Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices.

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances

Advanced Mathematical Modelling of Pancreatic β-Cells

Insulin-secreting pancreatic $\beta$-cells are responsible for maintaining the whole body glucose homeostasis. Dysfunction or loss of $\beta$-cell mass results in impaired insulin secretion and, in some cases, diabetes. Many of the factors that influence $\beta$-cell function or insulin exocytosis, however, are not fully understood. To support the investigation, mathematical models have been developed and used to design experiments. In this dissertation, we present the Integrated Oscillator Model (IOM) that is one of the mathematical models used for the investigation of the mechanism behind the bursting activity that underlies intracellular Ca$^{2+}$ oscillations and pulsatile insulin secretion. The IOM describes the interaction of the cellular electrical activity and intracellular Ca$^{2+}$ with glucose metabolism via numerous feedforward and feedback pathways. These interactions, in turn, produce metabolic oscillations with a sawtooth or pulsatile time course, reflecting different oscillation mechanisms. We determine conditions favorable to each type of oscillations, and show that the model accounts for key experimental findings of $\beta$-cell activity. We propose several extensions of the model to include all the main elements involved in the insulin secretion. The latest and most sophisticated model describes the complex metabolism in the mitochondria and the several biological processes in the insulin exocytosis cascade. The model, also, captures the changes in the $\beta$-cell activity and the resulting amount of secreted insulin in response to different concentrations of glucose in the blood. The model predictions, in agreement with findings reported in the experimental literature, show an increase of insulin secretion when the glucose level is high and a basal-low insulin concentration when the glucose level decreases. Finally, we use the new model to simulate the interaction among $\beta$-cells (through gap junction) within the same islet. The simulations show that the electrical coupling is sufficient to synchronize the $\beta$-cells within an islet. We also show that the amplitude of the oscillations in the insulin secretion rate is bigger when the $\beta$-cells synchronize. This suggests a more efficient secretion of insulin in the bloodstream when the cells burst in unison, as it has been observed experimentally

Nitric oxide signalling in astrocytes

Dans le cerveau, les astrocytes sont les cellules gliales les plus abondantes et elles jouent divers rôles, y compris le maintien des synapses tripartites et la régulation du débit sanguin cérébral (DSC). Le monoxyde d’azote (NO) est une molécule de signal endogène qui a un impact sur la régulation de l'activité synaptique et du DSC. Des études antérieures ont démontré que le NO est produit dans les cellules endothéliales et les neurones par la synthase du monoxyde d’azote endothéliale (eNOS) et neuronale (nNOS), respectivement. Cependant, la source de production de NO dans les astrocytes reste incertaine. Par conséquent, nous proposons que la voie de signalisation NOS constitutive puisse coexister dans les astrocytes et puisse être activée par différents neurotransmetteurs. L'objectif de cette thèse est d'identifier les sources et les activateurs de la production de NO dans les astrocytes corticaux de la souris. L'identification des isoformes constitutives de NOS effectuée au moyen de la microscopie électronique et d'immunohistochimie a révélé l’expression des eNOS et nNOS dans les astrocytes. Des préparations de culture d'astrocytes et de tranches de cerveau marquées avec du diacétate de 4-amino-5-méthylamino-2',7'-difluorescéine (DAF-FM), un indicateur de NO perméable aux cellules qui devient imperméable une fois à l’intérieur ont été réalisées. Cette fonctionnalité a été mise à profit pour évaluer la production de NO exclusivement dans les astrocytes en utilisant la microscopie confocale à uni- et multi-photons. De plus, des agonistes cholinergiques ou glutamatergiques qui ont la capacité d’augmenter la concentration de Ca2+ intracellulaire peuvent induire une production du NO in vitro et ex vivo dans les astrocytes, qui est supprimée en présence de l'inhibiteur de NOS non sélectif, L-NG -Nitro-arginine. Fait intéressant, la réponse NO à l’acétylcholine était absente chez les souris eNOS-/-, tandis que l'acide trans-1-aminocyclopentane-1,3-dicarboxylique (t-ACPD) a peu affecté la production de NO chez les souris nNOS-/-. Ces résultats impliquent que les eNOS et nNOS astrocytaires peuvent être déclenchés par des cascades d'activation distinctes (cholinergique et glutamatergique métabotrope). En outre, les études sur la mobilisation cytosolique du Ca2+ indiquent l'importance du réticulum endoplasmique comme réservoir de Ca2+ pour la production de NO, et suggèrent aussi une voie de signalisation astrocytaire qui, une fois activée par le t- ACPD, provoque l'efflux de Ca2+ médié par le récepteur à la ryanodine, qui à son tour active les nNOS adjacents et conduit à la production de NO. Par ailleurs, la superfusion de préparations in vitro et ex vivo avec du N-Méthyl-D-aspartate (NMDA) a provoqué une augmentation du NO tant dans les souris eNOS-/- que nNOS-/-, ce qui indique l'implication des eNOS et nNOS astrocytaires. La production de NO a été atténuée par l'inhibition du complexe PSD-95 / nNOS ce qui suggère que le récepteur NMDA astrocytaire rend fonctionnelle la cassette de signalisation NR2B/PSD-95/nNOS. En conclusion, nos résultats démontrent que : i) les astrocytes corticaux expriment à la fois eNOS et nNOS; ii) la nNOS cytosolique colocalise avec les récepteurs 2 et 3 de la ryanodine, alors que les nNOS membranaires colocalisent avec le récepteur NMDA contenant le NR2B; iii) la stimulation neuronale a la capacité d'induire la production de NO par les eNOS et nNOS astrocytaires par des voies de signalisation différentes; iv) l'activation des nNOS cytosoliques nécessite une activation des récepteurs à la ryanodine. Collectivement, ces données suggèrent une production de NO compartimentée et spécifique après une stimulation neuronale probablement dans le but de réguler finement et de façon polarisée les fonctions astrocytaires. Ce travail fournit un nouvel aperçu des conséquences physiologiques pour les fonctions neuronales et vasculaires et améliore notre compréhension de la fonction NO astrocytaire dans le cerveau.In the brain, astrocytes are the most abundant glial cells and play various roles including maintenance of tripartite synapses and regulation of CBF. An endogenous signal molecule that has a potential to have an effect on regulation of both synaptic activity and CBF is nitric oxide (NO). Previous studies have demonstrated that NO is produced in endothelial cells and neurons by endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), respectively. However, the source of NO production in astrocyte remains uncertain. Therefore, we propose that constitutive NOS signalling pathways may exist in astrocyte and can be activated by different neurotransmitters. The aim of this thesis is to identify the sources and activators of NO production in mouse cortical astrocytes. Identification of constitutive NOS isoforms done by means of electron microscopy and immunohistochemistry revealed the expression of both eNOS and nNOS in astrocytes. All preparations were performed in astrocyte cultures and brain slice preparations labeled with 4- amino-5-methylamino-2',7'-difluorescein (DAF-FM) diacetate, a cell-permeant NO indicator that becomes cell-impermeable once inside cells. Therefore, I took advantage of this feature to evaluate NO production exclusively in astrocytes using single and multi-photon confocal microscopy. We then tested whether cholinergic and glutamatergic agonists that have the capacity to increase intracellular Ca2+ concentration can induce an increase in astrocytic NO. Both in vitro and ex vivo, NO production levels indicate that cholinergic and glutamatergic stimulations can induce astrocytic NO increases, which was abolished by the non-selective NOS inhibitor L- NG -Nitro-arginine. Moreover, the NO response to ACh was absent in eNOS-/- mice, while trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) barely affected NO production in nNOS-/- mice. These results imply that astrocytic eNOS and nNOS can be triggered discretely by distinct activation cascades (cholinergic and metabotropic glutamatergic). Furthermore, studies on cytosolic Ca2+ mobilization point out the importance of the endoplasmic reticulum (ER) Ca2+ as key in the mechanism of NO production, and suggests a signalling pathway that t-ACPD causes IP3Rs to elicit RyRs-mediated Ca2+ efflux, which in turn, activates adjacent nNOS and leads to NO production. Furthermore, superfusion of in vitro and ex vivo preparations with N-Methyl-D-aspartate (NMDA) evoked an increase in NO in eNOS-/- and nNOS-/- mice. The NO production was attenuated through removal of PSD-95/nNOS complex. This result posits that astrocytic NMDA receptor may comprise the functional NR2B/PSD- 95/nNOS signalling cassette. In conclusion, our findings demonstrate that: i) cortical astrocytes express both eNOS and nNOS; ii) nNOS colocalizes with ryanodine receptor 2 and 3, whereas membrane nNOS colocalizes with NR2B-containing NMDA receptor; iii) neuronal stimulation has the capacity of inducing eNOS- and nNOS-produced NO in astrocytes via different activation signalling; iv) activation of cytosolic nNOS requires the activation of ryanodine receptors. Collectively, these data suggest a compartmentalized and specific NO production following neuronal stimulation probably for a fine and polarized regulation of astrocytic functions. This work provides new insight into physiological consequences for neuronal and vascular functions and ameliorates our understanding of astrocytic NO function in the brain

Miro-1 links mitochondria and microtubule dynein motors to control lymphocyte migration and polarity

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.This study was supported by SAF2011-25834 from the Spanish Ministry of Science and Innovation, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII), and ERC-2011-AdG 294340- GENTRIS. J.M.G.-G. received salary support from the Miguel Servet (CP11/00145) ISCIII program. R.V.-B. was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Economy and Competiveness (JCI-2011-09663

Investigating the Role of Mechanical Properties in Biological Systems

There is considerable interest in measuring, with nanoscale spatial resolution, the physical and material properties of biological membranes and whole cells because of their role in the physiology of living systems. The atomic force microscope (AFM) has proven to be particularly well-suited for biological studies because samples can be maintained in near physiological conditions to preserve sample integrity (such as in a buffer solution at a physiologically relevant pH or temperature), and the imaging process is relatively nondestructive. In this work, the mechanical properties of supported lipid bilayers and neurons were examined using AFM-based techniques. Specifically, tapping mode AFM-based techniques were used to investigate the influence of lipid composition and temperature fluctuations on the physicomechanical properties of supported lipid bilayers. Topographic images were produced by tapping mode AFM, while additional compositional contrast and mechanical information was gained from phase contrast imaging, higher harmonic imaging, and scanning probe acceleration microscopy (SPAM). Importantly, all of this data was acquired simultaneously during the tapping mode AFM imaging process and the same imaging parameters were used for each experiment so that fair mechanical comparisons could be made across experiments. Lastly, force-distance curves and force volume imaging were used to better understand the effect of microtubule disruption or stabilization on the toxic ability of amyloid-beta1-42 aggregates, which are implicated in the development of Alzheimer\u27s disease. Overall, the work presented in this dissertation improved techniques for studies in mechanobiology and examined how biologically relevant factors affect the mechanical properties of lipid membranes or whole cells

Analyzing the Functional Properties of the Creatine Kinase System with Multiscale 'Sloppy' Modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The ‘phosphocreatine shuttle’ hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using ‘sloppy modeling’ ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 µM*s(−1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 µM*s(−1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 µM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect