Mitochondrial Calcium Dynamics Research Articles

TBC1D15-regulated mitochondria–lysosome membrane contact exerts neuroprotective effects by alleviating mitochondrial calcium overload in seizure

Mitochondrial calcium overload plays an important role in the neurological insults in seizure. The Rab7 GTPase-activating protein, Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15), is involved in the regulation of mitochondrial calcium dynamics by mediating mitochondria–lysosome membrane contact. However, whether TBC1D15-regulated mitochondria–lysosome membrane contact and mitochondrial calcium participate in neuronal injury in seizure is unclear. We aimed to investigate the effect of TBC1D15-regulated mitochondria–lysosome membrane contact on epileptiform discharge-induced neuronal damage and further explore the underlying mechanism. Lentiviral vectors (Lv) infection and stereotaxic adeno-associated virus (AAV) injection were used to regulate TBC1D15 expression before establishing in vitro epileptiform discharge and in vivo status epilepticus (SE) models. TBC1D15’s effect on inter-organellar interactions, mitochondrial calcium levels and neuronal injury in seizure was evaluated. The results showed that abnormalities in mitochondria–lysosome membrane contact, mitochondrial calcium overload, mitochondrial dysfunction, increased levels of reactive oxygen species, and prominent neuronal damage were partly relieved by TBC1D15 overexpression, whereas TBC1D15 knockdown markedly deteriorated these phenomena. Further examination revealed that epileptiform discharge-induced mitochondrial calcium overload in primary hippocampal neurons was closely associated with abnormal mitochondria–lysosome membrane contact. This study highlights the crucial role played by TBC1D15-regulated mitochondria–lysosome membrane contact in epileptiform discharge-induced neuronal injury by alleviating mitochondrial calcium overload.

454 A Human 3D Model of Duchenne Muscular Dystrophy Cardiomyopathy to Investigate Calcium Regulation and Mitochondrial Dysfunction

OBJECTIVES/GOALS: We will use control- and DMD-engineered heart tissues to better model and investigate DMD cardiomyopathy. We will primarily assess cardiac calcium handling, mitochondrial function, and mitochondrial calcium handling, as calcium regulation and mitochondrial function are known to be affected in DMD. METHODS/STUDY POPULATION: We will use patient-derived stem cells, differentiated into cardiomyocytes in bioprinted 3D heart tissue muscle chambers to better model DMD cardiomyopathy. We will look at calcium handling and general mitochondrial function, as well as mitochondrial calcium handling, using a novel multifunctional genetic probe I previously developed allowing for simultaneous observation of cytosolic and mitochondrial calcium in real time. Optical mapping will also be used for tissue-level analysis. We will establish the functional differences at baseline, and then progress heart failure in the tissues to see how the abnormalities seen in the DMD tissues may get worse. Finally, we will investigate the effects of early restoration of dystrophin function on the effects of DMD cardiomyopathy development. RESULTS/ANTICIPATED RESULTS: We anticipate that DMD tissues will show more irregular/abnormal calcium handling, as seen in 2D hiPSC-CMs, as well as disruptions to mitochondrial function and ultrastructural development, as well as a decreased synchronization between cytosolic and mitochondrial calcium dynamics. We anticipate that these abnormalities will be exacerbated as the disease state progresses, but at least partially ameliorated with the restoration of dystrophin function. DISCUSSION/SIGNIFICANCE: DMD is a fatal disease with no known cure. Patients develop heart failure in their teens and die in their 20s, so any new insight that may prolong life and improve quality of life for patients is drastically needed. This would be the most accurate preclinical model of DMD cardiomyopathy to date and would investigate yet-untapped aspects of the disease state.

Mild Traumatic Brain Injury Induces Mitochondrial Calcium Overload and Triggers the Upregulation of NCLX in the Hippocampus

Traumatic brain injury (TBI) is brain damage due to external forces. Mild TBI (mTBI) is the most common form of TBI, and repeated mTBI is a risk factor for developing neurodegenerative diseases. Several mechanisms of neuronal damage have been described in the cortex and hippocampus, including mitochondrial dysfunction. However, up until now, there have been no studies evaluating mitochondrial calcium dynamics. Here, we evaluated mitochondrial calcium dynamics in an mTBI model in mice using isolated hippocampal mitochondria for biochemical studies. We observed that 24 h after mTBI, there is a decrease in mitochondrial membrane potential and an increase in basal matrix calcium levels. These findings are accompanied by increased mitochondrial calcium efflux and no changes in mitochondrial calcium uptake. We also observed an increase in NCLX protein levels and calcium retention capacity. Our results suggest that under mTBI, the hippocampal cells respond by incrementing NCLX levels to restore mitochondrial function.

Dinuclear nitrido-bridged osmium complexes inhibit the mitochondrial calcium uniporter and protect cortical neurons against lethal oxygen-glucose deprivation.

Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os2(μ-N)(NH3)8Cl2]3+) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os2(μ-N)(NH3)8(OH2)2]5+) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 (k obs = 4.92 × 10-3 s-1), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower (k obs = 1.63 × 10-5 s-1) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC50 for MCU inhibition = 103 nM; Os245' IC50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg-1), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors.

Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

ADP and ATP Promote Mitochondrial Calcium Capacity Through Distinct Roles

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state, as such these analytes have become prominent factors in metabolism research. Beyond mitochondrial oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to understanding the role of each individual adenylate in the realm of mitochondrial function. Mitochondrial Adenylate Kinase activity dismutates ADP into ATP and AMP via a reversible reaction. Through the use of an Adenylate Kinase inhibitor we have noted that this enzymatic activity drives stark differences in the adenylate profile of isolated mitochondria when measured by 31-P magnetic resonance spectroscopy. These differences are further noted when the Adenylate Kinase reverse reaction is blocked by calcium. Additionally, by utilizing endogenous mitochondrial Adenylate Kinase activity, we identified distinct roles for ADP and ATP in promoting mitochondrial calcium capacity. These roles are such that ADP provides a defense against permeability transition, while ATP contributes to calcium storage through the formation of dense calcium phosphate granules. Our findings showcase how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics were all affected by Adenylate Kinase activity. Of particular note, it was revealed that ATP is required for a particularly dense form of mitochondrial amorphous calcium phosphate, which in the future may further entangle mitochondria with physiological calcium events.

Adenylate kinase derived ATP shapes respiration and calcium storage of isolated mitochondria

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state and thus a prominent analyte in metabolism research. Beyond adenylate interconversion via oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to successfully predict their abundance in an experimental system and to correctly interpret many aspects of mitochondrial function.We showcase here how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Parameters affected include cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics. Study of the latter revealed that the presence of ATP is required for mitochondrial calcium to be shaped into a particularly dense form of mitochondrial amorphous calcium phosphate.

Sleep/wake calcium dynamics, respiratory function, and ROS production in cardiac mitochondria

IntroductionIncidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events. ObjectivesHere we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods. MethodsWe assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points. ResultsOur results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration. ConclusionsTaken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

Cyclophilin D counterbalances mitochondrial calcium uniporter-mediated brain mitochondrial calcium uptake

Mitochondria play an essential role in maintaining intraneuronal calcium homeostasis. Mitochondrial calcium uniporter (MCU) is a determined major brain mitochondrial calcium entry pathway. Activated MCU-mediated mitochondrial calcium overloading has been linked with brain mitochondrial pathology in disease conditions. Cyclophilin D (CypD)-mediated mitochondrial permeability transition (mPT) favors mitochondrial calcium efflux. The physiological function of CypD-mediated mPT has received increasing recognition. However, the regulatory role of CypD-mediated mPT in brain mitochondrial calcium dynamics in response to mitochondrial calcium accumulation via MCU has not been comprehensively studied. Here, by adopting purified brain mitochondria, we have determined an effect of CypD and CypD-mediated mPT against mitochondrial calcium overloading. In addition, blockade of CypD pharmaceutically or genetically blunts brain mitochondrial MCU’s sensitivity to its inhibitor. Therefore, our findings suggest that CypD-mediated mPT is a mitochondrial compensatory response to MCU-mediated excess mitochondrial calcium accumulation. Moreover, CypD may potentially modulate MCU function in calcium-stressed mitochondria.

Cardiolipin is essential for early embryonic viability and mitochondrial integrity of neurons in mammals

Cardiolipin(CL) is a hallmark phospholipid of mitochondria and plays a significant role in maintaining the mitochondrial structure and functions. Despite the physiological importance of CL, mutant organisms, yeast, Arabidopsis, C elegans, and Drosophila, which lack CL synthase (Crls1) gene and consequently are deprived of CL, are viable. Here we report conditional Crls1-deficient mice using targeted insertion of loxP sequences flanking the functional domain of CRLS1 enzyme. Homozygous null mutant mice exhibited early embryonic lethality at the peri-implantation stage. We generated neuron-specific Crls1 knockout (cKO) mice by crossing with Camk2α-Cre mice. Neuronal loss and gliosis were gradually manifested in the forebrains, where CL levels were significantly decreased. In the surviving neurons, malformed mitochondria with bubble-like or onion-like inner membrane structures were observed. We showed decreased supercomplex assembly and reduced enzymatic activities of electron transport chain complexes in the forebrain of cKO mice, resulting in affected mitochondrial calcium dynamics, a slower rate of Ca2+ uptake and a smaller calcium retention capacity. These observations clearly demonstrate indispensable roles of CL as well as of Crls1 gene in mammals.

Abstract 545: A Multifunctional Genetic Probe for Investigating Mitochondrial Calcium Dynamics in Cardiac Cells

Introduction: Calcium cycling plays a critical role in regulating function of cardiomyocytes (CMs) under both physiological and pathological conditions. Among their many important functions, mitochondria are known to involve in calcium handling. However, little is known about the calcium dynamics of human stem cell-derived cardiomyocytes (hiPSC-CM) and what little is known shows much greater variability than what is seen in adult cardiomyocytes. Methods: We produced adenovirus encoding the red Ca 2+ indicator RCaMP prepended with a mitochondrial targeting sequence fused to the green Ca 2+ indicator GCaMP with a cleavable linker. We then used this virus to induce expression of this calcium probe in CMs & recorded calcium activity with a confocal microscope. Results & Discussion: Mitochondrial targeting was confirmed by confocal imaging showing GCaMP (green), mRCaMP (red), and costaining with Mitotracker Deep Red (Magenta) (Figure 1A, 1B). Probe functionality was validated by observing beat-by-beat calcium dynamics in paced neonatal rat CMs with and without blocking mitochondrial calcium uptake with the mitochondrial calcium uniport inhibitor Ru360. Recording GCaMP and mRCaMP dynamics in adult CMs (Figure 1C) showed synchronous beat-by-beat calcium activity in the cytosol and mitochondria. However, iPSC-CMs (Figure 1D) showed variable mitochondrial activity, with some synchronous mitochondria and some showing inverse activity. Conclusions: hiPSC-CMs and adult CMs show differences in beat-by-beat mitochondrial calcium dynamics, offering a potential investigatory target for improving the therapeutic effect of stem cell-derived heart therapies.

Restriction of mitochondrial calcium overload by mcu inactivation renders a neuroprotective effect in zebrafish models of Parkinson's disease.

ABSTRACTThe loss of dopaminergic neurons (DA) is a pathological hallmark of sporadic and familial forms of Parkinson's disease (PD). We have previously shown that inhibiting mitochondrial calcium uniporter (mcu) using morpholinos can rescue DA neurons in the PTEN-induced putative kinase 1 (pink1)−/− zebrafish model of PD. In this article, we show results from our studies in mcu knockout zebrafish, which was generated using the CRISPR/Cas9 system. Functional assays confirmed impaired mitochondrial calcium influx in mcu−/− zebrafish. We also used in vivo calcium imaging and fluorescent assays in purified mitochondria to investigate mitochondrial calcium dynamics in a pink1−/− zebrafish model of PD. Mitochondrial morphology was evaluated in DA neurons and muscle fibers using immunolabeling and transgenic lines, respectively. We observed diminished mitochondrial area in DA neurons of pink1−/− zebrafish, while deletion of mcu restored mitochondrial area. In contrast, the mitochondrial volume in muscle fibers was not restored after inactivation of mcu in pink1−/− zebrafish. Mitochondrial calcium overload coupled with depolarization of mitochondrial membrane potential leads to mitochondrial dysfunction in the pink1−/− zebrafish model of PD. We used in situ hybridization and immunohistochemical labeling of DA neurons to evaluate the effect of mcu deletion on DA neuronal clusters in the ventral telencephalon of zebrafish brain. We show that DA neurons are rescued after deletion of mcu in pink1−/− and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) zebrafish model of PD. Thus, inactivation of mcu is protective in both genetic and chemical models of PD. Our data reveal that regulating mcu function could be an effective therapeutic target in PD pathology.

Cyclophilin D regulates neuronal activity-induced filopodiagenesis by fine-tuning dendritic mitochondrial calcium dynamics.

Recent studies have highlighted the role of mitochondria in dendritic protrusion growth and plasticity. However, the detailed mechanisms that mitochondria regulate dendritic filopodia morphogenesis remain elusive. Cyclophilin D (CypD, gene name: Ppif) controls the opening of mitochondrial permeability transition pore. Although the pathological relevance of CypD has been intensively investigated, little is known about its physiological function in neurons. Here, we have found that genetic depletion of or pharmaceutical inhibition of CypD blunts the outgrowth of dendritic filopodia in response to KCl-stimulated neuronal depolarization. Further cell biological studies suggest that such inhibitory effect of CypD loss-of-function is closely associated with compromised flexibility of dendritic mitochondrial calcium regulation during neuronal depolarization, as well as the resultant changes in intradendritic calcium homeostasis, calcium signaling activation, dendritic mitochondrial motility and redistribution. Interestingly, loss of CypD attenuates oxidative stress-induced mitochondrial calcium perturbations and dendritic protrusion injury. Therefore, our study has revealed the physiological function of CypD in dendritic plasticity by acting as a fine-tuner of mitochondrial calcium homeostasis. Moreover, CypD plays distinct roles in neuronal physiology and pathology. Cover Image for this issue: doi: 10.1111/jnc.14189.

Measurement and Analysis of the Mitochondrial Calcium Sequestration and Effects on the Buffering System

Calcium is a key signaling molecule found to regulate many cellular processes, including but not limited to, cellular physiology, bioenergetics, transcriptional events, signal transduction, and reactive oxygen species (ROS) production. While mitochondrial calcium dynamics have been extensively studied, it is yet unclear how calcium is sequestered and buffered in a quantitatively and mechanistic manner. Mitochondrial‐calcium homeostasis plays a crucial role in cell fate by a tightly‐regulated buffering system that maintains sub‐lethal levels of free calcium. If the calcium buffering capacity is exceeded mitochondrial dysfunction can result in excessive ROS production, initiating cell death signaling pathways, and catastrophic energy failure. In this study, we quantitatively characterized the mitochondrial calcium buffering power under a variety of experimental conditions and two calcium loading protocols. This was done with isolated cardiac mitochondria from guinea pigs using calcium‐sensitive molecular dyes. The mitochondrial calcium buffering power was quantified by monitoring the calcium flux from both outside and inside the mitochondria. Here, we report the changes in calcium buffering power caused by oxidative stress and under conditions that promote calcium uptake and retention. Furthermore, we demonstrate that the buffering power is also affected by the way calcium is loaded into the mitochondria. These data provide further insights on the mitochondrial calcium loading capacity and raises the question as to what are the components that regulate free calcium in the system.Support or Funding InformationThis research is supported by the National Heart, Lung, and Blood Institute (R00‐HL121160).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New insights into the role of mitochondrial calcium homeostasis in cell migration

Mitochondria are dynamic organelles involved in numerous physiological functions. Beyond their function in ATP production, mitochondria regulate cell death, reactive oxygen species (ROS) generation, immunity and metabolism. Mitochondria also play a key role in the buffering of cytosolic calcium, and calcium transported into the matrix regulates mitochondrial metabolism. Recently, the identification of the mitochondrial calcium uniporter (MCU) and associated regulators has allowed the characterization of new physiological roles for calcium in both mitochondrial and cellular homeostasis. Indeed, recent work has highlighted the importance of mitochondrial calcium homeostasis in regulating cell migration. Cell migration is a property common to all metazoans and is critical to embryogenesis, cancer progression, wound-healing and immune surveillance. Previous work has established that cytoplasmic calcium is a key regulator of cell migration, as oscillations in cytosolic calcium activate cytoskeletal remodelling, actin contraction and focal adhesion (FA) turnover necessary for cell movement. Recent work using animal models and in cellulo experiments to genetically modulate MCU and partners have shed new light on the role of mitochondrial calcium dynamics in cytoskeletal remodelling through the modulation of ATP and ROS production, as well as intracellular calcium signalling. This review focuses on MCU and its regulators in cell migration during physiological and pathophysiological processes including development and cancer. We also present hypotheses to explain the molecular mechanisms by which MCU may regulate mitochondrial dynamics and motility to drive cell migration.

Fus1/Tusc2 Is a Novel Regulator of Mitochondrial Calcium Handling, Ca2+-Coupled Mitochondrial Processes, and Ca2+-Dependent NFAT and NF-κB Pathways in CD4+T Cells

Fus1 has been established as mitochondrial tumor suppressor, immunomodulator, and antioxidant protein, but molecular mechanism of these activities remained to be identified. Based on putative calcium-binding and myristoyl-binding domains that we identified in Fus1, we explored our hypothesis that Fus1 regulates mitochondrial calcium handling and calcium-coupled processes. Fus1 loss resulted in reduced rate of mitochondrial calcium uptake in calcium-loaded epithelial cells, splenocytes, and activated CD4(+) T cells. The reduced rate of mitochondrial calcium uptake in Fus1-deficient cells correlated with cytosolic calcium increase and dysregulation of calcium-coupled mitochondrial parameters, such as reactive oxygen species production, ΔμH(+), mitochondrial permeability transition pore opening, and GSH content. Inhibition of calcium efflux via mitochondria, Na(+)/Ca(2+) exchanger significantly improved the mitochondrial calcium uptake in Fus1(-/-) cells. Ex vivo analysis of activated CD4(+) T cells showed Fus1-dependent changes in calcium-regulated processes, such as surface expression of CD4 and PD1/PD-L1, proliferation, and Th polarization. Fus1(-/-) T cells showed increased basal expression of calcium-dependent NF-κB and NFAT targets but were unable to fully activate these pathways after stimulation. Our results establish Fus1 as one of the few identified regulators of mitochondrial calcium handling. Our data support the idea that alterations in mitochondrial calcium dynamics could lead to the disruption of metabolic coupling in mitochondria that, in turn, may result in multiple cellular and systemic abnormalities. Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters.

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.

The worm in the fruit of the mitochondrial DNA tree

The worm in the fruit of the mitochondrial DNA tree