Transcriptional regulation in the absence of inositol trisphosphate receptor calcium signaling.

The activation of IP receptor (IPR) Ca channels generates agonist-mediated Ca signals that are critical for the regulation of a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IPR isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca dependent transcription factors (NFAT, CREB and AP-1) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies.

MICU1 controls the sensitivity of the mitochondrial Ca uniporter to activators and inhibitors.

Mitochondrial Ca homeostasis loses its control in many diseases and might provide therapeutic targets. Mitochondrial Ca uptake is mediated by the uniporter channel (mtCU), formed by MCU and is regulated by the Ca-sensing gatekeeper, MICU1, which shows tissue-specific stoichiometry. An important gap in knowledge is the molecular mechanism of the mtCU activators and inhibitors.

MICU1 regulates mitochondrial cristae structure and function independently of the mitochondrial Ca uniporter channel.

MICU1 is a calcium (Ca)-binding protein that regulates the mitochondrial Ca uniporter channel complex (mtCU) and mitochondrial Ca uptake. knockout mice display disorganized mitochondrial architecture, a phenotype that is distinct from that of mice with deficiencies in other mtCU subunits and, thus, is likely not explained by changes in mitochondrial matrix Ca content. Using proteomic and cellular imaging techniques, we found that MICU1 localized to the mitochondrial contact site and cristae organizing system (MICOS) and directly interacted with the MICOS components MIC60 and CHCHD2 independently of the mtCU.

Functional determination of calcium-binding sites required for the activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IPRs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca) signals in response to various external cues. Notably, IPR channel activity is determined by several obligatory factors, including IP, Ca, and ATP. The critical basic amino acid residues in the N-terminal IP-binding core (IBC) region that facilitate IP binding are well characterized.

Fluorescence imaging detection of nanodomain redox signaling events at organellar contacts.

This protocol describes how to visualize, detect, and analyze redox signals (oxidative bursts) at the ER-mitochondrial interface. It uses drug-inducible crosslinking to target the genetically encoded glutathione redox sensor Grx1roGFP2 to organellar contact sites to measure local redox changes associated with transient depolarizations of the mitochondrial membrane potential (flickers). The strategy allows imaging of the oxidized to reduced glutathione ratio (GSSG:GSH) in subcellular regions below the diffraction limit with good temporal resolution and minimum phototoxicity.

Metabolic adaptation to the chronic loss of Ca signaling induced by KO of IP receptors or the mitochondrial Ca uniporter.

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IPRs) and the mitochondrial Ca uniporter (MCU) are key proteins that regulate intracellular Ca concentration. Mitochondrial Ca accumulation activates Ca-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells.

Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IPRs), we show that Ca channels directly sense oxidative bursts and respond with Ca transients adjacent to active mitochondria.

IP receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer.

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts.

Enhanced Monarchy Butterfly Optimization Technique for effective breast cancer diagnosis.

Breast cancer is the biggest curse for the women society in the world since the survival factor of the infected patients is ensured only when it is detected at the early localized stage. The majority of the intelligent schemes proposed for detecting the breast cancer relies on the human skill that helps in trustworthy determination of essential pattern that confirms the existence of the infected cancer cells for deciding upon the course of treatment. Further, most of the research works contributed in the literature for detecting breast cancer necessitates huge time and laborinvolved that increases the time of diagnosis.

Redox regulation of ER and mitochondrial Ca signaling in cell survival and death.

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca signaling at the ER/mitochondrial interface - notably, inositol trisphosphate receptors (IPRs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death.

MICU1 Interacts with the D-Ring of the MCU Pore to Control Its Ca Flux and Sensitivity to Ru360.

Proper control of the mitochondrial Ca uniporter's pore (MCU) is required to allow Ca-dependent activation of oxidative metabolism and to avoid mitochondrial Ca overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter.

Redox regulation of type-I inositol trisphosphate receptors in intact mammalian cells.

A sensitization of inositol 1,4,5-trisphosphate receptor (IPR)-mediated Ca release is associated with oxidative stress in multiple cell types. These effects are thought to be mediated by alterations in the redox state of critical thiols in the IPR, but this has not been directly demonstrated in intact cells. Here, we utilized a combination of gel-shift assays with MPEG-maleimides and LC-MS/MS to monitor the redox state of recombinant IPR1 expressed in HEK293 cells.

MSTO1 is a cytoplasmic pro-mitochondrial fusion protein, whose mutation induces myopathy and ataxia in humans.

The protein MSTO1 has been localized to mitochondria and linked to mitochondrial morphology, but its specific role has remained unclear. We identified a c.22G > A (p.

Mitochondrial Ca Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established.

Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle.

Control of myocardial energetics by Ca signal propagation to the mitochondrial matrix includes local Ca delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca transfer.

Subcellular ROS imaging methods: Relevance for the study of calcium signaling.

Recent advances in genetically encoded fluorescent probes have dramatically increased the toolkit available for imaging the intracellular environment. Perhaps the biggest improvements have been made in sensing specific reactive oxygen species (ROS) and redox changes under physiological conditions. The new generation of probes may be targeted to a wide range of subcellular environments.

Hormone-induced calcium oscillations depend on cross-coupling with inositol 1,4,5-trisphosphate oscillations.

Receptor-mediated oscillations in cytosolic Ca(2+) concentration ([Ca(2+)]i) could originate either directly from an autonomous Ca(2+) feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca(2+) feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca(2+) oscillations or could just be a secondary response to the [Ca(2+)]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range.

Isoform- and species-specific control of inositol 1,4,5-trisphosphate (IP3) receptors by reactive oxygen species.

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca(2+)] ([Ca(2+)]c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS.

Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events.

Identification of functionally critical residues in the channel domain of inositol trisphosphate receptors.

We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP(3)R) channel function. The residues selected were highly conserved in all three IP(3)R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis.

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear.

Calcium-dependent conformational changes in inositol trisphosphate receptors.

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca(2+)-induced conformational changes of IP(3)Rs in their native membrane environment. We found that Ca(2+) decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca(2+) induces a conformational change in the IP(3)R that allows trypsin to cleave the C-terminal epitope.

Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.

Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors.