Routes of Ca²⁺ shuttling during Ca²⁺ oscillations FOCUS ON THE ROLE OF MITOCHONDRIAL Ca²⁺ HANDLING AND CYTOSOLIC Ca²⁺ BUFFERS

In some cell types, Ca²⁺ oscillations are strictly dependent on Ca²⁺ influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca²⁺ influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca²⁺ influx played a pivotal role. However, when the Ca²⁺ transport across the plasma membrane by the “lanthanum insulation method” was blocked prior to the induction of the serum-induced Ca²⁺ oscillations, mitochondrial Ca²⁺ transport was found to be able to substitute for the plasmalemmal Ca²⁺ exchange function, thus rendering the oscillations independent of extracellular Ca²⁺. However, in a physiological situation, the Ca²⁺-buffering capacity of mitochondria was found not to be essential for Ca²⁺ oscillations. Moreover, brief spontaneous Ca²⁺ changes were observed in the mitochondrial Ca²⁺ concentration without apparent changes in the cytosolic Ca²⁺ concentration, indicating the presence of a mitochondrial autonomous Ca²⁺ signaling mechanism. In the presence of calretinin, a Ca²⁺-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca²⁺ ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca²⁺ shuttling pathways in primary mesothelial cells during Ca²⁺ oscillations: Ca²⁺ shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca²⁺ buffers

Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations

Sensory neuron subpopulations as well as breast and prostate cancer cells express functional transient receptor potential vanilloid type 1 (TRPV1) ion channels; however little is known how TRPV1 activation leads to biological responses. Agonist-induced activation of TRPV1 resulted in specific spatiotemporal patterns of cytoplasmic Ca²⁺ signals in breast and prostate cancer-derived cells. Capsaicin (CAPS; 50 μM) evoked intracellular Ca²⁺ oscillations and/or intercellular Ca²⁺ waves in all cell lines. As evidenced in prostate cancer Du 145 cells, oscillations were largely dependent on the expression of functional TRPV1 channels in the plasma membrane, phospholipase C activation and on the presence of extracellular Ca²⁺ ions. Concomitant oscillations of the mitochondrial matrix Ca²⁺ concentration resulted in mitochondria energization evidenced by increased ATP production. CAPS-induced Ca²⁺ oscillations also occurred in a subset of sensory neurons, yet already at lower CAPS concentrations (1 μM). Stimulation of ectopically expressed TRPV1 channels in CAPS-insensitive NIH- 3T3 cells didn't provoke CAPS-triggered Ca²⁺ oscillations; rather it resulted in low- magnitude, long-lasting elevations of the cytosolic Ca²⁺ concentration. This indicates that sole TRPV1 activation is not sufficient to generate Ca²⁺ oscillations. Instead the initial TRPV1-mediated signal leads to the activation of the inositol phospholipid pathway. This in turn suffices to generate a biologically relevant frequency-modulated Ca²⁺ signal

Biological noise and positional effects influence cell stemness

Biological (or cellular) noise is the random quantitative variability of proteins and other molecules in individual, genetically identical cells. As the result of biological noise in the levels of some transcription factors that determine a cell's differentiation status, differentiated cells may dedifferentiate to a stem cell state given a sufficiently long time period. Here, to provide direct evidence supporting this hypothesis, we used a live-cell monitoring system based on enhanced green fluorescent protein (eGFP) expression to continuously assess the “stemness” of individual human and murine malignant mesothelioma cells over a period of up to 3 months. Re-expression of the transcription factors, the top hierarchical stemness markers Sox2 (SRY-box 2) and Oct4 (octamer-binding transcription factor), monitored as cell eGFP expression was observed in a subpopulation of differentiated eGFP(−) malignant mesothelioma cells. However, we found that this transition was extremely rare. Of note, when it did occur, neighboring cells that were not direct descendants of a newly emerged eGFP(+) stem cell were more likely than non-neighboring cells to also become an eGFP(+) stem cell. This observation suggested a positional effect and led to a clustered “mosaic” reappearance of eGFP(+) stem cells. Moreover, stem cells reappeared even in cell cultures derived from one single differentiated eGFP(−) cell. On the basis of our experimental in vitro and in vivo findings, we developed a tumor growth model to predict the clustered localization of cancer stem cells within a tumor mass

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood

In vivo import of unspliced tRNATyr containing synthetic introns of variable length into mitochondria of Leishmania tarentolae

The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecul

Stem Cell Factor-Based Identification and Functional Properties of In Vitro-Selected Subpopulations of Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Stem cell factor-based identification and functional properties of in vitro-selected subpopulations of malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics

Parvalbumin expression in oligodendrocyte-like CG4 cells causes a reduction in mitochondrial volume, attenuation in reactive oxygen species production and a decrease in cell processes’ length and branching

Forebrain glial cells - ependymal cells and astrocytes -acquire upon injury- a “reactive” phenotype associated with parvalbumin (PV) upregulation. Since free radicals, e.g. reactive oxygen species (ROS) play a role in the pathogenesis of multiple sclerosis, and that PV-upregulation in glial cells is inversely correlated with the level of oxidative stress, we hypothesized that PV-upregulation might also protect oligodendrocytes by decreasing ROS production. Lentiviral transduction techniques allowed for PV overexpression in CG4 oligodendrocyte progenitor cells (OPCs). Depending on the growth medium CG4 cells can be maintained in an OPC-like state, or induced to differentiate into an oligodendrocyte (OLG)-like phenotype. While increased levels of PV had no effect on cell proliferation and invasiveness in vitro, PV decreased the mitochondria volume in CG4 cell bodies, as well as the mitochondrial density in CG4 processes in both OPC-like and OLG-like states. In line with the PV-induced global decrease in mitochondrial volume, elevated PV levels reduced transcript levels of mitochondrial transcription factors involved in mitochondria biogenesis. In differentiated PV-overexpressing CG4 cells with a decreased mitochondrial volume, UV-induced ROS production was lower than in control CG4 cells hinting towards a possible role of PV in counteracting oxidative stress. Unexpectedly, PV also decreased the length of processes in undifferentiated CG4 cells and moreover diminished branching of differentiated CG4 cell processes, strongly correlated with the decreased density of mitochondria in CG4 cell processes. Thus besides conferring a protective role against oxidative stress, PV in a cell autonomous fashion additionally affects process’ growth and branching in CG4 cells

Inducible and reversible silencing of the Pvalb gene in mice: An in vitro and in vivo study

Inducible and reversible regulation of gene expression is a powerful approach for unraveling gene functions. Here, we describe the generation of a system to efficiently downregulate in a reversible and inducible manner the Pvalb gene coding for the calcium‐binding protein parvalbumin (PV) in mice. We made use of an IPTG‐inducible short hairpin RNA to activate Pvalb transcript knockdown and subsequently downregulate PV. The downregulation was rapidly reversed after withdrawal of IPTG. In vitro and in vivo experiments revealed a decrease in PV expression of ≥50% in the presence of IPTG and full reversibility after IPTG removal. We foresee that the tightly regulated and reversible PV downregulation in mice in vivo will provide a new tool for the control of Pvalb transcript expression in a temporal manner. Because PV protein and PVALB transcript levels were found to be lower in the brain of patients with autism spectrum disorder and schizophrenia, the novel transgenic mouse line might serve as a model to investigate the putative role of PV in these neurodevelopmental disorders

Critical Behavior at the Chiral Phase Transition

Quantum chromodynamics with two zero mass flavors is expected to exhibit a phase transition with O(4) critical behavior. Fixing the universality class is important for phenomenology and for facilitating the extrapolation of simulation data to physical quark mass values. At Lattice '96 the Tsukuba and Bielefeld groups reported results from new simulations with dynamical staggered quarks at $N_t = 4$, which suggested a departure from the expected critical behavior. We report observations of similar deviations and discuss efforts in progress to understand this phenomenon.Comment: 3 pp, LaTeX with 6 encapsulated Postscript figures. Lattice '97 proceeding