Showing papers on "Cellular compartment published in 2020"

Organelle interplay—peroxisome interactions in health and disease

Abstract: Peroxisomes are multifunctional, dynamic, membrane-bound organelles with important functions in cellular lipid metabolism, rendering them essential for human health and development. Important roles for peroxisomes in signaling and the fine-tuning of cellular processes are emerging, which integrate them in a complex network of interacting cellular compartments. Like many other organelles, peroxisomes communicate through membrane contact sites. For example, peroxisomal growth, positioning, and lipid metabolism involves contacts with the endoplasmic reticulum (ER). Here, we discuss the most recent findings on peroxisome-organelle interactions including peroxisome-ER interplay at membrane contacts sites, and functional interplay with mitochondria, lysosomes, and lipid droplets in mammalian cells. We address tether proteins, metabolic cooperation, and the impact of peroxisome interactions on human health and disease.

Vitamin D receptor(s): In the nucleus but also at membranes?

Abstract: The genomic actions of the vitamin D are mediated via its biologically most potent metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) and the transcription factor vitamin D receptor (VDR). Activation of VDR by 1,25(OH)2 D3 leads to change in the expression of more 1000 genes in various human tissues. Based on (epi)genome, transcriptome and crystal structure data the molecular details of this nuclear vitamin D signalling pathway are well understood. Vitamin D is known for its role on calcium homeostasis and bone formation, but it also modulates energy metabolism, innate and adaptive immunity as well as cellular growth, differentiation and apoptosis. The observation of rapid, non-genomic effects of 1,25(OH)2 D3 at cellular membranes and in the cytosol initiated the question, whether there are alternative vitamin D-binding proteins in these cellular compartments. So far, the best candidate is the enzyme PDIA3 (protein disulphide isomerase family A member 3), which is found at various subcellular locations. Furthermore, also VDR seems to play a role in membrane-based responses to vitamin D. In this viewpoint, we will dispute whether these rapid, non-genomic pathways are a meaningful addition to the genome-wide effects of vitamin D.

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells

Abstract: Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2 Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle One-sentence summary SARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection

Lipids in the Physiopathology of Hereditary Spastic Paraplegias.

Abstract: Hereditary spastic paraplegias (HSP) are a group of neurodegenerative diseases sharing spasticity in lower limbs as common symptom. There is a large clinical variability in the presentation of patients, partly underlined by the large genetic heterogeneity, with more than 60 genes responsible for HSP. Despite this large heterogeneity, the proteins with known function are supposed to be involved in a limited number of cellular compartments such as shaping of the endoplasmic reticulum or endolysosomal function. Yet, it is difficult to understand why alteration of such different cellular compartments can lead to degeneration of the axons of cortical motor neurons. A common feature that has emerged over the last decade is the alteration of lipid metabolism in this group of pathologies. This was first revealed by the identification of mutations in genes encoding proteins that have or are supposed to have enzymatic activities on lipid substrates. However, it also appears that mutations in genes affecting endoplasmic reticulum, mitochondria, or endolysosome function can lead to changes in lipid distribution or metabolism. The aim of this review is to discuss the role of lipid metabolism alterations in the physiopathology of HSP, to evaluate how such alterations contribute to neurodegenerative phenotypes, and to understand how this knowledge can help develop therapeutic strategy for HSP.

On the move: redox-dependent protein relocation in plants.

Abstract: Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

β-Amyloid Peptide: the Cell Compartment Multi-faceted Interaction in Alzheimer’s Disease

Abstract: Alzheimer’s disease (AD) is the most widespread form of dementia, characterized by memory loss and reduction of cognitive functions that strongly interfere with normal daily life. Numerous evidences show that aggregates of the amyloid beta peptide, formed by 39 to 42 amino acid residues (Aβ39–43), from soluble small oligomers to large fibrils are characteristic markers of this pathology. However, AD is a complex disease and its neurodegenerative molecular mechanism is not yet fully understood. Growing evidence suggests a link between Aβ polymorphic nature, oligomers and fibrils, and specific mechanisms of neurodegeneration. The Aβ variable nature and its multiplicity of interactions with different proteins and organelles reflect the complexity of this pathology. In this review, we analyze the effects of the interaction between Aβ peptide and different cellular compartments in relation to the different kinds and sizes of amyloid aggregates. In particular, Aβ interaction with different cell structures such as the plasma membrane, mitochondria, lysosomes, nucleus, and endoplasmic reticulum is discussed. Further, we analyze the Aβ peptide ability to modify the structure and function of the target organelle, inducing alteration of its physiological role thus contributing to the pathological event. Dysfunction of cellular components terminating with the activation of the cellular death mechanism and subsequent neurodegeneration is also taken into consideration.

Organellar and Secretory Ribonucleases: Major Players in Plant RNA Homeostasis

Abstract: Organellar and secretory RNases, associated with different cellular compartments, are essential to maintain cellular homeostasis during development and in stress responses.

FOXM1 nuclear transcription factor translocates into mitochondria and inhibits oxidative phosphorylation.

Abstract: Forkhead box M1 (FOXM1), a nuclear transcription factor that activates cell cycle regulatory genes, is highly expressed in a majority of human cancers. The function of FOXM1 independent of nuclear transcription is unknown. In the present study, we found the FOXM1 protein inside the mitochondria. Using site-directed mutagenesis, we generated FOXM1 mutant proteins that localized to distinct cellular compartments, uncoupling the nuclear and mitochondrial functions of FOXM1. Directing FOXM1 into the mitochondria decreased mitochondrial mass, membrane potential, respiration, and electron transport chain (ETC) activity. In mitochondria, the FOXM1 directly bound to and increased the pentatricopeptide repeat domain 1 (PTCD1) protein, a mitochondrial leucine-specific tRNA binding protein that inhibits leucine-rich ETC complexes. Mitochondrial FOXM1 did not change cellular proliferation. Thus, FOXM1 translocates into mitochondria and inhibits mitochondrial respiration by increasing PTCD1. We identify a new paradigm that FOXM1 regulates mitochondrial homeostasis in a process independent of nuclear transcription.

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope.

Abstract: Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria.IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

Stochiometric quantification of the thiol redox proteome of macrophages reveals subcellular compartmentalization and susceptibility to oxidative perturbations.

Abstract: Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. Herein, we report the characterization of the cellular redox landscape in terms of quantitative, site-specific occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4000 cysteine sites were quantified, revealing a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis revealed that the average occupancies of SSG and total oxidation in specific cellular compartments correlate well with the expected redox potentials of respective organelles in macrophages, consistent with the notion of redox compartmentalization. The lowest average occupancies were observed in more reducing organelles such as the mitochondria (non-membrane) and nucleus, while the highest average occupancies were found in more oxidizing organelles such as endoplasmic reticulum (ER) and lysosome. Furthermore, a pattern of subcellular susceptibility to redox changes was observed under oxidative stress induced by exposure to engineered metal oxide nanoparticles. Peroxisome, ER, and mitochondria (membrane) are the organelles which exhibit the most significant redox changes; while mitochondria (non-membrane) and Golgi were observed as the organelles being most resistant to oxidative stress. Finally, it was observed that Cys residues at enzymatic active sites generally had a higher level of occupancy compared to non-active Cys residues within the same proteins, suggesting site occupancy as a potential indicator of protein functional sites. The raw data are available via ProteomeXchange with identifier PXD019913.

Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity.

Abstract: Proteasome inhibition is used therapeutically to induce proteotoxic stress and trigger apoptosis in cancer cells that are highly dependent on the proteasome. As a mechanism of resistance, inhibition of the cellular proteasome induces the synthesis of new, uninhibited proteasomes to restore proteasome activity and relieve proteotoxic stress in the cell, thus evading apoptosis. This evolutionarily conserved compensatory mechanism is referred to as the proteasome-bounce back response and is orchestrated in mammalian cells by nuclear factor erythroid derived 2-related factor 1 (NRF1), a transcription factor and master regulator of proteasome subunit genes. Upon synthesis, NRF1 is cotranslationally inserted into the endoplasmic reticulum (ER), then is rapidly retrotranslocated into the cytosol and degraded by the proteasome. In contrast, during conditions of proteasome inhibition or insufficiency, NRF1 escapes degradation, is proteolytically cleaved by the aspartyl protease DNA damage inducible 1 homolog 2 (DDI2) to its active form, and enters the nucleus as an active transcription factor. Despite these insights, the cellular compartment where the proteolytic processing step occurs remains unclear. Here we further probed this pathway and found that NRF1 can be completely retrotranslocated into the cytosol where it is then cleaved and activated by DDI2. Furthermore, using a triple-negative breast cancer cell line MDA-MB-231, we investigated the therapeutic utility of attenuating DDI2 function. We found that DDI2 depletion attenuated NRF1 activation and potentiated the cytotoxic effects of the proteasome inhibitor carfilzomib. More importantly, expression of a point-mutant of DDI2 that is protease-dead recapitulated these effects. Taken together, our results provide a strong rationale for a combinational therapy that utilizes inhibition of the proteasome and the protease function of DDI2. This approach could expand the repertoire of cancer types that can be successfully treated with proteasome inhibitors in the clinic.

Phosphatidylinositol 4,5-bisphosphate is regenerated by speeding of the PI 4-kinase pathway during long PLC activation.

Abstract: The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

Peroxisomal targeting of a protein phosphatase type 2C via mitochondrial transit

Abstract: Correct intracellular distribution of proteins is critical for the function of eukaryotic cells. Certain proteins are targeted to more than one cellular compartment, e.g. to mitochondria and peroxisomes. The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal mitochondrial presequence followed by a transmembrane domain, and has been detected in the mitochondrial intermembrane space. Here we show mitochondrial transit of Ptc5 to peroxisomes. Translocation of Ptc5 to peroxisomes depended both on the C-terminal peroxisomal targeting signal (PTS1) and N-terminal cleavage by the mitochondrial inner membrane peptidase complex. Indirect targeting of Ptc5 to peroxisomes prevented deleterious effects of its phosphatase activity in the cytosol. Sorting of Ptc5 involves simultaneous interaction with import machineries of both organelles. We identify additional mitochondrial proteins with PTS1, which localize in both organelles and can increase their physical association. Thus, a tug-of-war-like mechanism can influence the interaction and communication of two cellular compartments.

LAM Genes Contribute to Environmental Stress Tolerance but Sensibilize Yeast Cells to Azoles.

Abstract: Lam proteins transport sterols between the membranes of different cellular compartments. In Saccharomyces cerevisiae, the LAM gene family consists of three pairs of paralogs. Because the function of paralogous genes can be redundant, the phenotypes of only a small number of LAM gene deletions have been reported; thus, the role of these genes in yeast physiology is still unclear. Here, we surveyed the phenotypes of double and quadruple deletants of paralogous LAM2(YSP2)/LAM4 and LAM1(YSP1)/LAM3(SIP3) genes that encode proteins localized in the junctions of the plasma membrane and endoplasmic reticulum. The quadruple deletant showed increased sterol content and a strong decrease in ethanol, heat shock and high osmolarity resistance. Surprisingly, the quadruple deletant and LAM2/LAM4 double deletion strain showed increased tolerance to the azole antifungals clotrimazole and miconazole. This effect was not associated with an increased rate of ABC-transporter substrate efflux. Possibly, increased sterol pool in the LAM deletion strains postpones the effect of azoles on cell growth. Alternatively, LAM deletions might alleviate the toxic effect of sterols as Lam proteins can transport toxic sterol biosynthesis intermediates into membrane compartments that are sensitive to these compounds. Our findings reveal novel biological roles of LAM genes in stress tolerance and suggest that mutations in these genes may confer upregulation of a mechanism that provides resistance to azole antifungals in pathogenic fungi.

Mycobacterium tuberculosis Calcium Pump CtpF Modulates the Autophagosome in an mTOR-Dependent Manner

Abstract: Calcium is a very important second messenger, whose concentration in various cellular compartments is under tight regulation. A disturbance in the levels of calcium in these compartments can play havoc in the cell, as it regulates various cellular processes by direct or indirect mechanisms. Here, we have investigated the functional importance of a calcium transporting P2A ATPase, CtpF of Mycobacterium tuberculosis (Mtb) in the pathogen's interaction with the host. Among its uncanny ways of dealing with the host with umpteen strategies for survival and persistence in humans, CtpF is identified as a new player. The levels of ctpF are upregulated in macrophage stresses like hypoxia, high nitric oxide levels and acidic pH. Using confocal microscopy and fluorimetry, we show that CtpF effluxes calcium in macrophages in early stages of Mtb infection. Downregulation of ctpF expression by conditional knockdown resulted in perturbation of host calcium levels and consequent decreased activation of mTOR. We present a mechanism how calcium efflux by the pathogen inhibits mTOR-dependent autophagy and enhances bacterial survival. Our work highlights how Mtb engages its metal efflux pumps to exploit host autophagic process for its proliferation.

A Combinatorial Reporter Set to Visualize the Membrane Contact Sites Between Endoplasmic Reticulum and Other Organelles in Plant Cell.

Abstract: The membrane contact sites (MCSs) enable interorganelle communication by associating organelles at distances of tens of nanometers over extended membrane surfaces and serve to maintain cellular homeostasis through efficient exchange of metabolites, lipid, and calcium between organelles, organelle fission, and movement. Most MCSs and a growing number of tethering proteins especially those involved in mediating the junctions between endoplasmic reticulum (ER) and other organelles have been extensively characterized in mammal and yeast. However, the studies of plant MCSs are still at stages of infancy, at least one reason might be due to the lack of bona fide markers for visualizing these membrane junctions in plant cells. In this study, a series of genetically encoded reporters using split super-folder GFP protein were designed to detect the possible MCSs between ER and three other cellular compartments including chloroplast, mitochondria and plasma membrane (PM) in plant cell. By expressing these genetically encoded reporter in Arabidopsis protoplasts as well as Nicotiana benthamiana leaf, we could intuitively observe the punctate signal surrounding chloroplast upon expression of ER-chloroplast MCS reporter, punctate signal of ER-mitochondria MCS reporter and punctate signal close to the PM upon expression of ER-PM MCS reporter. We also showed that the ER-chloroplast MCSs were dynamic structures that undergo active remodeling with concomitant occurrence of chloroplast dysfunction inside plant cells. This study demonstrates that ER associates with various organelles in close proximity in plant cells and provides tools that might be applicable for visualizing MCSs in plants.

Joint inhibition of mitochondrial complex IV and alternative oxidase by genetic or chemical means represses chloroplast transcription in Arabidopsis.

Abstract: Changes in the functional state of mitochondria have profound effects on other cellular compartments. Genome-wide expression analysis of Arabidopsis rps10 mutants with an RNAi-silenced expression o...

A subcellular proteome atlas of the yeast Komagataella phaffii.

Abstract: The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

Integrating single-cell RNA-sequencing and functional assays to decipher mammary cell states and lineage hierarchies.

Abstract: The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER−) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER+) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit+ progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage.

Abstract: Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Protein kinase CK2 impact on intracellular calcium homeostasis in prostate cancer.

Abstract: Protein kinase CK2 plays multiple roles in cell function in normal and disease states. CK2 is elevated in numerous types of cancer cells, and CK2 suppression of apoptosis represents a key link to the cancer cell phenotype. CK2 regulation of cell survival and death involves diverse processes, and our previous work suggested that mitochondrial machinery is a key locus of this function. One of the earliest responses of prostate cells to inhibition of CK2 is a change in mitochondrial membrane potential, possibly associated with Ca2+ signaling. Thus, in the present work, we investigated early impact of CK2 on intracellular Ca2+ dynamics. Three prostate cancer (PCa) cell lines, PC3-LN4, C4-2B, and 22Rv1, were studied. PCa cells were treated with the CK2 small molecule inhibitors 4,5,6,7-tetrabrombenzotriazole and CX-4945 followed by analysis of Ca2+ levels in various cellular compartments over time. The results showed dose-dependent loss in cytosolic Ca2+ levels starting within 2 min and reaching maximal loss within 5–10 min. There was a concomitant increase in Ca2+ in the endoplasmic reticulum (ER) and mitochondrial compartments. The results suggest that inhibition of CK2 activity results in a rapid movement of Ca2+ out of the cytosol and into the ER and mitochondria, which may be among the earliest contributory factors for induction of apoptosis in cells subjected to inhibition of CK2. In cells with death-inducing levels of CK2 inhibition, total cellular Ca2+ levels dropped at 2 h post-treatment. These novel observations represent a potential mechanism underlying regulation of cell survival and death by CK2 activity.

Deuterium Tracing to Interrogate Compartment-Specific NAD(P)H Metabolism in Cultured Mammalian Cells.

Abstract: Oxidation-reduction (redox) reactions are ubiquitous in biology and typically occur in specific subcellular compartments. In cells, the electron transfer between molecules and organelles is commonly facilitated by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). While often taken for granted, these metabolic reactions are critically important for maintaining redox homeostasis and biochemical potentials across membranes. While 13C tracing and metabolic flux analysis (MFA) have emerged as powerful tools to study intracellular metabolism, this approach is limited when applied to pathways catalyzed in multiple cellular compartments. To address this issue, we and others have applied 2H (deuterium) tracers to observe transfer of labeled hydride anions, which accompanies electron transfer. Furthermore, we have developed a reporter system for indirectly quantifying NADPH enrichment in specific subcellular compartments. Here, we provide a detailed description of 2H tracing applications and the interrogation of mitochondrial versus cytosolic NAD(P)H metabolism in cultured mammalian cells. Specifically, we describe the generation of reporter cell lines that express epitope-tagged R132H-IDH1 or R172K-IDH2 and produce (D)2-hydroxyglutarate in a doxycycline-dependent manner. These tools and methods allow for quantitation of reducing equivalent turnover rates, the directionality of pathways present in multiple compartments, and the estimation of pathway contributions to NADPH pools.

Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway.

Abstract: Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells

Subcellular fractionation of frozen skeletal muscle samples.

Abstract: Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as the cytosol, mitochondria, and nuclei. Subcellular fractionation is us...

Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin.

Abstract: Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin's effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

Cellular localisation of structurally diverse diphenylacetylene fluorophores.

Abstract: Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor-acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed, compounds with high log D values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly lipophilic compounds (log D = 2-3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.