Ratiometric Pericam Research Articles

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca2+ uptake exerts dual effects on mitochondria. Ca2+ accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca2+ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca2+ ([Ca2+]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca2+]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca2+ indicators with a 20-fold difference in Ca2+ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca2+ indicator (D4cpv) calibrated against known Ca2+ levels. Matrix pH (pHm) and ΔΨm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca2+]m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca2+]m coincided with a rapid elevation in pHm and was followed by an after-stimulus ΔΨm hyperpolarization. However, pHm decreased and no ΔΨm hyperpolarization was observed in response to lower levels of [Ca2+]m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca2+]m are required to stimulate presynaptic mitochondrial energy metabolism.

Selectivity of Action of Pregabalin on Ca2+ Channels but Not on Fusion Pore, Exocytotic Machinery, or Mitochondria in Chromaffin Cells of the Adrenal Gland

The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.

Understanding calcium homeostasis in postnatal gonadotropin-releasing hormone neurons using cell-specific Pericam transgenics

The gonadotropin-releasing hormone (GnRH) neurons are the key output cells of a complex neuronal network controlling fertility in mammals. To examine calcium homeostasis in postnatal GnRH neurons, we generated a transgenic mouse line in which the genetically encodable calcium indicator ratiometric Pericam (rPericam) was targeted to the GnRH neurons. This mouse model enabled real-time imaging of calcium concentrations in GnRH neurons in the acute brain slice preparation. Investigations in GnRH-rPericam mice revealed that GnRH neurons exhibited spontaneous, long-duration (∼8s) calcium transients. Dual electrical-calcium recordings revealed that the calcium transients were correlated perfectly with burst firing in GnRH neurons and that calcium transients in GnRH neurons regulated two calcium-activated potassium channels that, in turn, determined burst firing dynamics in these cells. Curiously, the occurrence of calcium transients in GnRH neurons across puberty or through the estrous cycle did not correlate well with the assumption that GnRH neuron burst firing was contributory to changing patterns of pulsatile GnRH release at these times. The GnRH-rPericam mouse was also valuable in determining differential mechanisms of GABA and glutamate control of calcium levels in GnRH neurons as well as effects of G-protein-coupled receptors for GnRH and kisspeptin. The simultaneous measurement of calcium levels in multiple GnRH neurons was hampered by variable rPericam fluorescence in different GnRH neurons. Nevertheless, in the multiple recordings that were achieved no evidence was found for synchronous calcium transients. Together, these observations show the great utility of transgenic targeting strategies for investigating the roles of calcium with specified neuronal cell types.

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Superoxide Flashes in Mouse Skeletal Muscle Are Produced by Discrete Arrays of Active Mitochondria Operating Coherently

Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.

Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin

The dynamics of mitochondrial [Ca2+] ([Ca2+]M) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca2+]M are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods. We have made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF, and two Ca2+-sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca2+ changes. Instead, fluorescent dyes failed to follow Ca2+ changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca2+-dependent inhibition of mitochondrial Ca2+ uptake, but data obtained with aequorin or pericam under the same conditions did not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced changes in mitochondrial morphology and membrane potential, which were small and reversible at low concentrations (1–2μM), but produced large and prolonged damage at higher concentrations. In addition, cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology after light excitation. Our results suggest that [Ca2+]M data obtained with these dyes should be taken with care.

Mitochondrial Ca 2+ signalling in hippocampal neurons

High-resolution fluorescent imaging of mitochondrial-targeted probes was used to examine the ability of mitochondria to decode complex spatial and temporal Ca 2+ signals evoked in synaptically active networks of hippocampal neurons. Green-to-red photoconversion of the mitochondrial-targeted probe, mito-Kaede, demonstrated that mitochondria were present as discrete organelles 2–6 μm in length. Real-time imaging of mitochondrial-targeted ratiometric pericam (2 mtRP) visualised rapid, repetitive, transient mitochondrial Ca 2+ fluxes in response to periods of synaptic activation. Mitochondrial Ca 2+ fluxes within cellular compartments were dependent on the extent of synaptic recruitment, but independent of cross-talk with the endoplasmic reticulum or the presence of an interconnected mitochondrial network. Mitochondria in dendritic regions demonstrated a greater sensitivity to synaptic activation compared with somatic mitochondria. Temporal decoding of synaptic signals was rate-limited by the activity of the mitochondrial Na +/Ca 2+ exchanger. Spatial regulation of mitochondrial Ca 2+ uptake was determined by the magnitude of the cytosolic Ca 2+ rise in each cellular compartment.

Cell Type-Specific Expression of a Genetically Encoded Calcium Indicator Reveals Intrinsic Calcium Oscillations in Adult Gonadotropin-Releasing Hormone Neurons

The gonadotropin-releasing hormone (GnRH) neurons exhibit a unique pattern of episodic activity to control fertility in all mammals. To enable the measurement of intracellular calcium concentration ([Ca2+]i) in adult GnRH neurons in situ, we generated transgenic mice in which the genetically encodable calcium indicator ratiometric Pericam was expressed by approximately 95% of GnRH neurons. Real-time monitoring of [Ca2+]i within adult male GnRH neurons in the acute brain slice revealed that approximately 70% of GnRH neurons exhibited spontaneous, 10-15 s duration [Ca2+]i transients with a mean frequency of 7 per hour. The remaining 30% of GnRH neurons did not exhibit calcium transients nor did a population of non-GnRH cells located within the lateral septum that express Pericam. Pharmacological studies using antagonists to the inositol-1,4,5-trisphosphate receptor (InsP3R) and several calcium channels, demonstrated that [Ca2+]i transients in GnRH neurons were generated by an InsP3R-dependent store-release mechanism and were independent of plasma membrane ligand- or voltage-gated calcium channels. Interestingly, the abolition of action potential-mediated transmission with tetrodotoxin reduced the number of [Ca2+]i transients in GnRH neurons by 50% (p < 0.05), suggesting a modulatory role for synaptic inputs on [Ca2+]i transient frequency. Using a novel transgenic strategy that enables [Ca2+]i to be examined in a specific neuronal phenotype in situ, we provide evidence for spontaneous [Ca2+]i fluctuations in adult GnRH neurons. This represents the initial description of spontaneous [Ca2+]i transients in mature neurons and shows that they arise from an InsP3R-generating mechanism that is further modulated by synaptic inputs.

Identification of Mitochondrial DNA Polymorphisms That Alter Mitochondrial Matrix pH and Intracellular Calcium Dynamics

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.

Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1

Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca(2+) homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca(2+) released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca(2+) entering across plasma membrane channels, because the Ca(2+) ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca(2+) entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by approximately 35%. These observations indicate that close contact to Ca(2+) influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca(2+) propagation within interconnected mitochondria.

Stable Interactions between Mitochondria and Endoplasmic Reticulum Allow Rapid Accumulation of Calcium in a Subpopulation of Mitochondria

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

Intracellular transport of calcium from plasma membrane to mitochondria in adrenal H295R cells: implication for steroidogenesis.

Angiotensin II and extracellular potassium stimulate aldosterone production in adrenal glomerulosa cells by mobilizing the calcium messenger system. This response requires calcium influx across the plasma membrane, followed by calcium uptake into the mitochondria. It has been proposed that calcium is transported to the mitochondria via the lumen of the endoplasmic reticulum, acting as a kind of intracellular calcium pipeline. This hypothesis has been tested in the present study by measuring intramitochondrial calcium variations in H295R cells with a new fluorescent calcium probe, ratiometric pericam. Calyculin A, a protein phosphatase inhibitor, induced the formation of a large cortical layer of actin filaments, removing the peripheral endoplasmic reticulum away from the plasma membrane and thereby physically uncoupling the calcium channels from the pipeline. The mitochondrial calcium response to potassium was markedly reduced after calyculin treatment, but that of AngII was unaffected. Under the same conditions, potassium-stimulated pregnenolone and aldosterone production was significantly reduced, whereas the steroidogenic response to AngII remained unchanged. The inhibitory action of calyculin A on the responses to potassium was not mediated by a modification of the calcium channel activity and was not accompanied by a reduction of the cytosolic calcium response. It therefore appears that, in H295R cells, the organization of the actin cytoskeleton at the cell periphery influences the steroidogenic action of potassium, but not the response to angiotensin II. The response to potassium is proposed to be dependent on the endoplasmic reticulum-mediated transfer of calcium entering through plasma membrane calcium channels to the mitochondria.