Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Producción CientíficaThe photoprotein aequorin was the first probe used to measure specifically the [Ca2+] inside the lumen of the endoplasmic reticulum ([Ca2+]ER) of intact cells and it provides values for the steady-state [Ca2+]ER, around 500 M, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca2+]ER offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca2+-affinity aequorin allows covering a large dynamic range of [Ca2+], from 10−5 to 10−3 M; (iii) aequorin is nearly insensitive to changes in Mg2+ or pH, has a high signal-to-noise ratio and calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (iv) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine requires previous complete depletion of Ca2+ of the ER for 1–2 h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca2+]ER, only relatively brief experiments can be performed; and (iv) expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. Choosing aequorin or other techniques to measure [Ca2+]ER will depend of the correct balance between these properties in a particular problem

Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Producción CientíficaChanges in the free calcium concentration of the endoplasmic reticulum ([Ca 2+],,) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported that recombinant aequorin targeted to the endoplasmic reticulum (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB J 1997; 11: 881-8861 can be used to monitor selectively [Ca2+le, in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH, and PC12 cells). Functional studies showed that the steady state lumenal [Ca*+],, ranged from around 300 pM in granule cells to 800 ).rM in GH,cells. InsP,-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH,cells) produced a very rapid decrease in lumenal [Ca’+],,. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+lerin all the excitable cells (anterior pituitary, GH,, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+le, in several primary non-dividing cells

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Producción CientíficaWe use here a new very low-Ca2+-affinity targeted aequorin to measure the [Ca2+] in the endoplasmic reticulum ([Ca2+]ER). The new aequorin chimera has the right Ca2+-affinity to make long-lasting measurements of [Ca2+]ER in the millimolar range. Moreover, previous Ca2+-depletion of the ER is no longer required. The steady-state [Ca2+]ER obtained is 1–2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca2+]ER among different regions of the ER. About half of the ER had a [Ca2+]ER of 1 mM or below, and the rest had [Ca2+]ER values above 1 mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca2+]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca2+ of the ER was almost linearly dependent on the extracellular [Ca2+] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca2+] microdomains connecting the store operated Ca2+ channels with the ER Ca2+ pumps may be important during refilling

Mitochondrial Ca2+ dynamics in MCU knockout C. elegans worms

Producción CientíficaMitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.Ministerio de Economía y Competitividad - (Proyecto BFU2017-83509-R)Fondo Europeo de Desarrollo Regional (FEDER) y Junta de Castilla y León - (Projecto VA011G18

The role of Ca2+ signaling in aging and neurodegeneration: Insights from caenorhabditis elegans models

Producción CientíficaCa2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration

Modulation of secretion by the endoplasmic reticulum in mouse chromaffin cells

Producción CientíficaThe endoplasmic reticulum (ER) has been suggested to modulate secretion either behaving as a Ca2+ sink or as a Ca2+ source in neuronal cells. Working as a Ca2+ sink, through ER-Ca2+ pumping, it may reduce secretion induced by different stimuli. Instead, working as a Ca2+ source through the Ca2+ induced Ca2+ release (CICR) phenomenon, it may potentiate secretion triggered by activation of plasma membrane Ca2+ channels. We have previously demonstrated the presence of CICR in bovine chromaffin cells, but we now find that mouse chromaffin cells almost lack functional caffeine-sensitive ryanodine receptors in the ER and, consistently, no CICR from the ER could be observed. In addition, inhibition of ER Ca2+ pumping with ciclopiazonic acid or thapsigargin strongly stimulated high-K+-evoked catecholamine secretion and cytosolic [Ca2+] ([Ca2+]c) transients. Surprisingly, 5 mM caffeine reduced high-K+-induced [Ca2+]c peaks but considerably potentiated secretion induced by high-K+ stimulation. However, this potentiation was insensitive to ryanodine and additive to that induced by emptying the ER of Ca2+ with thapsigargin, suggesting that it is unrelated to the activation of ryanodine receptors. We conclude that, in mouse chromaffin cells, CICR is not functional and the ER strongly inhibits secretion by acting as a damper of the [Ca2+]c signal.2015-09-1

Mitochondrial free [Ca2+] levels and the permeability transition

Producción CientíficaMitochondrial Ca2+ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca2+] ([Ca2+]M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca2+]M from increasing above 2–3 M. Instead, using low-Ca2+-affinity aequorin probes, we have measured [Ca2+]M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca2+]M to levels of 0.5–1mM was actually observed at any phosphate concentration (0–10mM) during continuous perfusion of 3.5–100 MCa2+-buffers. In spite of this high and maintained (>10 min) [Ca2+]M, mitochondria retained functionality and the [Ca2+]M drop induced by a protonophore was fully reversible. In addition, this high [Ca2+]M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca2+]M. In conclusion [Ca2+]M levels of 0.5–1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators

A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes

Producción CientíficaSecretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2- EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH3 cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques

Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion

Producción CientíficaActivation of calcium-ion (Ca2+) channels on the plasma membrane and on intracellular Ca2+ stores, such as the endoplasmic reticulum, generates local transient increases in the cytosolic Ca2+ concentration that induce Ca2+ uptake by neighbouring mitochondria. Here, by using mitochondrially targeted aequorin proteins with different Ca2+ affinities, we show that half of the chromaffin-cell mitochondria exhibit surprisingly rapid millimolar Ca2+ transients upon stimulation of cells with acetylcholine, caffeine or high concentrations of potassium ions. Our results show a tight functional coupling of voltage-dependent Ca2+ channels on the plasma membrane, ryanodine receptors on the endoplasmic reticulum, and mitochondria. Cell stimulation generates localized Ca2+ transients, with Ca2+ concentrations above 20–40 mM, at these functional units. Protonophores abolish mitochondrial Ca2+ uptake and increase stimulated secretion of catecholamines by three- to fivefold. These results indicate that mitochondria modulate secretion by controlling the availability of Ca2+ for exocytosis.2015-03-1

Ca2+ Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

Producción CientíficaWe have investigated the dynamics of the free [Ca2+] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca2+-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2+] ([Ca2+]SG] was around 20–40 lM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca2+ pump inhibitor benzohydroquinone induced a rapid release of Ca2+ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2+ pumps is essential to maintain the steady-state [Ca2+]SG. Both inositol 1,4, 5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+ release from the granules, suggesting the presence of InsP3 and ryanodine receptors in the granules. The response to high-K+ depolarization was different in both cell types, a decrease in [Ca2+]SG in PC12 cells and an increase in [Ca2+]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca2+]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2+ through SERCA-type Ca2+ pumps and can release it through InsP3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca2+ may be released during cell stimulation and contribute to secretion