Try a new search

Format these results:

Searched for:


Total Results:


Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition

Neginskaya, Maria A; Morris, Sally E; Pavlov, Evgeny V
The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.
PMID: 37566029
ISSN: 2073-4409
CID: 5599212

Mitochondrial Permeability Transition [Editorial]

Bernardi, Paolo; Pavlov, Evgeny
The mitochondrial permeability transition (PT) is a phenomenon that can be broadly defined as an increase in the permeability of the mitochondrial inner membrane [...].
PMID: 36497124
ISSN: 2073-4409
CID: 5378942

Both ANT and ATPase are essential for mitochondrial permeability transition but not depolarization

Neginskaya, M A; Morris, S E; Pavlov, E V
An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.
PMID: 36388971
ISSN: 2589-0042
CID: 5388592

Overexpression of RCAN1, a Gene on Human Chromosome 21, Alters Cell Redox and Mitochondrial Function in Enamel Cells

Li, Yi; Costiniti, Veronica; Souza Bomfim, Guilherme H; Neginskaya, Maria; Son, Ga-Yeon; Rothermel, Beverly; Pavlov, Evgeny; Lacruz, Rodrigo S
The regulator of calcineurin (RCAN1) has been implicated in the pathogenesis of Down syndrome (DS). Individuals with DS show dental abnormalities for unknown reasons, and RCAN1 levels have been found to be elevated in several tissues of DS patients. A previous microarray analysis comparing cells of the two main formative stages of dental enamel, secretory and maturation, showed a significant increase in RCAN1 expression in the latter. Because the function of RCAN1 during enamel formation is unknown, there is no mechanistic evidence linking RCAN1 with the dental anomalies in individuals with DS. We investigated the role of RCAN1 in enamel by overexpressing RCAN1 in the ameloblast cell line LS8 (LS8+RCAN1). We first confirmed that RCAN1 is highly expressed in maturation stage ameloblasts by qRT-PCR and used immunofluorescence to show its localization in enamel-forming ameloblasts. We then analyzed cell redox and mitochondrial bioenergetics in LS8+RCAN1 cells because RCAN1 is known to impact these processes. We show that LS8+RCAN1 cells have increased reactive oxygen species (ROS) and decreased mitochondrial bioenergetics without changes in the expression of the complexes of the electron transport chain, or in NADH levels. However, LS8+RCAN1 cells showed elevated mitochondrial Ca2+ uptake and decreased expression of several enamel genes essential for enamel formation. These results provide insight into the role of RCAN1 in enamel and suggest that increased RCAN1 levels in the ameloblasts of individuals with DS may impact enamel formation by altering both the redox environment and mitochondrial function, as well as decreasing the expression of enamel-specific genes.
PMID: 36429004
ISSN: 2073-4409
CID: 5374552

Mitochondria modulate ameloblast Ca2+ signaling

Costiniti, Veronica; Bomfim, Guilherme H S; Neginskaya, Maria; Son, Ga-Yeon; Mitaishvili, Erna; Giacomello, Marta; Pavlov, Evgeny; Lacruz, Rodrigo S
The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.
PMID: 35084775
ISSN: 1530-6860
CID: 5157132

Correction: Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation

Angelova, Plamena R; Choi, Minee L; Berezhnov, Alexey V; Horrocks, Mathew H; Hughes, Craig D; De, Suman; Rodrigues, Margarida; Yapom, Ratsuda; Little, Daniel; Dolt, Karamjit S; Kunath, Tilo; Devine, Michael J; Gissen, Paul; Shchepinov, Mikhail S; Sylantyev, Sergiy; Pavlov, Evgeny V; Klenerman, David; Abramov, Andrey Y; Gandhi, Sonia
PMID: 33041329
ISSN: 1476-5403
CID: 4890182

Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis

Solesio, Maria E; Xie, Lihan; McIntyre, Brendan; Ellenberger, Mathew; Mitaishvili, Erna; Bhadra-Lobo, Siddharth; Bettcher, Lisa F; Bazil, Jason N; Raftery, Daniel; Jakob, Ursula; Pavlov, Evgeny V
Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical to those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.
PMID: 33843973
ISSN: 1470-8728
CID: 4845732

C subunit of the ATP synthase is an amyloidogenic calcium dependent channel-forming peptide with possible implications in mitochondrial permeability transition

Amodeo, Giuseppe Federico; Lee, Brenda Yasie; Krilyuk, Natalya; Filice, Carina Teresa; Valyuk, Denis; Otzen, Daniel Erik; Noskov, Sergey; Leonenko, Zoya; Pavlov, Evgeny V
The c subunit is an inner mitochondrial membrane (IMM) protein encoded by three nuclear genes. Best known as an integral part of the F0 complex of the ATP synthase, the c subunit is also present in other cytoplasmic compartments in ceroid lipofuscinoses. Under physiological conditions, this 75 residue-long peptide folds into an α-helical hairpin and forms oligomers spanning the lipid bilayer. In addition to its physiological role, the c subunit has been proposed as a key participant in stress-induced IMM permeabilization by the mechanism of calcium-induced permeability transition. However, the molecular mechanism of the c subunit participation in IMM permeabilization is not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy and black lipid membrane methods to gain insights into the structural and functional properties of unmodified c subunit protein that might make it relevant to mitochondrial toxicity. We discovered that c subunit is an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a Ca2+-dependent manner. C subunit oligomers exhibited ion channel activity in lipid membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic properties and are driven by mechanisms similar to those of neurodegenerative polypeptides such as Aβ and α-synuclein.
PMID: 33888826
ISSN: 2045-2322
CID: 4847492

Tetra-arylborate lipophilic anions as targeting groups

Gaddale Devanna, Kishore K; Gawel, Justyna M; Prime, Tracy A; Cvetko, Filip; Benincá, Cristiane; Caldwell, Stuart T; Negoda, Alexander; Harrison, Andrew; James, Andrew M; Pavlov, Evgeny V; Murphy, Michael P; Hartley, Richard C
Tetraphenylborate (TPB) anions traverse membranes but are excluded from mitochondria by the membrane potential (Δψ). TPB-conjugates also distributed across membranes in response to Δψ, but surprisingly, they rapidly entered cells. They accumulated within lysosomes following endocystosis. This pH-independent targeting of lysosomes makes possible new classes of probe and bioactive molecules.
PMID: 33634803
ISSN: 1364-548x
CID: 4795092

The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function

Strubbe-Rivera, Jasiel O; Schrad, Jason R; Pavlov, Evgeny V; Conway, James F; Parent, Kristin N; Bazil, Jason N
Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves cristae despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.
PMID: 33441863
ISSN: 2045-2322
CID: 4747052