Engineering new limits to magnetostriction through metastability in iron-gallium alloys.

Magnetostrictive materials transduce magnetic and mechanical energies and when combined with piezoelectric elements, evoke magnetoelectric transduction for high-sensitivity magnetic field sensors and energy-efficient beyond-CMOS technologies. The dearth of ductile, rare-earth-free materials with high magnetostrictive coefficients motivates the discovery of superior materials. Fe1-xGax alloys are amongst the highest performing rare-earth-free magnetostrictive materials; however, magnetostriction becomes sharply suppressed beyond x = 19% due to the formation of a parasitic ordered intermetallic phase. Here, we harness epitaxy to extend the stability of the BCC Fe1-xGax alloy to gallium compositions as high as x = 30% and in so doing dramatically boost the magnetostriction by as much as 10x relative to the bulk and 2x larger than canonical rare-earth based magnetostrictors. A Fe1-xGax - [Pb(Mg1/3Nb2/3)O3]0.7-[PbTiO3]0.3 (PMN-PT) composite magnetoelectric shows robust 90° electrical switching of magnetic anisotropy and a converse magnetoelectric coefficient of 2.0 × 10-5 s m-1. When optimally scaled, this high coefficient implies stable switching at ~80 aJ per bit.

Calcium misregulation and the pathogenesis of muscular dystrophy.

Although the exact nature of the relationship between calcium and the pathogenesis of Duchenne muscular dystrophy (DMD) is not fully understood, this is an important issue which has been addressed in several recent reviews (Alderton and Steinhardt, 2000a, Gailly, 2002, Allen et al., 2005). A key question when trying to understand the cellular basis of DMD is how the absence or low level of expression of dystrophin, a cytoskeletal protein, results in the slow but progressive necrosis of muscle fibres. Although loss of cytoskeletal and sarcolemmal integrity which results from the absence of dystrophin clearly plays a key role in the pathogenesis associated with DMD, a number of lines of evidence also establish a role for misregulation of calcium ions in the DMD pathology, particularly in the cytoplasmic space just under the sarcolemma. A number of calcium-permeable channels have been identified which can exhibit greater activity in dystrophic muscle cells, and exIsting evidence suggests that these may represent different variants of the same channel type (perhaps the transient receptor potential channel, TRPC). In addition, a prominent role for calcium-activated proteases in the DMD pathology has been established, as well as modulation of other intracellular regulatory proteins and signaling pathways. Whether dystrophin and its associated proteins have a direct role in the regulation of calcium ions, calcium channels or intracellular calcium stores, or indirectly alters calcium regulation through enhancement of membrane tearing, remains unclear. Here we focus on areas of consensus or divergence amongst the existing literature, and propose areas where future research would be especially valuable.

A decrease in membrane tension precedes successful cell-membrane repair.

We hypothesized that the requirement for Ca(2+)-dependent exocytosis in cell-membrane repair is to provide an adequate lowering of membrane tension to permit membrane resealing. We used laser tweezers to form membrane tethers and measured the force of those tethers to estimate the membrane tension of Swiss 3T3 fibroblasts after membrane disruption and during resealing. These measurements show that, for fibroblasts wounded in normal Ca(2+) Ringer's solution, the membrane tension decreased dramatically after the wounding and resealing coincided with a decrease of approximately 60% of control tether force values. However, the tension did not decrease if cells were wounded in a low Ca(2+) Ringer's solution that inhibited both membrane resealing and exocytosis. When cells were wounded twice in normal Ca(2+) Ringer's solution, decreases in tension at the second wound were 2.3 times faster than at the first wound, correlating well with twofold faster resealing rates for repeated wounds. The facilitated resealing to a second wound requires a new vesicle pool, which is generated via a protein kinase C (PKC)-dependent and brefeldin A (BFA)-sensitive process. Tension decrease at the second wound was slowed or inhibited by PKC inhibitor or BFA. Lowering membrane tension by cytochalasin D treatment could substitute for exocytosis and could restore membrane resealing in low Ca(2+) Ringer's solution.

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line.

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.

Increased activity of calcium leak channels caused by proteolysis near sarcolemmal ruptures.

Dystrophin, a 427 kD membrane-associated structural protein in muscle cells, is thought to confer strength to the myofiber sarcolemma and protect the membrane from rupture during the stresses of contraction. Dystrophin is absent in muscle cells from Duchenne muscular dystrophy (DMD) patients and mdx mice, a DMD model. Dystrophic muscle membranes undergo more frequent transient, nonlethal tears than normal cell membranes, especially during exercise. In addition, the mean open probability of a background ("leak") calcium channel is higher in dystrophic muscle cells, which leads to higher intracellular free calcium levels. Because elevated calcium levels may contribute to the eventual necrosis of muscle cells in DMD, we examined the possibility that the history of sarcolemmal rupture at a specific location on the membrane affects the open probability of nearby calcium leak channels. Membrane ruptures left by the excision of cell-attached patch-clamp electrodes were used to mimic natural tears. Patches made within 5 microns of excision sites contained channels with a fourfold greater mean open probability than channels in patches 50 microm away from ruptures. The increased leak channel activity near ruptures was seen continuously through the duration of the recordings and was not seen if the rupture was made in the presence of the protease inhibitor leupeptin. Calcium background channels proteolytically activated near ruptures, perhaps in a calcium-dependent manner, may thus be the lasting consequence of the weaker dystrophic sarcolemma, leading to chronically raised intracellular free calcium, increased calcium-dependent proteolysis and, eventually, necrosis.

Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction.

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.

Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes.

To estimate calpain proteolysis, we measured the hydrolysis rate of a fluorogenic calpain substrate in individual resting normal and dystrophic mdx mouse myotubes in culture. Hydrolysis rates were high during myoblast and myotube alignment and fusion. After alignment and fusion ceased, hydrolysis rates declined. For normal myotubes, hydrolysis remained low after the development of contractile activity. In contrast, after the development of contractile activity, dystrophic mdx myotubes had abnormally high levels of hydrolysis that were dependent on external calcium and that could be abolished by calpeptin, an inhibitor of calpain. We eliminated the direct effects of contraction during measurements of hydrolysis by the addition of tetrodotoxin. Substrate hydrolysis by lysosomes or proteosomes was controlled for using NH(4)Cl and clasto-lactacystin beta-lactone, respectively. Increased activity of the calcium-activated protease in mature mdx myotubes was linked to the abnormal activity of calcium-specific leak channels because an antagonist of these channels reduced the higher levels of hydrolysis in dystrophic myotubes to nearly normal levels. The abnormal activity of these channels is linked to an increased frequency of transient sarcolemmal disruptions in the more fragile mdx myotubes (, ). Treatment of mdx myotubes with a pro-drug of methylprednisolone also reduced calpain substrate hydrolysis to nearly normal levels. However, this inhibition only required 2.5 h of pretreatment, which was not long enough to act by the known effects of prednisolone on calcium homeostasis.

How calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes.

Duchenne muscular dystrophy patients lack the protein dystrophin which is an essential link in the complex of proteins that connect the cytoskeleton to the extracellular matrix. In mechanically stressed tissues such as muscle, transient sarcolemmal microdisruptions are normal, but in dystrophic muscle cells the frequency of these microdisruptions is greatly increased. Although both normal and dystrophic cells are able to actively repair these microdisruptions, calcium entry through the more frequent sarcolemmal microdisruptions of dystrophic cells results in an increased calcium-dependent proteolysis that alters the activity of the calcium leak channel. The accumulation of abnormally active calcium leak channels over time results in a gradual loss of calcium homeostasis and eventual cell death.

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.

The mechanism of facilitated cell membrane resealing.

Disruption of the plasma membrane evokes an exocytotic response that is required for rapid membrane resealing. We show here in Swiss 3T3 fibroblasts that a second disruption at the same site reseals more rapidly than the initial wound. This facilitated response of resealing was inhibited by both low external Ca2+ concentration and specific protein kinase C (PKC) inhibitors, bisindolylmaleimide I (BIS) and Gö-6976. In addition, activation of PKC by phorbol ester facilitated the resealing of a first wound. BIS and Gö-6976 suppressed the effect of phorbol ester on resealing rate. Fluorescent dye loss from a FM1-43 pre-labeled endocytotic compartment was used to investigate the relationship between exocytosis, resealing and the facilitation of resealing. Exocytosis of endocytotic compartments near the wounding site was correlated with successful resealing. The destaining did not occur when exocytosis and resealing were inhibited by low external Ca2+ concentration or by injected tetanus toxin. When the dye loaded cells were wounded twice, FM1-43 destaining at the second wound was less than at the first wound. Less destaining was also observed in cells pre-treated with phorbol ester, suggesting newly formed vesicles, which were FM1-43 unlabeled, were exocytosed in the resealing at repeated woundings. Facilitation was also blocked by brefeldin A (BFA), a fungal metabolite that inhibits vesicle formation at the Golgi apparatus. Lowering the temperature below 20 degrees C also blocked facilitation as expected from a block of Golgi function. BFA had no effect on the resealing rate of an initial wound. The facilitation of the resealing by phorbol ester was blocked by pre-treatment with BFA. These results suggest that at first wounding the cell used the endocytotic compartment to add membrane necessary for resealing. At a second wounding, PKC, activated by Ca2+ entry at the first wound, stimulated vesicle formation from the Golgi apparatus, resulting in more rapid resealing of the second membrane disruption. Since vesicle pools were implicated in both membrane resealing and facilitation of membrane resealing, we reasoned that artificial decreases in membrane surface tension would have the same result. Decreases in surface tension induced by the addition of a surfactant (Pluronic F68 NF) or cytochalasin D facilitated resealing at first wounding. Furthermore, Pluronic F68 NF restored resealing when exocytosis was blocked by tetanus toxin. These results suggest that membrane resealing requires a decrease in surface tension and under natural conditions this is provided by Ca2+-dependent exocytosis of new membrane near the site of disruption.

Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.

Lipofection of a cDNA plasmid containing the dystrophin gene lowers intracellular free calcium and calcium leak channel activity in mdx myotubes.

Muscle cells from Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse lack the protein dystrophin. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myofibers and cultured myotubes and is correlated with abnormally active calcium-specific leak channels. Higher [Ca2+]i results in greater calcium-dependent proteolysis, which may eventually lead to necrosis. We performed liposome-mediated transfection of a cDNA plasmid containing the full-length dystrophin gene into mdx myoblasts and examined the resting [Ca2+]i and leak channel activity of the resulting differentiated myotubes. Many myotubes from transfected cultures expressed dystrophin at levels similar to normal myotubes as determined by immunostaining. The intracellular free calcium, measured by emission ratio microfluorimetry using the calcium indicator fura-PE3, was significantly lower in the dystrophin-positive mdx myotubes than in untransfected control mdx myotubes. The mean open probability of the calcium leak channel was also reduced to a level similar to normal myotubes and significantly less than that for untransfected mdx myotubes. These results show that introduction of extrachromosomal copies of the full dystrophin gene to originally dystrophic muscle cells can correct the defect in calcium homeostasis that is hypothesized to lead to the muscle cell necrosis seen in DMD.

A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle.

There are conflicting reports regarding whether resting free calcium levels ([Ca2+]i) are elevated in dystrophic mouse (mdx) myotubes and adult myofibers. We reinvestigated this question and found several lines of evidence supporting the hypothesis that increased calcium influx via leak channels leads to increases in resting [Ca2+]i. 1) Step calibration of fura 2/free acid in myofibers with use of microinjected Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers revealed greater [Ca2+]i in dystrophic cells. Careful calibration of fura PE3-AM, a compartmentalization-resistant derivative of fura 2, also showed elevated [Ca2+]i in mdx myotubes. 2) Chronic, but not acute, application of tetrodotoxin reduced resting [Ca2+]i in dystrophic myotubes, suggesting that elevated resting [Ca2+]i is a consequence of previous long-term contractile activity. 3) Rates of manganese quenching of fura 2 fluorescence, an indirect indicator of calcium influx, were significantly higher in mdx myotubes and were increased by nifedipine, a calcium leak channel agonist. 4) Calcium leak channel activity, measured using patch clamping, was greater in the sarcolemma of adult non-enzyme-treated mdx myofibers.

A capacitative calcium current in cultured skeletal muscle cells is mediated by the calcium-specific leak channel and inhibited by dihydropyridine compounds.

Calcium stores from cultured skeletal muscle cells were depleted using cyclopiazonic acid (CPA), a reversible inhibitor of Ca2+-ATPases at the sarcoplasmic reticulum. Store depletion led to activation of the calcium-specific leak channel, as assayed using single-channel patch clamp analysis and rates of manganese influx and quenching of fura-2 fluorescence. Two novel dihydropyridine compounds inhibited this single-channel leak channel activity, the resting and depletion-induced manganese influx, and refilling of the CPA-depleted intracellular calcium store. These compounds represent the first antagonists for a calcium leak channel and for a channel that mediates a capacitative current. The development of the skeletal muscle capacitative current was inhibited by genistein, a tyrosine kinase inhibitor, but was not affected by okadaic acid, a phosphatase inhibitor, or econazole. Thus, the capacitative current in cultured skeletal muscle cells was mediated by the calcium leak channel and was inhibited by pharmacological antagonists and may provide a model system for uncovering the complete set of signals leading from store depletion to channel activation.

Calcium-regulated exocytosis is required for cell membrane resealing.

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

An increase in intracellular pH during neural induction in Xenopus.

In this paper, we show that an intracellular alkalinization of the dorsal ectoderm cells is among the earliest responses to neural induction in Xenopus. Planar explants of the dorsal marginal zone were prepared from embryos that had been microinjected during cleavage stages with the fluorescent pH indicator bis-carboxyethyl-carboxyfluorescein-dextran (BCECF-dextran), and intracellular pH (pHi) was monitored continuously by emission ratio microfluorimetry. During stage 10.5, the dorsal ectoderm cells undergo a sustained intracellular alkalinization of approximately 0.1 pH units in response to neural induction; in the absence of the inductive signal, the pH of the dorsal ectoderm cells decreases slightly. Ectoderm cells within planar explants of the ventral marginal zone show little change in pH during a similar period. This increase in intracellular pH is inhibited by 4, 4'-dihydrodiisothiocyanatostilbene-2, 2'-disulfonate (H2DIDS) or a low Na+/high Cl- medium, treatments that presumably affect anion transport. Under these conditions, expression of the anterior neural-specific homeobox gene engrailed is not detected, while the notochord-specific epitope recognized by the Tor-70 antibody is expressed in the presence of H2DIDS. This characteristic alkalinization is not evoked by pharmacological agents that reportedly alter ectodermal developmental pathways in Xenopus embryos, such as NH4Cl, phorbol esters, or cAMP-dependent protein kinase agonists. Our results suggest that an ionic regulatory event may participate in the regulation of gene expression in response to neural induction.

Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release.

After injury to the cell membrane, rapid resealing of the membrane occurs with little loss of intracellular contents. This process has been studied by measurement of the rate of dye loss after membrane puncture in both the sea urchin embryo and 3T3 fibroblasts. Resealing of disrupted cell membranes requires external calcium that can be antagonized by magnesium. Block of multifunctional calcium/calmodulin kinase, which regulates exocytotic vesicle availability at synapses, and of kinesin, which is required for outward-directed transport of vesicles, inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins B and A, suggesting that the two synaptosomal-associated proteins synaptobrevin and SNAP-25 also participate in resealing. This pattern of inhibition indicates that the calcium-dependent mechanisms for cell membrane resealing may involve vesicle delivery, docking, and fusion, similar to the exocytosis of neurotransmitters.