μM ATP Research Articles

Fast Ca2+ Transients of Inner Hair Cells Arise Coupled and Uncoupled to Ca2+ Waves of Inner Supporting Cells in the Developing Mouse Cochlea.

Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca2+ action potentials (APs), which stimulate the auditory pathway and guide maturation of neuronal circuits. During these early postnatal days, intercellular propagating Ca2+ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca2+ APs independently or require a trigger by an ISC Ca2+ wave. To identify the Ca2+ transients of IHCs underlying Ca2+ APs and to analyze their dependence on ISC Ca2+ waves, we performed fast Ca2+ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca2+ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca2+ waves in ISCs. ISC Ca2+ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca2+ elevations of small amplitude appeared to be triggered by ISC Ca2+ waves in IHCs of Cav1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca2+ influx through Cav1.3 channels. The Ca2+ wave-triggered Ca2+ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca2+ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca2+ elevations in adjacent ISCs. PPADS irreversibly abolished Ca2+ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca2+ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2–5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca2+ waves. Neonatal IHCs thus spontaneously generate electrical and Ca2+ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker’s organ indicating two sources of spontaneous activity in the developing auditory system.

Increased ATP and ADO Overflow From Sympathetic Nerve Endings and Mesentery Endothelial Cells Plus Reduced Nitric Oxide Are Involved in Diabetic Neurovascular Dysfunction.

Since the mechanism of human diabetic peripheral neuropathy and vascular disease in type 1 diabetes mellitus remains unknown, we assessed whether sympathetic transmitter overflow is altered by this disease and associated to vascular dysfunction. Diabetes was induced by streptozotocin (STZ)-treatment and compared to vehicle-treated rats. Aliquots of the ex vivo perfused rat arterial mesenteric preparation, denuded of the endothelial layer, were collected to quantify analytically sympathetic nerve co-transmitters overflow secreted by the isolated mesenteries of both groups of rats. Noradrenaline (NA), neuropeptide tyrosine (NPY), and ATP/metabolites were detected before, during, and after electrical field stimulation (EFS, 20 Hz) of the nerve terminals surrounding the mesenteric artery. NA overflow was comparable in both groups; however, basal or EFS-secreted ir-NPY was 26% reduced (p < 0.05) in diabetics. Basal and EFS-evoked ATP and adenosine (ADO) overflow to the arterial mesentery perfusate increased twofold and was longer lasting in diabetics; purine tissue content was 37.8% increased (p < 0.05) in the mesenteries from STZ-treated group of rats. Perfusion of the arterial mesentery vascular territory with 100 μM ATP, 100 nM 2-MeSADP, or 1 μM UTP elicited vasodilator responses of the same magnitude in controls or diabetics, but the increase in luminally accessible NO was 60–70% lower in diabetics (p < 0.05). Moreover, the concentration–response curve elicited by two NO donors was displaced downwards (p < 0.01) in diabetic rats. Parallel studies using primary cultures of endothelial cells from the arterial mesentery vasculature revealed that mechanical stimulation induced a rise in extracellular nucleotides, which in the cells from diabetic rats was larger and longer-lasting when comparing the extracellular release of ATP and ADO values to those of vehicle-treated controls. A 5 min challenge with purinergic agonists elicited a cell media NO rise, which was reduced in the endothelial cells from diabetic rats. Present findings provide neurochemical support for the diabetes-induced neuropathy and show that mesenteric endothelial cells alterations in response to mechanical stimulation are compatible with the endothelial dysfunction related to vascular disease progress.

Purinergic Signaling Controls Fibroblast Growth Factor‐21 Expression in Skeletal Muscle through Akt/mTOR Pathway.

Fibroblast Growth Factor‐21 (FGF‐21) is a myokine which induce fiber type transformation and metabolic adaptations in muscle. However, little is known about the molecular mechanisms involved in the regulation of FGF‐21 expression in skeletal muscle in non‐pathological conditions. We have previously described that purinergic signaling is related with muscle adaptations and IL6‐myokine production through the “excitation‐transcription coupling” (ETC) mechanism, in which extracellular ATP (eATP) promotes gene expression in skeletal muscle. We have also demonstrated that eATP activates the PI3K/Akt signaling pathway in myotubes. Considering that Akt has been proposed as a master regulator for FGF‐21 expression control, the aim of this work is to determine the role of extracellular ATP over the FGF‐21 production in adult mouse skeletal muscle, and their dependence on the Akt/mTOR signaling pathway. Mice FDB‐isolated fibers or whole‐muscle were used in this study. The mRNA levels of FGF‐21 after electrical (20 Hz, 270 pulses, 0.3 msec each) or exogenous ATP (100 μM) stimulation were assessed by qPCR. FGF‐21 protein levels were assessed by immunoblot. We used pharmacological inhibitors of purinergic receptors (100 μM Suramin) and Akt/mTOR pathway inhibitors (50 μM LY294002; 1 μM Akt inhibitor VIII; 100 nM rapamycin), to turn off different interactors of the putative signaling pathway. ATP stimulation (100 μM) evoked an 8‐fold increase in FGF‐21 mRNA levels in isolated FDB fibers at 30 min post stimulation. The increase in FGF‐21 mRNA levels evoked by eATP was inhibited by suramin, a general antagonist of P2Y/P2X nucleotide receptors. Electrical stimulation also evokes an increase in FGF‐21 mRNA levels, which was inhibited by both nifedipine (a DHPR blocker) and suramin. Exogenous ATP increased protein levels of FGF‐21 in whole FDB‐muscles at 120 min, in a concentration‐dependent manner, with a maximal increase of 3.5 folds with 3 μM ATP. The increase in FGF‐21 protein level evoked by ATP, was abolished by LY294002 (a PI3K inhibitor), Akt inhibitor VIII, and rapamycin (mTOR inhibitor). In this work, we demonstrated for the first time that extracellular ATP evokes an increase in the mRNA and protein levels of FGF‐21 in skeletal muscle. The increase on FGF‐21 levels evoked by ATP is dependent on Akt/mTOR pathway activation. These results suggest that ETC mechanism could be involved in regulation of FGF‐21 expression in skeletal muscle.Support or Funding InformationFunded by CONICYT PCHA 21151035(MA‐C), 63140009(CM) and Fondecyt 1151353(SB), 1151293(EJ)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Guanine and inosine nucleotides/nucleosides suppress murine T cell activation

Damaged tissues and cells release intracellular purine nucleotides, which serve as intercellular signaling factors. We previously showed that exogenously added adenine nucleotide (250 μM ATP) suppressed the activation of murine splenic T lymphocytes. Here, we examined the effects of other purine nucleotides/nucleosides on mouse T cell activation. First, we found that pretreatment of mouse spleen T cells with 250 μM GTP, GDP, GMP, guanosine, ITP, IDP, IMP or inosine significantly reduced the release of stimulus-inducible cytokine IL-2. This suppression of IL-2 release was not caused by induction of cell death. Further studies with GTP, ITP, guanosine and inosine showed that pretreatment with these nucleotides/nucleosides also suppressed release of IL-6. However, these nucleotides/nucleosides did not suppress stimulus-induced phosphorylation of ERK1/2, suggesting that the suppression of the release of inflammatory cytokines does not involve inhibition of ERK1/2 signaling. In contrast to ATP pretreatment at the same concentration, guanine or inosine nucleotides/nucleosides did not attenuate the expression of CD25. Our findings indicate that exogenous guanine or inosine nucleotides/nucleosides can suppress inflammatory cytokine release from T cells, and may be promising candidates for use as supplementary agents in the treatment of T cell-mediated immune diseases.

Adenine Nucleotides Attenuate Murine T Cell Activation Induced by Concanavalin A or T Cell Receptor Stimulation.

Extracellular ATP and its metabolites affect various cellular immune responses, including T cell function, but there are apparently conflicting reports concerning the effects of adenine nucleotides on T cells. For example, it has been reported that ATP-mediated activation of P2 receptor is involved in T cell activation; activation of adenosine receptors suppresses T cell function; and 1 mM ATP induces T cell death via activation of P2X7 receptor. Therefore, in this work we investigated in detail the effects of 100–250 μM ATP, ADP, or AMP on murine T cell activation. First, an in vitro study showed that pretreatment of murine splenic T cells with 100–250 μM ATP, ADP, or AMP significantly suppressed the concanavalin A (ConA)-induced release of cytokines, including IL-2. This suppression was not due to induction of cell death via the P2X7 receptor or to an immunosuppressive effect of adenosine. ATP attenuated the expression of CD25, and decreased the cell proliferation ability of activated T cells. The release of IL-2 by ConA-stimulated lymphocytes was suppressed by post-treatment with ATP, as well as by pretreatment. These results suggest that exogenous ATP suppresses the activation of T cells. Secondly, we evaluated the effect of ATP in a ConA-treated mice. Treatment with ATP attenuated the increase of IL-2 concentration in the blood. Overall, these results suggest that adenine nucleotides might have potential as supplemental therapeutic agents for T cell-mediated immune diseases, by suppressing T cell activation.

Catalytic Activity Profile of Polyphosphate Kinase 1 from Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) catalyzes reverse transfer of the terminal phosphate from ATP to form polyphosphate (polyP) and from polyP to form ATP, and is responsible for the synthesis of most of cellular polyPs. When Ppk1 from Myxococcus xanthus was incubated with 0.2mM polyP60-70 and 1mM ATP or ADP, the rate of ATP synthesis was approximately 1.5-fold higher than that of polyP synthesis. If in the same reaction the proportion of ADP in the ATP/ADP mixture exceeded one-third, the equilibrium shifted to ATP synthesis, suggesting that M. xanthus Ppk1 preferentially catalyzed ATP formation. At the same time, GTP and GDP were not recognized as substrates by Ppk1. In the absence of polyP, Ppk1 generated ATP and AMP from ADP, and ADP from ATP and AMP, suggesting that the enzyme catalyzed the transfer of a phosphate group between ADP molecules yielding ATP and AMP, thus exhibiting adenylate kinase activity.

Kca3.1 Activation Via P2y2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

Disorders in cell signaling mediated by ATP or histamine, activating specific membrane receptors, have been frequently associated with tumorigenesis. Among the elements of response to purinergic (and histaminergic) signaling, ion channel activation controls essential cellular processes in cancer, such as cell proliferation, motility, and death. Here, we studied the effects that ATP had on electrical properties of human ovarian adenocarcinoma cells named SKOV-3. ATP caused increase in intracellular Ca2+ concentration ([Ca2+]i) and, concurrently, it evoked a complex electrical response with a conspicuous outward component. This current was generated through P2Y2 receptor activation and opening of K+ channels, KCa3.1, as indicated by electrophysiological and pharmacological analysis, as well as by immunodetection and specific silencing of P2Y2 or KCa3.1 gene by esiRNA transfection. Low µM ATP concentration increased SKOV-3 cell migration, which was strongly inhibited by KCa3.1 channel blockers and by esiRNA-generated P2Y2 or KCa3.1 downregulation. Finally, in human ovarian tumors, the P2Y2 and KCa3.1 proteins are expressed and co-localized in neoplastic cells. Thus, stimulation of P2Y2 receptors expressed in SKOV-3 cells promotes motility through KCa3.1 activation. Since P2Y2 and KCa3.1 are co-expressed in primary tumors, our findings suggest that they may play a role in cancer progression.

Transcriptomic profiling of a chicken lung epithelial cell line (CLEC213) reveals a mitochondrial respiratory chain activity boost during influenza virus infection.

Avian Influenza virus (AIV) is a major concern for the global poultry industry. Since 2012, several countries have reported AIV outbreaks among domestic poultry. These outbreaks had tremendous impact on poultry production and socio-economic repercussion on farmers. In addition, the constant emergence of highly pathogenic AIV also poses a significant risk to human health. In this study, we used a chicken lung epithelial cell line (CLEC213) to gain a better understanding of the molecular consequences of low pathogenic AIV infection in their natural host. Using a transcriptome profiling approach based on microarrays, we identified a cluster of mitochondrial genes highly induced during the infection. Interestingly, most of the regulated genes are encoded by the mitochondrial genome and are involved in the oxidative phosphorylation metabolic pathway. The biological consequences of this transcriptomic induction result in a 2.5- to 4-fold increase of the ATP concentration within the infected cells. PB1-F2, a viral protein that targets the mitochondria was not found associated to the boost of activity of the respiratory chain. We next explored the possibility that ATP may act as a host-derived danger signal (through production of extracellular ATP) or as a boost to increase AIV replication. We observed that, despite the activation of the P2X7 purinergic receptor pathway, a 1mM ATP addition in the cell culture medium had no effect on the virus replication in our epithelial cell model. Finally, we found that oligomycin, a drug that inhibits the oxidative phosphorylation process, drastically reduced the AIV replication in CLEC213 cells, without apparent cellular toxicity. Collectively, our results suggest that AIV is able to boost the metabolic capacities of its avian host in order to provide the important energy needs required to produce progeny virus.

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells.

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10μM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y1 or adenosine A2A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

Abstract P5-05-03: Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas

Abstract The composition of the extracellular tumor microenvironment differs from that of most other tissues and is thought to provide cancer cells with a growth and survival advantage compared to normal cells. In solid tumors, the extracellular concentration of ATP can be elevated to ~100 µm and extracellular pH can be as low as 6.5. In the current project, we investigate the consequences of purinergic signaling in human and murine breast carcinomas: we study intracellular Ca2+ signals and associated changes in cell proliferation during stimulation with extracellular nucleotides. We employ biopsies of human and murine primary breast carcinomas and compare them with matched normal breast tissue. Human biopsies are obtained with written informed consent from patients undergoing breast conserving surgery at Aarhus University Hospital or Regional Hospital Randers in Denmark. Murine biopsies are from mice overexpressing unactivated ErbB2 specifically in the breast tissue. We isolate epithelial organoids (~150 µm diameter) from tissue biopsies by partial digestion with collagenase III. Organoids loaded with the Ca2+-sensitive fluorophore Fura-2 are studied by fluorescence microscopy. In separate experiments, cell proliferation is quantified by detecting newly synthesized DNA using immunofluorescence imaging of organoids incubated with the thymidine analogue bromodeoxyuridine (BrdU). We find that intracellular Ca2+ responses during stimulation with extracellular ATP are elevated 2- to 10-fold in breast carcinomas from mice and humans, respectively, compared to matched normal breast tissue. We observe similar differences between breast cancer tissue and normal breast tissue in response to stimulation with the P2Y2/P2Y4-agonist UTP, whereas virtually no rise in the intracellular concentration of Ca2+ is observed in response to the P2X7-agonist 3'-O-(4-benzoyl)benzoyl-ATP. Application of cyclopiazonic acid – an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase – also cause exaggerated intracellular Ca2+ responses in breast cancer compared to normal breast tissue. Consistent with the elevated Ca2+ responses, stimulation with 100 µm ATP or 100 µm UTP increases the rate of cell proliferation (i.e., fraction of BrdU-positive cells) by ~2-fold in the breast cancer tissue. In conclusion, we find that purinergic signaling is upregulated in human and murine breast carcinomas compared to normal breast tissue. Activation of purinergic receptors – most likely P2Y2 and/or P2Y4 – enhances cell proliferation in breast cancer tissue. We propose that the high ATP levels in the tumor microenvironment promote breast cancer development or progression and that the associated signaling pathways represent promising targets for therapy. Citation Format: Mele M, McWhan K, Henningsen M, Vahl P, Jensen V, Johansen T, Pedersen H, Christiansen P, Bødtkjer E. Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-03.

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

P2X7 receptor as a novel drug delivery system to increase the entrance of hydrophilic drugs into cells during photodynamic therapy.

The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5μMMB for 15min promoted the rate of entry of MB into the cytoplasm to 4.7%. However, treatment with 5μMMB and 1mM ATP for the same amount of time increased this rate to 90.2%. However, this effect was inhibited by pretreatment with a P2X7 antagonist. We used peritoneal macrophages and a cell line that does not express P2X7R as controls. These cells were more resistant to PDT with MB under the same experimental conditions. Taken together, these results suggest the use of the pore associated with P2X7R as a drug delivery system to increase the passage of hydrophilic drugs into cells that express this receptor, thus facilitating PDT.

Magnesium inhibits the calcification of the extracellular matrix in tendon-derived stem cells via the ATP-P2R and mitochondrial pathways

Tendon calcification has been widely regarded by researchers to result from the osteogenic differentiation of Tendon-Derived Stem Cells (TDSCs) and ectopic mineralization caused by the calcification of cellular matrix. Recent studies have revealed a correlation between the Mg2+/Ca2+ balance and the degeneration or calcification of tendon tissues. Furthermore, the ATP–P2X/P2Y receptor pathway has been shown to play a decisive role in the process of calcification, with calcium exportation from mitochondria and calcium oscillations potentially representing the cohesive signal produced by this pathway. Our previous study demonstrated that matrix calcification is inhibited by magnesium. In this study, we examined the effects of extracellular Mg2+ on the deposition of calcium phosphate matrix and cellular pathways in TDSCs. The suppression of the export of calcium from mitochondria has also been detected. We found that a high concentration of extracellular Mg2+ ([Mg2+]e) inhibited the mineralization of the extracellular matrix in TDSCs and that 100 μM ATP reversed this inhibitory effect in vitro. In addition, the spontaneous release of ATP was inhibited by high [Mg2+]e levels. A high [Mg2+]e suppressed the expression of P2X4, P2X5 and P2X7 and activated the expression of P2Y1, P2Y2, P2Y4 and P2Y14. The interaction between Mg2+ and Ca2+ is therefore contradictory, Mg2+ inhibits mitochondrial calcium concentrations, meanwhile it reverses the opening of mPTP that is induced by Ca2+. JC-1 staining verified the protective effect of Mg2+ on mitochondrial membrane potential and the decrease induced by Ca2+. Taken together, these results indicate that high [Mg2+]e interferes with the expression of P2 receptors, resulting in decreased extracellular mineralization. The balance between Mg2+ and Ca2+ influences mitochondrial calcium exportation and provides another explanation for the mechanism underlying matrix calcification in TDSCs.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes

The collagen type I segment long spacing (SLS) crystallite is a well-ordered rod-like molecular aggregate, ∼300nm in length, which is produced in vitro under mildly acidic conditions (pH 2.5–3.5) in the presence of 1mM ATP. The formation of the SLS crystallite amplifies the inherent linear structural features of individual collagen heterotrimers, due to the punctate linear distribution and summation of the bulkier amino acid side chains along the length of individual collagen heterotrimers. This can be correlated structurally with the 67nm D-banded collagen fibril that is found in vivo, and formed in vitro. Although first described many years ago, the range of conditions required for ATP-induced SLS crystallite formation from acid-soluble collagen have not been explored extensively. Consequently, we have addressed biochemical parameters such as the ATP concentration, pH, speed of formation and stability so as to provide a more complete structural understanding of the SLS crystallite. Treatment of collagen type I with 1mM ATP at neutral and higher pH (6.0–9.0) also induced the formation of D-banded fibrils. Contrary to previous studies, we have shown that the polysulphonated diazo dyes Direct red (Sirius red) and Evans blue, but not Congo red and Methyl blue, can also induce the formation of SLS-like aggregates of collagen, but under markedly different ionic conditions to those employed in the presence of ATP. Specifically, pre-formed D-banded collagen fibrils, prepared in a higher than the usual physiological NaCl concentration (e.g. 500mM NaCl, 20mM Tris-HCl pH7.4 or x3 PBS), readily form SLS aggregates when treated with 0.1mM Direct red and Evans blue, but this did not occur at lower NaCl concentrations. These new data are discussed in relation to the anion (Cl−) and polyanion (phosphate and sulphonate) binding by the collagen heterotrimer and their likely role in collagen fibrillogenesis and SLS formation.

The Acidosis-Induced Slowing of Regulated Thin Filament Velocity in a Motility Assay Disappears at Low ATP

Acidosis (low pH) is a putative agent of muscle fatigue that slows contraction velocity but the mechanisms underlying this effect are unclear. It may directly affect myosin by slowing a step associated with detachment or it may act indirectly by affecting the ability of Ca++ to activate the thin filament, through effects on the regulatory proteins troponin/tropmyosin. We measured the effect of manipulating Ca++ and the lifetime rigor binding (by lowering the [ATP]) to examine the relative role of each of these mechanisms. At saturating Ca++ (pCa 4) and ATP concentrations (1mM) decreasing the pH from 7.4 to 6.8 decreased regulated actin filament velocity (Vrtf) by 50%, however at low [ATP] (<200 μM) Vrtf was unaffected by acidosis. Repeating these experiments in the absence of Ca++ (pCa 9) confirmed the phenomenon of rigor activation at normal pH (7.4), with filaments moving fastest at 50 μM ATP (0.5 μm/s). At low pH the filaments moved slower but still demonstrated rigor activation at [ATP] <75 μM. Single molecule laser trap assay experiments are under way to determine whether the slowed velocity was due slowed attachment or detachment kinetics. The acidosis-induced decrease in Vrtf at saturating Ca++ and ATP are consistent with a slowing of the steps in the cross-bridge cycle associated with ADP-release. However this effect disappears at low ATP where the attached lifetime is more dependent on the duration of rigor. Therefore the data suggest that acidosis does not affect the formation of a rigor bond or its ability to activate the thin filament. Inhibition of Ca++ binding to troponin might still contribute to slowing at saturating ATP and submaximal Ca++ levels.

Mechanism of Cooperative Force Generations between Skeletal Myosins

To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosin-rod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of power strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple power stroke states further enhance a chance of power stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of power stroke synchronization, since the strain-dependent transitions accompanied by the first or second power stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of power strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 μM ATP. These results were consistent with experimental results, supporting our conclusions.

Separating Ciliary Beating Force into Dynein-Driven Force and Axoneme-Intrinsic Bending Force By 3-D Tracking Microscopy

Highly coordinated dyneins generate asymmetric ciliary beating composed of clearly distinguished effective and recovery strokes. We herein aim to understand the mechanism of asymmetric beating of a single cilium by combining multiple optical-microscope techniques: 3-D tracking; optical tweezers; DIC adjusted to the different focal planes. A fluorescent bead was attached to the tip of the surface-immobilized mouse-tracheal cilium that was reactivated with a 100 µM ATP-containing solution, and trapped at various points of the beating stroke to measure the 3-D force (F). Next, after the solution was replaced with 100 µM ATP and 50 µM Vanadate, we measured 2-D structural bending energy in a relaxed state at the same cilium. With the estimation of the 3-D force driven by dyneins (FD) by subtraction of the effect of bending energy from F, the following were obtained: 1) FD periodically changed in the range from −75 to 35 pN, which is about 10 times larger than the force of viscous drag. 2) As the trapping point was displaced from the position of post-effective stroke, maximum values of FD (FDM) of the effective stroke increased from 3 to 35 pN and FDM of the recovery stroke increased from −1 to −75 pN, suggesting that the change in the force depends on the bending deformation of the cilium. 3) Under the trapping condition, in spite of the small curvature change, the direction of FD was spontaneously switched from the recovery direction to the effective direction, and vice versa. This result supports that FD in the effective and the recovery stroke direction, were generated by the different dynein-bridge sets on each side of the axoneme.

Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging.

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca2+ release from intracellular stores and subsequent Ca2+ entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca2+ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca2+ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca2+ responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca2+ responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca2+ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.