Polarity of T Cell Shape, Motility, and Sensitivity to Antigen

AbstractT cell activation requires contact with APCs. We used optical techniques to demonstrate T cell polarity on the basis of shape, motility, and localized sensitivity to antigen. An intracellular Ca2+ clamp showed that T cell shape and motility are extremely sensitive to changes in [Ca2+]i (Kd = 200 nM), with immobilization and rounding occurring via a calcineurin-independent pathway. Ca2+-dependent immobilization prolonged T cell contact with the antigen-presenting B cell; buffering the [Ca2+]i signal prevented the formation of stable cell pairs. Optical tweezers revealed spatial T cell sensitivity to antigen by controlling placement on the T cell surface of either B cells or α-CD3 MAb-coated beads. T cells were 4-fold more sensitive to contact made at the leading edge of the T cell compared with the tail. We conclude that motile T cells are polarized antigen sensors that respond physically to [Ca2+]i signals to stabilize their interaction with APCs

From Pinocytosis to Methuosis—Fluid Consumption as a Risk Factor for Cell Death

The volumes of a cell [cell volume (CV)] and its organelles are adjusted by osmoregulatory processes. During pinocytosis, extracellular fluid volume equivalent to its CV is incorporated within an hour and membrane area equivalent to the cell’s surface within 30 min. Since neither fluid uptake nor membrane consumption leads to swelling or shrinkage, cells must be equipped with potent volume regulatory mechanisms. Normally, cells respond to outwardly or inwardly directed osmotic gradients by a volume decrease and increase, respectively, i.e., they shrink or swell but then try to recover their CV. However, when a cell death (CD) pathway is triggered, CV persistently decreases in isotonic conditions in apoptosis and it increases in necrosis. One type of CD associated with cell swelling is due to a dysfunctional pinocytosis. Methuosis, a non-apoptotic CD phenotype, occurs when cells accumulate too much fluid by macropinocytosis. In contrast to functional pinocytosis, in methuosis, macropinosomes neither recycle nor fuse with lysosomes but with each other to form giant vacuoles, which finally cause rupture of the plasma membrane (PM). Understanding methuosis longs for the understanding of the ionic mechanisms of cell volume regulation (CVR) and vesicular volume regulation (VVR). In nascent macropinosomes, ion channels and transporters are derived from the PM. Along trafficking from the PM to the perinuclear area, the equipment of channels and transporters of the vesicle membrane changes by retrieval, addition, and recycling from and back to the PM, causing profound changes in vesicular ion concentrations, acidification, and—most importantly—shrinkage of the macropinosome, which is indispensable for its proper targeting and cargo processing. In this review, we discuss ion and water transport mechanisms with respect to CVR and VVR and with special emphasis on pinocytosis and methuosis. We describe various aspects of the complex mutual interplay between extracellular and intracellular ions and ion gradients, the PM and vesicular membrane, phosphoinositides, monomeric G proteins and their targets, as well as the submembranous cytoskeleton. Our aim is to highlight important cellular mechanisms, components, and processes that may lead to methuotic CD upon their derangement

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Brain Research / Progesterone-associated increase in ERP amplitude correlates with an improvement in performance in a spatial attention paradigm

Ovarian sex hormones modulate neuronal circuits not directly involved in reproductive functions. In the present study, we investigated whether endogenous fluctuations of estradiol and progesterone during the menstrual cycle are associated with early cortical processing stages in a cued spatial attention paradigm. EEG was monitored while young women responded to acoustically cued visual stimuli. Women with large mean amplitude of the event-related potential (ERP) (80120 ms following visual stimuli) responded faster to visual stimuli. In luteal women, mean amplitude of the ERP as well as alpha amplitude, an indicator of attentional modulation, correlated positively with progesterone. Further, cerebral asymmetry in ERP amplitude in the alpha frequency band following target presentation was restricted to luteal women. Critically, early follicular women responded slower to right hemifield compared to left hemifield targets. In late follicular or luteal women, we did not detect a right hemifield disadvantage. Progesterone correlated negatively with RTs in luteal women. Therefore, whereas our behavioral data indicate a functional cerebral asymmetry in early follicular women, EEG recording reveal a physiological cerebral hemisphere asymmetry in the alpha frequency band in luteal women. We assume that a progesterone-associated enhancement in synchronization of synaptic activity in the alpha frequency band in luteal women improves early categorization of visual targets in a cued spatial attention paradigm.FWF-W1233(VLID)223153

Polyvalent Cations as Permeant Probes of MIC and TRPM7 Pores

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg2+-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg2+ to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg2+ each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg2+ are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 μM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg2+ induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside

Distinct properties of CRAC and MIC channels in RBL cells.

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels

Polyvalent Cations as Permeant Probes of MIC and TRPM7 Pores

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg2+-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg2+ to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg2+ each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg2+ are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 μM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg2+ induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside

Distinct Properties of CRAC and MIC Channels in RBL Cells

abstract In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca 2 � release–activated Ca 2 � (CRAC) channels open in response to passive Ca 2 � store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg 2 � exposes an outwardly rectifying current (Mg 2 �-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg 2 � , spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg 2 �. Removal of internal Mg 2 � induced MIC current despite widely varying Ca 2 � and EGTA levels, suggesting that Ca 2 �-store depletion is not involved in activation of MIC channels. Increasing internal Mg 2 � from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg 2 � and Cs � carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg 2 � blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels. key words: store-operated channel • CRAC channel • Ca 2 � channel • TRPM7 • cation channe

cAMP-Dependent chloride conductance evokes ammonia-induced blebbing in the microglial cell line, BV-2

Cell blebbing is a key feature in apoptosis. Because blebbing dynamically alters cell volume and regulatory volume changes have been linked to chloride (Cl) channels, we evaluated an association between blebbing and Cl channels activity. We used scanning electron microscopy, confocal laser microscopy, and cell sorting to quantify cell volume and blebbing and whole-cell recording to characterize Cl(-) currents. We found that blockade of Cl channel activity as well as inhibition of adenylyl cyclase or protein kinase A (PKA) activity suppressed ammonia-induced blebbing in the microglia cell line, BV-2. In further experiments, we elucidated the common mechanism of Cl channel activity and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway on cell blebbing. These experiments indicated that perfusion of cells with cAMP or the catalytic subunit of PKA activated a Cl(-) current under normotonic conditions. The pharmacological profile (sensitivity to 5-nitro-2-(3-phenylpropylamino)benzoic acid [NPPB], flufenamic acid, and [(dihydroindenyl)oxy]alkanoic acid [DIOA]), outward rectification, and kinetic of the current were identical to the swelling-activated Cl channel. Superfusion of cells with ammonia elicited an outwardly rectifying current sensitive to Cl channel blockers. We propose that ammonia induces a PKA-dependent phosphorylation of Cl channels. Localized influx of Cl(-) is followed by influx of water, required for bleb expansion.(VLID)220917

Distinct Properties of CRAC and MIC Channels in RBL Cells

abstract In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca 2 � release–activated Ca 2 � (CRAC) channels open in response to passive Ca 2 � store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg 2 � exposes an outwardly rectifying current (Mg 2 �-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg 2 � , spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg 2 �. Removal of internal Mg 2 � induced MIC current despite widely varying Ca 2 � and EGTA levels, suggesting that Ca 2 �-store depletion is not involved in activation of MIC channels. Increasing internal Mg 2 � from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg 2 � and Cs � carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg 2 � blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels. key words: store-operated channel • CRAC channel • Ca 2 � channel • TRPM7 • cation channel Downloaded fro