reduced plasma membrane Ca[sup]2+[/sup] efflux on intracellular Ca[sup]2+[/sup] oscillations in hepatocytesMarko Marhl, Marko Gosak, Matjaž Perc, C. Jane Dixon, Anne K. Green, 2008, izvirni znanstveni članek Opis: In many non-excitable eukaryotic cells, including hepatocytes, ▫$Ca^{2+}$▫ 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 ▫$Ca^{2+}$▫ plasma membrane efflux on ▫$Ca^{2+}$▫ oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane ▫$Ca^{2+}$▫ efflux increases the frequency of ▫$Ca^{2+}$▫ 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 ▫$Ca^{2+}$▫ oscillations in the cell. Here we divide the cell into a grid of elements and treat the ▫$Ca^{2+}$▫ 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.
Ključne besede: cellular signalling, calcium oscillations, intracellular oscilations, spatio-temporal dynamics, hepatocytes, stochastic simulations
Objavljeno v DKUM: 07.06.2012; Ogledov: 1651; Prenosov: 57
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10. MLC-kinase/phosphatase control of Ca[sup]2+ signal transduction in airway smooth musclesAleš Fajmut, Milan Brumen, 2008, izvirni znanstveni članek Opis: In airway smooth muscles, kinase/phosphatase-dependent phosphorylation and dephosphorylation of the myosin light chain (MLC) have been revealed by many authors as important steps in calcium ▫$(Ca^{2+})$▫ signalling pathway from the variation of ▫$Ca^{2+}$▫ concentration in cytosol to the force development. Here, a theoretical analysis of the control action of MLC-kinase (MLCK) and MLC-phosphatase (MLCP) in ▫$Ca^{2+}$▫ signalling is presented and related to the general control principles of these enzymes, which were previously studied by Reinhart Heinrich and his co-workers. The kinetic scheme of the mathematical model considers interactions among ▫$Ca^{2+}$▫, calmodulin (CaM) and MLCK and the well-known 4-state actomyosin latch bridge model, whereby a link between them is accomplished by the conservation relation of all species of MLCK. The mathematical model predicts the magnitude and velocity of isometric force in smooth muscles upon transient biphasic ▫$Ca^{2+}$▫ signal. The properties of signal transduction in the system such as the signalling time, signal duration and signal amplitude, which are reflected in the properties of force developed, are studied by the principles of the metabolic control theory. The analysis of our model predictions confirms as shown by Reinhart Heinrich and his co-workers that MLCK controls the amplitude of signal more than its duration, whereas MLCP controls both. Finally, the simulations of elevated total content of MLCK, a typical feature of bronchial muscles of asthmatic subjects and spontaneously hypertensive rats as well as potentiation of MLCP catalytic activity, are carried out and are discussed in view of an increase in the force magnitude.
Ključne besede: cells, calcium, calcium oscillations, myosin light chains, enzyme activities, mathematical models
Objavljeno v DKUM: 07.06.2012; Ogledov: 2348; Prenosov: 35
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