WHOAS : Browsing BioCurrents Research Center by Issue Date

Article

A non-invasive method for measuring preimplantation embryo physiology

(Cambridge University Press, 2000-02) Trimarchi, James R. ; Liu, Lin ; Porterfield, D. Marshall ; Smith, Peter J. S. ; Keefe, David L.

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration ([similar]0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 [mu]m from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocysts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.

Article

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion

(Company of Biologists Limited, 2003) Dumollard, Remi ; Hammar, Katherine M. ; Porterfield, D. Marshall ; Smith, Peter J. S. ; Cibert, Christian ; Rouviere, Christian ; Sardet, Christian

Fertilization increases both cytosolic Ca2+ concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca2+ waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P3-induced Ca2+ transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca2+ during sperm-triggered Ca2+ waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca2+ wave pacemaker PM2. Strikingly, the activity of the Ca2+ wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca2+, the egg's mitochondria are stimulated by Ins(1,4,5)P3-mediated Ca2+ signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca2+ wave pacemaker PM2.

Article

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

(American Society for Biochemistry and Molecular Biology, 2004-06-23) Graf, Solomon A. ; Haigh, Sarah E. ; Corson, Erica D. ; Shirihai, Orian S.

ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36–70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1–35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable {alpha}-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106–715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.

Article

Modulation of the actin cytoskeleton via gelsolin regulates aacuolar H+-ATPase recycling

(American Society for Biochemistry and Molecular Biology, 2004-12-09) Beaulieu, Valerie ; Da Silva, Nicolas ; Pastor-Soler, Nuria ; Brown, Christopher R. ; Smith, Peter J. S. ; Brown, Dennis ; Breton, Sylvie

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H+-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.

Article

Calcium-independent, cGMP-mediated light adaptation in invertebrate ciliary photoreceptors

(Society for Neuroscience, 2005-02-23) Gomez, Maria del Pilar ; Nasi, Enrico

Calcium is thought to be essential for adaptation of sensory receptor cells. However, the transduction cascade of hyperpolarizing, ciliary photoreceptors of the scallop does not use IP3-mediated Ca release, and the light-sensitive conductance is not measurably permeable to Ca2+. Therefore, two typical mechanisms that couple the light response to [Ca]i changes seem to be lacking in these photoreceptors. Using fluorescent indicators, we determined that, unlike in their microvillar counterparts, photostimulation of ciliary cells under voltage clamp indeed evokes no detectable change in cytosolic Ca. Notwithstanding, these cells exhibit all of the hallmarks of light adaptation, including response range compression, sensitivity shift, and photoresponse acceleration. A possible mediator of Ca-independent sensory adaptation is cGMP, the second messenger that regulates the light-sensitive conductance; cGMP and 8-bromo cGMP not only activate light-dependent K channels but also reduce the amplitude of the light response to an extent greatly in excess of that expected from simple occlusion between light and chemical stimulation. In addition, these substances accelerate the time course of the photocurrent. Tests with pharmacological antagonists suggest that protein kinase G may be a downstream effector that controls, in part, the cGMP-triggered changes in photoresponse properties during light adaptation. However, additional messengers are likely to be implicated, especially in the regulation of response kinetics. These observations suggest a novel feedback inhibition pathway for signaling sensory adaptation.

Article

A direct signaling role for phosphatidylinositol 4,5-bisphosphate (PIP2) in the visual excitation process of microvillar receptors

(American Society for Biochemistry and Molecular Biology, 2005-03-01) Gomez, Maria del Pilar ; Nasi, Enrico

In microvillar photoreceptors the pivotal role of phospholipase C in light transduction is undisputed, but previous attempts to account for the photoresponse solely in terms of downstream products of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis have proved wanting. In other systems PIP2 has been shown to possess signaling functions of its own, rather than simply serving as a precursor molecule. Because illumination of microvillar photoreceptors cells leads to PIP2 break-down, a potential role for this phospholipid in phototransduction would be to help maintain some element(s) of the transduction cascade in the inactive state. We tested the effect of intracellular dialysis of PIP2 on voltage-clamped molluscan photoreceptors and found a marked reduction in the amplitude of the photocurrent; by contrast, depolarization-activated calcium and potassium currents were unaffected, thus supporting the notion of a specific effect on light signaling. In the dark, PIP2 caused a gradual outward shift of the holding current; this change was due to a decrease in membrane conductance and may reflect the suppression of basal openings of the light-sensitive conductance. The consequences of depleting PIP2 were examined in patches of light-sensitive microvillar membrane screened for the exclusive presence of light-activated ion channels. After excision, superfusion with anti-PIP2 antibodies induced the appearance of single-channel currents. Replenishment of PIP2 by exogenous application reverted the effect. These data support the notion that PIP2, in addition to being the source of inositol trisphosphate and diacylglycerol, two messengers of visual excitation, may also participate in a direct fashion in the control of the light-sensitive conductance

Article

On the gating mechanisms of the light-dependent conductance in Pecten hyperpolarizing photoreceptors : does light remove inactivation in voltage-dependent K channels?

(Rockefeller University Press, 2005-04-11) Gomez, Maria del Pilar ; Nasi, Enrico

The hyperpolarizing receptor potential of ciliary photoreceptors of scallop and other mollusks is mediated by a cGMP-activated K conductance; these cells also express a transient potassium current triggered by depolarization. During steady illumination, the outward currents elicited by voltage steps lose their decay kinetics. One interesting conjecture that has been proposed is that the currents triggered by light and by depolarization are mediated by the same population of channels, and that illumination evokes the receptor potential by removing their steady-state inactivation. Exploiting the information that has become available on the phototransduction cascade of ciliary photoreceptors, we demonstrated that the same downstream signaling elements are implicated in the modulation of voltage-elicited currents: direct chemical stimulation both at the level of the G protein and of the final messenger that controls the light-dependent channels (cGMP) also attenuate the falling phase of the voltage-activated current. Application of a protein kinase G antagonist was ineffective, suggesting that a cGMP-initiated phosphorylation step is not implicated. To ascertain the commonality of ionic pathways we used pharmacological blockers. Although millimolar 4-aminopyridine (4-AP) suppressed both currents, at micromolar concentrations only the photocurrent was blocked. Conversely, barium completely and reversibly antagonized the transient voltage-activated current with no detectable effect on the light-evoked current. These results rule out that the same ionic pores mediate both currents; the mechanism of light modulation of the depolarization-evoked K current was elucidated as a time-dependent increase in the light-sensitive conductance that is superimposed on the inactivating K current.

Preprint

Electrokinetic measurements of membrane capacitance and conductance for pancreatic β-cells

( 2005-10-31) Pethig, Ronald ; Jakubek, L. M. ; Sanger, R. H. ; Heart, E. ; Corson, Erica D. ; Smith, Peter J. S.

Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary β-cells and INS-1 insulinoma cells) determined using the methods of dielectrophoresis and electrorotation. The membrane capacitance value of 12.57 (± 1.46) mF/m2 obtained for β-cells, and the values 9.96 (± 1.89) mF/m2 to 10.65 (± 2.1) mF/m2 obtained for INS-1 cells, fall within the range expected for mammalian cells. The electrorotation results for the INS-1 cells lead to a value of 36 (± 22) S/m2 for the membrane conductance associated with ion channels, if values in the range 2nS to 3 nS are assumed for the membrane surface conductance. This membrane conductance value falls within the range reported for INS cells obtained using the whole-cell patch-clamp technique. However, the total ‘effective’ membrane conductance value of 601 (± 182) S/m2 obtained for the INS-1 cells by dielectrophoresis is significantly larger (by a factor of around three-fold) than the values obtained by electrorotation. This could result from an increased membrane surface conductance, or increased passive conduction of ions through membrane pores, induced by the larger electric field stresses experienced by cells in the dielectrophoresis experiments.

Article

Paraquat increases cyanide-insensitive respiration in murine lung epithelial cells by activating an NAD(P)H:paraquat oxidoreductase : identification of the enzyme as thioredoxin reductase

(American Society for Biochemistry and Molecular Biology, 2007-01-17) Gray, Joshua P. ; Heck, Diane E. ; Mishin, Vladimir ; Smith, Peter J. S. ; Hong, Jun-Yan ; Thiruchelvam, Mona ; Cory-Slechta, Deborah A. ; Laskin, Debra L. ; Laskin, Jeffrey D.

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10–100 µM) was found to cause a 2–4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial- but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 µM), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.

Article

Modulation of extracellular proton fluxes from retinal horizontal cells of the catfish by depolarization and glutamate

(Rockefeller University Press, 2007-07-30) Kreitzer, Matthew A. ; Collis, Leon P. ; Molina, Anthony J. A. ; Smith, Peter J. S. ; Malchow, Robert Paul

Self-referencing H+-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-D-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 µM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca2+/H+ ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.

Preprint

Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs

( 2007-11-14) Pethig, Ronald ; Menachery, Anoop ; Heart, E. ; Sanger, R. H. ; Smith, Peter J. S.

Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10x10 array of threedimensional cell constructs. Some of these constructs, measuring approximately 150 μm in diameter and 120 μm in height, and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these ‘pseudo-islets’ was also demonstrated. The footprint of the 10x10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.

Article

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

(BioMed Central, 2008-03-26) Salvador-Recatala, Vicenta ; Schneider, Toni ; Greenberg, Robert M.

The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory β subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the α1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two β subunit subtypes: a structurally conventional β subunit and a variant β subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavβ subunit. Here, we focus on the modulatory phenotype of the conventional Cavβ subunit (SmCavβ) using the human Cav2.3 channel as the substrate for SmCavβ and the whole-cell patch-clamp technique. The conventional Schistosoma mansoni Cavβ subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavβ run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavβ lends the Cav2.3/SmCavβ complex sensitivity to Na+ ions. A mutant version of the Cavβ subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavβ subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.

Preprint

Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients

( 2008-11) Messerli, Mark A. ; Collis, Leon P. ; Smith, Peter J. S.

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 μm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane.

Preprint

Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility

( 2009-04-28) Messerli, Shanta M. ; Kasinathan, Ravi S. ; Morgan, William ; Spranger, Stefani ; Greenberg, Robert M.

One potential physiological target for new antischistosomals is the parasite's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sublethal concentrations (100 - 500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance.

Preprint

Windows to cell function and dysfunction : signatures written in the boundary layers

( 2010-01-26) Smith, Peter J. S. ; Collis, Leon P. ; Messerli, Mark A.

The medium surrounding cells either in culture or in tissues contains a chemical mix varying with cell state. As solutes move in and out of the cytoplasmic compartment they set up characteristic signatures in the cellular boundary layers. These layers are complex physical and chemical environments whose profiles both reflect cell physiology and provide conduits for intercellular messaging. Here we review some of the most relevant characteristics of the extracellular/intercellular space. Our initial focus is primarily with cultured cells but we extend our consideration to the far more complex environment of tissues and discuss how chemical signatures in the boundary layer can or may affect cell function. Critical to the entire essay are the methods used, or being developed, to monitor chemical profiles in the boundary layers. We review recent developments in ultramicro electrochemical sensors and tailored optical reporters suitable for the task in hand.

Preprint

Dielectrophoretic tweezer for isolating and manipulating target cells

( 2010-06-03) Menachery, Anoop ; Graham, David M. ; Messerli, Shanta M. ; Pethig, Ronald ; Smith, Peter J. S.

The ability to isolate and accurately position single cells in three dimensions is becoming increasingly important in many areas of biological research. We describe the design, theoretical modeling and testing of a novel dielectrophoretic (DEP) tweezer for picking out and relocating single target cells.. The device is constructed using facilities available in most electrophysiology laboratories, without the requirement of sophisticated and expensive microfabrication technology, and offers improved practical features over previously reported DEP tweezer designs. The DEP tweezer has been tested using transfected HEI 193 human schwannoma cells, with visual identification of the target cells being aided by labeling the incorporated gene product with a green fluorescent protein.

Preprint

Construction, Theory, and Practical Considerations for using Self-referencing of Ca2+-Selective Microelectrodes for Monitoring Extracellular Ca2+ Gradients

( 2010-10) Messerli, Mark A. ; Smith, Peter J. S.

Ca2+ signaling in the extra- and intracellular domains is linked to Ca2+ transport across the plasma membrane. Non-invasive monitoring of these resulting extracellular Ca2+ gradients with self-referencing of Ca2+-selective microelectrodes is used for studying Ca2+ signaling across Kingdoms. The quantitated Ca2+ flux enables comparison with changes to intracellular [Ca2+] measured with other methods and determination of Ca2+ transport stoichiometry. Here we review the construction of Ca2+-selective microelectrodes, their physical characteristics and their use in self-referencing mode to calculate Ca2+ flux. We also discuss potential complications when using them to measure Ca2+ gradients near the boundary layers of single cells and tissues.

Article

Arrestin in ciliary invertebrate photoreceptors : molecular identification and functional analysis in vivo

(Society for Neuroscience, 2011-02-02) Gomez, Maria del Pilar ; Espinosa, Lady ; Ramirez, Nelson ; Nasi, Enrico

Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. Arrestin was detected by Western blot analysis of retinal lysates, and localized in ciliary photoreceptors by immunostaining of whole-eye cryosections and dissociated cells. Two arrestin isoforms were molecularly identified by PCR; these present the canonical N- and C-arrestin domains, and are identical at the nucleotide level over much of their sequence. A high degree of homology to various β-arrestins (up to 70% amino acid identity) was found. In situ hybridization localized the two transcripts within the retina, but failed to reveal finer spatial segregation, possibly because of insufficient differences between the riboprobes. Intracellular dialysis of anti arrestin antibodies into voltage-clamped ciliary photoreceptors produced a gradual slow-down of the photocurrent falling phase, leaving a tail that decayed over many seconds after light termination. The antibodies also caused spectrally neutral flashes to elicit prolonged aftercurrents in the absence of large metarhodopsin accumulation; such aftercurrents could be quenched by chromatic illumination that photoconverts metarhodopsin back to rhodopsin. These observations indicate that the antibodies depleted functionally available arrestin, and implicate this molecule in the deactivation of the photoresponse at the rhodopsin level.

Preprint

Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase

( 2011-06) Alavian, Kambiz N. ; Li, Hongmei ; Collis, Leon P. ; Bonanni, Laura ; Zeng, Lu ; Sacchetti, Silvio ; Lazrove, Emma ; Nabili, Panah ; Flaherty, Benjamin ; Graham, Morven ; Chen, Yingbei ; Messerli, Shanta M. ; Mariggio, Maria A. ; Rahner, Christoph ; McNay, Ewan ; Shore, Gordon ; Smith, Peter J. S. ; Hardwick, J. Marie ; Jonas, Elizabeth A.

Anti-apoptotic BCL-2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence suggests that Bcl-xL interacts directly with the beta subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases ATPase activity of purified synthase complexes, while inhibition of endogenous Bcl-xL decreases F1FO enzymatic activity. Our findings suggest that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-xL expressing neurons.

Article

Regeneration in the era of functional genomics and gene network analysis

(Marine Biological Laboratory, 2011-08) Smith, Joel ; Morgan, Jennifer R. ; Zottoli, Steven J. ; Smith, Peter J. S. ; Buxbaum, Joseph D. ; Bloom, Ona E.

What gives an organism the ability to regrow tissues and to recover function where another organism fails is the central problem of regenerative biology. The challenge is to describe the mechanisms of regeneration at the molecular level, delivering detailed insights into the many components that are cross-regulated. In other words, a broad, yet deep dissection of the system-wide network of molecular interactions is needed. Functional genomics has been used to elucidate gene regulatory networks (GRNs) in developing tissues, which, like regeneration, are complex systems. Therefore, we reason that the GRN approach, aided by next generation technologies, can also be applied to study the molecular mechanisms underlying the complex functions of regeneration. We ask what characteristics a model system must have to support a GRN analysis. Our discussion focuses on regeneration in the central nervous system, where loss of function has particularly devastating consequences for an organism. We examine a cohort of cells conserved across all vertebrates, the reticulospinal (RS) neurons, which lend themselves well to experimental manipulations. In the lamprey, a jawless vertebrate, there are giant RS neurons whose large size and ability to regenerate make them particularly suited for a GRN analysis. Adding to their value, a distinct subset of lamprey RS neurons reproducibly fail to regenerate, presenting an opportunity for side-by-side comparison of gene networks that promote or inhibit regeneration. Thus, determining the GRN for regeneration in RS neurons will provide a mechanistic understanding of the fundamental cues that lead to success or failure to regenerate.

BioCurrents Research Center

Permanent URI for this collection

Browse

Browse

Browsing BioCurrents Research Center by Issue Date

Results Per Page

Sort Options