Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Voltage,reactive oxygen species and the influx of calcium

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca²⁺ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca²⁺ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca²⁺ influx, suggesting that these enzymes are involved in sensing Ca²⁺ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) encodes plant cell signals.¹ Reactive oxygen species (ROS) are potent regulators of the PM Ca²⁺ channels implicated in signalling and developmental increases in [Ca²⁺]_cyt.^1,2 Plasma membrane (PM) voltage (V_m) also plays a significant part in generating specific [Ca²⁺]_cyt elevations through the opening of voltage-gated Ca²⁺-permeable channels, allowing Ca²⁺ influx.^1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca²⁺ channels (HACCs),⁴ ROS-activated HACCs⁵ and depolarisation-activated Ca²⁺ channels (DACCs).⁶ The DACC characterisation pointed to the presence of a Cl⁻-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.⁶ This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.⁷ Previous studies have shown that root hair PM also harbors K⁺ channels (mediating inward or outward flux)^8–10 and a H⁺-ATPase.¹¹ A key problem to address now is how these transporters interact to generate and be influenced by PM V_m, thus gating and in turn being regulated by their companion Ca²⁺ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between V_m and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.¹² In the “pump state” the H⁺-ATPase predominates, there is net H⁺ efflux and the hyperpolarized V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 and oscillations between these states may explain the temporal changes in H⁺ flux recently observed at the apex of growing Arabidopsis root hairs.¹⁵ Peaks of H⁺ influx may reflect a depolarized V_m that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca²⁺ influx it permits could be amplified through intracellular release.The PM H⁺-ATPase traditionally lies at the core of models of voltage and ionic flux^14,16 but in terms of [Ca²⁺]_cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.⁵ AtrbohC is implicated in the transition to polar growth at normal extracellular pH⁵ and also osmoregulation.¹⁷ NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e⁻ efflux systems,¹⁸ their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.¹⁹ Two H⁺ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e⁻ efflux by animal NADPH oxidases is fairly constant over negative V_m and decreases at very depolarized V_m.²⁰ AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H⁺ flux oscillations.¹⁵ The latter suggests that AtrbohC function does in some way affect V_m, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.²¹NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency^22,23 to signal stress conditions. Blockade of PM Ca²⁺ channels by lanthanides increases superoxide production in tobacco suspension cells.²⁴ This suggests that NADPH oxidases are involved in sensing the cell''s Ca²⁺ status and the prediction would be that extracellular Ca²⁺ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).^25,26 Lowering extracellular Ca²⁺ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca²⁺ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca²⁺]_cyt²⁷ (Fig. 1). However, reduction of Ca²⁺ influx should promote voltage hyperpolarisation (just as block of K⁺ influx causes hyperpolarisation in root hairs²⁸) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity⁵ to increase Ca²⁺ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl₂, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ to increase [Ca²⁺]_cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to V_m, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca²⁺]_cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing V_m and [Ca²⁺]_cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.²⁹ While there is still much to do on the “inventory” of PM transporters involved in Ca²⁺ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action.     

Control of depolarization-evoked presynaptic neurotransmitter release by Cav2.1 calcium channel: old story, new insights

Depolarization-evoked synaptic transmission relies on the Ca²⁺-regulated release of quantal packets of neurotransmitters following the fusion of synaptic vesicles with the presynaptic plasma membrane. It is well known that neuronal voltage-gated Ca²⁺ channels (VGCC), mainly of the Ca_V2.1 and Ca_V2.2 subtypes, play a key role in the first steps of this process, by controlling extracellular Ca²⁺ influx into active zones of the synapse. These channels are in close association with the vesicle machinery and interact with several members of SNARE proteins (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) including syntaxin 1A/1B and SNA P-25 (Q-SNARE s), and synaptotagmin 1 and synaptobrevin 2 (R-SNARE s) (reviewed in ref. ¹). All bind to the synprint (synaptic protein interaction) motif within the intracellular II -III linker of Ca_V2.1 and Ca_V2.2 channels and are responsible for a bidirectional coupling (i) linking the Ca²⁺ influx with the synaptic vesicle release machinery, which is essential for efficient, fast and spatially delimited neurotransmitter release² and (ii) providing regulation of Ca²⁺ channel activity and thus of Ca²⁺ influx.³Key words: calcium channel, Ca_V2.1 channel, P/Q channel, syntaxin, synaptotagmin, SNAP25, exocytosis, synaptic transmissionSeveral studies have proposed that synaptotagmin 1 is the Ca²⁺ sensor for release, linking Ca²⁺ influx to vesicle fusion (reviewed in ref. ⁴). Synaptotagmin 1 has two repeating domains that are rich in negative charges (C2A and C2B), each capable of binding Ca²⁺ ions. It is commonly thought that following Ca²⁺ entry through VGCCs, Ca²⁺ ions bind to C2A and C2B domains, allowing insertion of the Ca²⁺ binding loops of C2A domain in the target bilayer. This then pins the vesicle to the plasma membrane to trigger exocytotic fusion. This view was supported by a point mutation in the C2A domain of synaptotagmin 1 that caused a decrease in Ca²⁺ affinity with a concomitant decrease of neurotransmitter release.⁵ However, despite the fact that synaptotagmin 1 represents the most popular candidate for Ca²⁺ sensor, the initial Ca²⁺ binding event, which occurs during the dynamic process of release is at the EEEE locus within the Ca²⁺ channel itself. This makes the Ca²⁺ channel an excellent candidate for serving as a Ca²⁺ sensor of secretion.⁶Over the past few years, the group of Daphne Atlas has performed extensive studies to differentiate the role of Ca²⁺ binding at the pore of the channel from Ca²⁺ binding to intracellular proteins during evoked-neurotransmitter release. Substituting extracellular Ca²⁺ by lanthanum (La³⁺), a trivalent cation that effectively binds to the EEEE locus of VGCCs but is unable to permeate through the channel, is sufficient to support depolarization-evoked release of catecholamine in PC12 and primary chromaffin cells, as well as insulin release in pancreatic and insulinoma cells. These results led to the suggestion that evoked release may be dependent on ion channel pore occupancy as opposed to cation influx and elevation of intracellular Ca²⁺ concentration.^7–9 This model was further supported by experiments in which depolarization-evoked secretion of catecholamine in chromaffin cells was supported by Ca²⁺ bound at the selectivity filter of a non-conducting Ca_V1.2 channel.¹⁰ These studies are consistent with the proposal that conformational changes subsequent to Ca²⁺ binding at the selectivity filter of the channel are the primary trigger of secretion, whereas synaptotagmin 1 is associated with the channel and acts as a vesicle docking protein (reviewed in ref. ¹¹).In a recent issue of Channels, Cohen-Kutner et al. extended this concept to the neuronal Ca_V2.1 channel.¹² Using the two-electrode voltage-clamp technique on BAPTA-injected Xenopus oocyte expressing the human Ca_V2.1 channel (in combination with β₃ and α₂δ auxiliary subunits), the authors show that overexpression of syntaxin 1A (Stx1A) depresses whole-cell inward barium (Ba²⁺) current in a dose-dependent manner (Fig. 1, reviewed in ref. ¹²). As previously reported by Bezprozvanny et al.³ this effect is mainly due to a hyperpolarized shift of the steady-state inactivation curve, which decreases the number of available channels at typical resting membrane potentials. A recovery of channel activity is observed following co-expression of botulinium neurotoxin C1 (BoNT/C1) (Fig. 3, reviewed in ref. ¹²). In contrast, expression of the other Q-SNARE protein SNAP-25 drastically increases inward Ba²⁺ current (Fig. 2, reviewed in ref. ¹²). However, when both Q-SNARE proteins are co-expressed, Ca_V2.1 channel recovers wild-type P/Q kinetics and current amplitude (Fig. 2, reviewed in ref. ¹²). Similarly, increases in P/Q currents by expressing the R-SNARE synaptobrevin (VAMP-2) are reversed by the Q-SNARE proteins (Fig. 4, reviewed in ref. ¹²). Taken together these results suggest that: (i) when expressed in BAPTA injected Xenopus oocyte, each of the SNARE proteins is able to modulate the kinetic properties of Ca_V2.1 channel and (ii) when co-expressed, SNARE proteins no longer affect channel activity but rather form a Ca²⁺-independent excitosome complex with a fully functional channel. These data fit nicely with previous work from the Catterall laboratory on P/Q-type channels,¹³ and with previous work on N-type channels.¹⁴To investigate the relevance of Ca_V2.1 channel interaction with SNARE proteins for depolarization-evoked secretion, membrane capacitance changes induced in Xenopus oocytes were monitored in the presence of extracellular Ca²⁺, as previously shown for Ca_V1.2 and Ca_V2.2.¹⁵ While expression of Ca_V2.1 alone in this reconstituted release assay produced only a small change in capacitance, coexpression with the SNARE proteins efficiently induced a BoNT/C- and BoNT/A-sensitive membrane fusion, particularly when all SNARE proteins were co-expressed, i.e., when all members of the excitosome complex are present (Fig. 5, reviewed in ref. ¹²). Hence, increasing the amount of excitosome promotes the capability of Ca_V2.1 channels to produce evoked-secretion, probably by increasing the number of functional excitosome complexes (Fig. 6, reviewed in ref. ¹²).In summary, Cohen-Kutner et al. provide evidence that when expressed in Xenopus oocyte (and possibly in other cellular systems), Ca_V2.1 channels could associate with SNARE proteins at resting intracellular Ca²⁺ concentrations, resulting in tethering the vesicle to the channel and thereby generating docked but non-releasable vesicles. Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Syt1 C2 domains. The fusion of releasable vesicles requires a conformational change of the complex that occurs within the channel itself, during an incoming action potential (Fig. 1).Open in a separate windowFigure 1A putative model of functional coupling between Ca_V2.1 channel and vesicle release machinery. At resting membrane potential, Ca_V2.1 channel associates with SNARE proteins to form an excitosome complex, in turn generating docked but non-releasable vesicle (A). Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Synaptotagmin 1 C2 domains (B). The fusion of the releasable vesicle requires a conformational change of the excitosome complex that occurs within the channel itself, during an incoming action potential (C).The concept that Ca_V2.1 channels, besides sustaining Ca²⁺ influx, could also work as a molecular on/off-switch of secretion by controlling the ultimate stage of the process (i.e., the conformational change of the releasing complex) is intriguing and is worthy of further investigation. To better dissociate secretion events linked to Ca²⁺ entry through Ca_V2.1 channel from those induced by conformational changes of the channel, it would be necessary to measure secretion in the presence of a non-permeant cation such as La³⁺. Furthermore, one would also need to evaluate mediation of secretion by a non-conducting Ca_V2.1 channel, as already done for L-type channels (Ca_V1.2).^7,9,10 Moreover, the possibility that Ca_V2.1 channels could control secretion via a conformational change of the releasing complex raises questions concerning the preferential channel-gating mode controlling this process. It was recently shown that application of the gating modifier BayK 8644 to non-conducting Ca_V1.2 channels modifies secretion kinetics of catecholamine in chromaffin cells.¹⁶ It is also well known that the auxiliary β-subunit of VGCCs modulates Ca_V2.1 gating modes.¹⁷ Therefore, comparing secretion mediated by a non-conducting Ca_V2.1 channel in the presence of different types of β-subunits would provide important information on the molecular mechanisms through which Ca_V2.1 channels control evoked-secretion, both at the fundamental and physiopathological levels.In conclusion, since the pioneering work by Katz and Miledi in 1967 on the importance of the extracellular Ca²⁺ in the “electro-secretory” process,¹⁸ the identification of the calcium channel as the Ca²⁺ sensor of secretion is one of the most recent and exciting steps that have been made in the understanding of the molecular aspects of the mechanisms involved in the control of depolarization-evoked neurotransmitter release.     

Step by Step: Deciphering Ion Transport in the Root Xylem Parenchyma

Proton pumps produce electrical potential differences and differences in pH across the plasma membrane of cells which drive secondary ion transport through sym- and antiporters. We used the patch-clamp technique to characterize an H⁺-pump in the xylem parenchyma of barley roots. This cell type is of special interest with respect to xylem loading. Since it has been an ongoing debate whether xylem loading is a passive or an active process, the functional characterization of the H⁺-pump is of major interest in the context of previous work on ion channels through which passive salt efflux into the xylem vessels could occur. Cell-type specific features like its Ca²⁺ dependence were determined, that are important to interpret its physiological role and eventually to model xylem loading. We conclude that the electrogenic pump in the xylem parenchyma does not participate directly in the transfer of KCl and KNO₃ to the xylem but, in combination with short-circuiting conductances, plays a crucial role in controlling xylem unloading and loading through modulation of the voltage difference across the plasma membrane. Here, our recent results on the H⁺ pump are put in a larger context and open questions are highlighted.Key Words: plant nutrition, H⁺-ATPase, anion conductance, K⁺ channel, electrophysiology, signaling networkThe root xylem parenchyma is of major interest with respect to nutrient (and signal) traffic between root and shoot. One of its main functions appears to be xylem loading. However, the cell walls of the vascular tissue provide apoplastic paths between xylem and phloem that represent the upward and downward traffic lanes, allowing nutrient circulation¹ (Fig. 1). Therefore mechanisms for ion uptake and for ion release must exist side by side. In the last 15 years major progress has been made in the investigation of transport properties of xylem-parenchyma cells, and both uptake and release channels and transporters were identified. Today, we have good knowledge on the role of K⁺ and anion conductances in xylem loading with salts.² Note, that from the functionally well characterized conductances only the molecular structure of K⁺ channels is known. In contrast, many transporters are identified on the molecular level, but functional data are scarce.Open in a separate windowFigure 1Distribution of tissues in the periphery of the stele. The stippled area marks the region from which early metaxylem protoplasts originated. E, Endodermis with Casparian strip; eMX, ‘early’ metaxylem vessel; IMX, ‘late’ metaxylem vessel; Mph, metaphloem (sieve tube); Pph, protophloem (sieve tube); P, pericycle; Cx, cortex. Symplasmic and apoplasmic transport routes are indicated in red and black, respectively. The Casparian strip prevents apoplastic transport into the stele. Plasmodesmata are shown exemplarily for the indicated symplastic pathway. All cells of the symplast are connected via plasmodesmata. Sites of active uptake into the root symplast and of release into the stelar apoplast are indicated by a black and an orange arrow. Modified from Wegner and Raschke, 1994.³A challenging question to deal with was the dispute about xylem loading with ions being a passive or active process. While it is clear that energy through electrogenic H⁺ efflux is needed to take up nutrient ions from the soil against their electrochemical gradient into the cortical symplast, it has been a matter of debate if ion release into xylem vessels also is energy-linked or if the electrochemical potentials of ions are raised high enough to allow a thermodynamically passive flux.^2,3 The Casparian strip prohibits apoplastic transport of nutrients into the stele and electrically insulates the stelar from the cortical apoplast. Therefore the electrical potential difference of the cells in the xylem parenchyma could be independent from the cortical potential difference but be subject to control, for instance, from the shoot.⁴ Indeed, evidence points to xylem loading as a second control point in nutrient transfer to the shoot.^5,6 The identification and characterization of K⁺ and anion conductances clearly showed that release of KCl and KNO₃ into the xylem can be passive through voltage-dependent ion channels.^2,3,7–9 No need appeared for a pump energizing the transfer of salts to the xylem.However, H⁺ pumps are ubiquitous. H⁺-ATPases are encoded by a multigene family and heterologous expression in yeast showed that isoforms have distinct enzymatic properties.^10,11 As the example of the amino acid transporter AAP6 from the xylem parenchyma shows, a cell-type specific functional characterization of transporters is essential to draw conclusions on their physiological role. AAP6 is the only member of a multigene family with an affinity for aspartate in the physiologically relevant range. The actual apoplastic concentration of amino acids and the pH will determine what is transported in vivo.^12,13 Xylem-parenchyma cells of barley roots were strongly labelled by antibodies against the plasma membrane H⁺-ATPase.¹⁴ In a recent publication in Physiologia Plantarum we report the functional analysis of the electrogenic pump from the plasma membrane of xylem parenchyma from barley roots that was done with the patch-clamp technique after specific isolation of protoplasts from this cell type. It displayed characteristics of an H⁺-ATPase: current-voltage relationships were characteristic for a ‘rheogenic’ pump¹⁵ and currents were stimulated by fusicoccin or by an enlarged transmembrane pH gradient and inhibited by dicyclohexylcarbodiimide (DCCD). Importantly, it also showed distinct characteristics. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Noteworthy, K⁺ and anion conductances from the same cell type are controlled by intracellular [Ca²⁺]^7,9 (Fig. 2). It was proposed that the effect of abscisic acid (ABA) on anion conductances is mediated via an increase in the cytosolic Ca²⁺ concentration.¹⁶ Very likely stelar H⁺ pumps are stimulated by ABA.¹⁷ Thus, a Ca²⁺ independent control has to be hypothesized in this case.Open in a separate windowFigure 2Control of ion conductances in the plasma membrane of xylem-parenchyma cells. Arrowheads indicate stimulation and bars indicate inhibition by an increase in cytosolic [Ca²⁺],^7,9,16 by ABA,^16,17,21 by cytosolic and apoplastic acidification,^4,22 by G-proteins²³ and by an increase in apoplastic [K⁺]⁷ and [NO₃⁻].²⁴ Apoplastic [K⁺] and [NO₃⁻] modify the voltage dependence exerting negative feedback on K⁺ efflux and a positive feedback on NO₃⁻ efflux. Abscisic acid has an immediate effect on ion channel activity, most likely via [Ca²⁺], and causes a change in gene expression as indicated by circles (up) and bars (down). ABA perception is not clear. A Ca²⁺ influx could occur through a hyperpolarization activated cation conductance (HACC).^16,25 Cation transporters are NORC, nonselective cation conductance, KORC, K⁺-selective outwardly rectifying conductance (=SKOR⁸), and KIRC, K⁺-selective inwardly rectifying conductance, and anion conductances with different voltage-dependencies and gating characteristics are X-QUAC, quickly activating anion conductance, X-SLAC, slowly activating anion conductance, and X-IRAC, inwardly rectifying anion channel.^2,3,9,16,26 Transported ions and direction of flux are plotted.To date, we know that besides Ca²⁺ and abscisic acid also the pH, nonhydrolyzable GTP analogs and extracellular NO₃⁻ and K⁺ affect membrane transport capacities of root xylem-parenchyma cells (Fig. 2). Other control mechanisms by metabolites, the redox potential and phytohormones have to be included, especially if they represent signals in xylem loading or root-shoot communication. The composition of the xylem sap changes during the course of a day, depending on nutrient supply and various stresses, and the apoplastic ion concentration is considered to be an important factor in ion circulation.^6,18,19 ABA is such a signal. It is known to increase solute accumulation within the root by inhibiting release of ions into the xylem.¹⁷ Any change in transport activity has an impact on the membrane potential. This again determines whether salt release or uptake takes place. Passive salt release is restricted to a limited range of membrane potentials in which conductances for anions and cations are active simultaneously, that is with depolarization. Negative membrane voltages will be required for reabsorption of NO₃⁻ by a putative NO₃⁻/H⁺-symporter and for the uptake of K⁺ and amino acids.^3,13 As shown in our recent paper, the balance between the activities of the H⁺-pump and the anion conductances could affect the position between a depolarized and a hyperpolarized state of the parenchymal membrane. Thus, H⁺ pump activity is crucial in membrane voltage control. Furthermore, the simultaneous activities of H⁺ pumps and anion conductances make the generation of a high pH gradient possible, whilst maintaining electroneutrality. The proton gradient could be used for ion transport through cotransporters and antiporters as suggested for the loading of borate into the xylem through the boron transporter BOR1.²⁰ So we are on the way to decipher xylem loading in roots and this exciting field will also provide information about small-scale nutrient cycling and root-shoot communication. To determine how the activities of pumps, channels and transporters are adjusted among each other is the next challenge. Further insight has to be obtained by experimentation as well as by biophysical modeling.     

Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca2+ exchangers

Tonoplast-localised proton-coupled Ca²⁺ transporters encoded by cation/H⁺ exchanger (CAX) genes play a critical role in sequestering Ca²⁺ into the vacuole. These transporters may function in coordination with Ca²⁺ release channels, to shape stimulus-induced cytosolic Ca²⁺ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant''s stress response. A common feature of these mutants was the perturbation of H⁺-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca²⁺/H⁺ exchangers and H⁺ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca²⁺ flux. These results suggest cautious interpretation of mutant Ca²⁺/H⁺ exchanger phenotypes that may be due to either perturbed Ca²⁺ or H⁺ transport.Key words: abiotic stress, Ca²⁺ transport, Ca²⁺/H⁺ exchanger, H⁺-ATPase, Na⁺ transport, pH, salt stress, vacuoleCa²⁺ plays a fundamental role in the plant cell, functioning as a highly versatile second messenger controlling a multitude of cellular reactions and adaptive responses.^1,2 Ca²⁺ dynamics are maintained by precise interplay among transporters involved in its release from or uptake into Ca²⁺ stores. The vacuole, as the largest internal Ca²⁺ pool, is assumed to play a major role in Ca²⁺ signalling, and has been shown to be the source of Ca²⁺ release following various abiotic stresses such as cold and osmotic stress.^3,4 Rapid, stimulus-induced release of Ca²⁺ from the vacuole is attributable to selectively permeable Ca²⁺ channels, however, the identity of candidate genes encoding this mechanism remains contested.^5,6 Better understood, are the two major vacuolar uptake mechanisms; P-type Ca²⁺ pumps, including ACA4 and ACA11, which mediate high-affinity Ca²⁺ uptake, and a family of cation/H⁺ exchangers (CAX), responsible for lower-affinity but high-capacity Ca²⁺ uptake.^7,8 While Ca²⁺ pumps rely directly on the hydrolysis of ATP to drive Ca²⁺ uptake, Ca²⁺/H⁺ exchangers are energized indirectly by the pH gradient generated by electrogenic H⁺ pumps located on the tonoplast, including the vacuolar-type H⁺-ATPase (V-ATPase).⁹With the aim of further understanding the role of specific CAX isoforms in Arabidopsis, we and others have recently characterized CAX mutants and overexpression lines and observed a variety of phenotypes, including altered response to abiotic stresses.^10–14 While some phenotypes are identical among different CAX mutants, others are specific to individual lines.¹⁴ Moreover, these analyses have highlighted the interplay of these transporters with H⁺ pumps at both the tonoplast and the plasma membrane. Overexpression of CAX1 in Arabidopsis results in increased activity of the V-ATPase, whereas mutations in CAX1 cause a concomitant decrease in measured V-ATPase activity (Fig. 1).¹¹ Similar reductions in V-ATPase activity are also observed in cax2 and cax3 mutant plants but to a lesser extent,^12,13 and a significant reduction is observed in a cax1 cax3 double knockout line.¹³ At the plasma membrane, P-type H⁺-ATPase (P-ATPase) activity is increased in cax1 but decreased in cax3 (Fig. 1).¹⁴ Indeed cax3 lines appeared more sensitive to changes in the pH of the growth media.¹⁴ This implies that unlike cax1, cax3 is less efficient at cytoplasmic pH adjustment. Another intriguing observation is that activity of the H⁺-pyrophosphatase (H⁺-PPase) at the tonoplast is largely unaltered following CAX gene deletion. While overexpression of the Arabidopsis H⁺-PPase AVP1 leads to increased Ca²⁺/H⁺ exchange activity,^15,16 there is little alteration in H⁺-PPase activity following perturbed expression of CAX1 or CAX2.^11,12 Thus, this feedback interplay appears to exist only between exchangers and H⁺-ATPases.Open in a separate windowFigure 1Tonoplast H⁺-ATPase (V-ATPase) activity and plasma membrane H⁺-ATPase (P-ATPase) activity in wild type Arabidopsis (ecotype Col-0) and Arabidopsis lines with manipulated tonoplast Ca²⁺/H⁺ exchange activity. 35S::CAX1 and 35S::CAX2 denote lines that overexpress a constitutively active N-terminally truncated CAX1 or CAX2 construct driven by the CaMV 35S promoter in the cax1-1 or cax2-1 mutant background, respectively. V-ATPase H⁺-transport activity was measured by the ATP-dependent quenching of quinacrine fluorescence, and rates of bafilomycin-sensitive, vanadate-resistant hydrolytic activity of the V-ATPase were determined in isolated tonoplast membranes, as described in refs. ¹¹ and ¹³. Rates of vanadate-sensitive, bafilomycin- and azide-resistant hydrolytic activity of the P-ATPase were determined in isolated plasma membranes, as described in ref. ¹⁴. Results are shown as % of wild type (Col-0) ATPase activity and are means ± SE of 3–4 independent experiments. Data taken and modified from refs. ^11–14.The V-ATPase is important not only for maintenance of a pH gradient across the tonoplast, but also in maintenance of Golgi structure, endocytosis and secretory trafficking.^17,18 The V-ATPase is localised at the Golgi, endoplasmic reticulum and endosomes, in addition to the tonoplast.⁹ The det3 mutant, with a mutation in subunit C (VHA-C), has a 40–60% reduction in V-ATPase activity, but numerous severe developmental phenotypes.¹⁹ In contrast, the cax1 and cax1 cax3 mutants have a reduction in V-ATPase activity equivalent to det3 (Fig. 1), but the morphological phenotypes are not as pronounced.¹³ It is therefore likely that reduction of tonoplast Ca²⁺/H⁺ exchange primarily affects tonoplast V-ATPase activity, while V-ATPase activity in the secretory pathway is unperturbed. The V-ATPase is a multi-subunit protein and some of these subunit gene products appear to be either tonoplast-specific or tonoplast-enriched. Mutations in tonoplast subunits may cause defective V-ATPase activity only at the tonoplast.⁹ It will be of interest to see whether such tonoplast-specific V-ATPase mutants phenocopy the cax mutants, and possess perturbed Ca²⁺/H⁺ exchange activity and altered abiotic stress responses.CAX-mediated transport may alter both cytoplasmic and lumenal pH, as well as intracellular Ca²⁺ gradients. In the case of the V-ATPase, evidence is emerging for a role not only in the generation of a pH gradient across membranes, but also in the direct sensing of pH within the compartment,^20,21 creating a feedback mechanism which regulates pump activity. Thus, in cax1 lines, abnormal acidification of the lumen is detected by the V-ATPase resulting in a dampening of its activity. This would conserve ATP, which we postulate could be utilized to drive the tonoplast Ca²⁺ pump which itself is upregulated in cax1 as a compensatory mechanism to correct perturbations in the Ca²⁺ gradient.¹¹ In the case of cax1, this in turn may signal the P-ATPase to remove surplus H⁺ from the cytoplasm, triggering its upregulation (Fig. 1). However, not all CAX mutants show this complex H⁺ feedback mechanism.Co-ordinate downregulation of the V-ATPase in the cax1 mutant lines may also be a result of activity of the SOS2 kinase. This Ser/Thr kinase, which specifically interacts with the N-terminus of CAX1 resulting in Ca²⁺/H⁺ exchange activation,²² upregulates V-ATPase activity through interactions with the VHA-B regulatory subunit.²³ Loss of CAX1 may be signalling to the V-ATPase through changes in SOS2 activity resulting in a compensatory downregulation of the pump. It is tempting to speculate that SOS2 may signal the alteration in P-ATPase activity, as it is known to regulate other plasma membrane proteins, notably the Na⁺/H⁺ exchanger SOS1.²⁴ It will be interesting to determine if SOS2 and the P-ATPase interact directly. It is notable, however, that SOS2 does not appear to interact with CAX3,²² while P-ATPase activity is reduced in cax3 plants.¹⁴Our recent results indicate there are at least two modes by which Ca²⁺/H⁺ exchangers can mediate adaptive responses to stress: direct manipulation of cytosolic Ca²⁺ and indirect feedback of H⁺ flux (Fig. 2). For example, salt stress responses are likely controlled via the generation of a specific cytosolic Ca²⁺ signature, which mediates a downstream signalling pathway. CAX3 appears to be the principle isoform providing tonoplast Ca²⁺/H⁺ exchange in response to salt stress.¹⁴ Disruption of CAX3-mediated tonoplast Ca²⁺ transport and the alteration of cytosolic Ca²⁺ dynamics may therefore alter the plant''s normal response to salt stress (Fig. 2). Maintenance of H⁺ gradients at both the vacuole and plasma membrane are also critical for salt tolerance, such that salt treatment upregulates V-ATPase and P-ATPase activity.²⁵ This energizes Na⁺ efflux from the cytosol mediated by Na⁺/H⁺ exchangers at the plasma membrane and the tonoplast.^24,26 Therefore downregulation of H⁺ pumps at both membranes in the cax3 mutant is likely to perturb the ability of the cell to remove Na⁺ (Fig. 2). Further analysis of cax mutants, P-ATPase mutants, and tonoplast-specific V-ATPase mutants will be required to determine whether many of the phenotypes resulting from lack of Ca²⁺/H⁺ exchange activity are due to altered Ca²⁺ transport or H⁺ transport.Open in a separate windowFigure 2Model of tonoplast Ca²⁺/H⁺ exchanger interaction with H⁺ pumps in response to salt stress. (A) In response to NaCl treatment, an elevation in cytosolic Ca²⁺ will occur, possibly due to vacuolar Ca²⁺ release.³ Increased CAX3-mediated Ca²⁺/H⁺ exchange activity¹⁴ will sequester excess Ca²⁺ into the vacuole. CAX3 may be involved in the generation of a specific Ca²⁺ signature that is recognised by the cell to mediate downstream stress responses. In addition, salt stress will lead to upregulation of H⁺ pumps at both the plasma membrane and the tonoplast (P-ATPase and V-ATPase)²⁵ which will in turn energize Na⁺/H⁺ exchange activity encoded by SOS1 and NHX1, promoting Na⁺ efflux from the cell. Increased V-ATPase activity may also upregulate Ca²⁺/H⁺ exchange. Activity of SOS1 requires activation by the kinase SOS2²⁴ which may also regulate tonoplast Na⁺/H⁺ exchange and V-ATPase activity.^23,24 (B) In a cax3 knockout mutant experiencing salt stress, the cytosolic Ca²⁺ elevation may be sustained due to reduced vacuolar Ca²⁺ sequestration and normal salinity-induced Ca²⁺ signalling pathways may be perturbed. Lack of CAX3 downregulates both P-ATPase and V-ATPase activity¹⁴ thereby reducing energization of the plasma membrane and tonoplast Na⁺/H⁺ exchangers and reducing Na⁺ efflux from the cell. Some energization of H⁺-coupled processes at the vacuole may be maintained by residual H⁺-pyrophosphatase (V-PPase) activity.The phenomenon observed between tonoplast Ca²⁺/H⁺ exchangers and H⁺ pumps at both the tonoplast and plasma membranes, suggesting a co-ordinate regulation between several transporters, is not solely restricted to this family of transporters. It is a common observation emerging from recent research on the manipulation of tonoplast transporters. Several labs have reported unpredictable phenotypes associated with ectopic expression of tonoplast proteins.^26–28 Until we understand the significance of these types of unexpected interactions, it is naïve to believe that engineering plants will provide predictable results.     

Induction as well as suppression: How aphid saliva may exert opposite effects on plant defense

Aphids ingest from the sieve tubes and by doing so they are confronted with sieve-tube occlusion mechanisms, which are part of the plant defense system. Because aphids are able to feed over longer periods, they must be able to prevent occlusion of the sieve plates induced by stylet penetration. Occlusion probably depends upon Ca²⁺-influx into the sieve element (SE) lumen. Aphid behavior, biochemical tests and in vitro experiments demonstrated that aphid''s watery saliva, injected during initial phase of a stylet penetration into the SE lumen, contains proteins that are able to bind calcium and prevent calcium-induced SE occlusion. In this addendum, we speculate on the consequences of saliva secretion for plant resistance. (a) The release of elicitors (e.g., oligogalacturonides) due to cell wall digestion by gel saliva enzymes may increase the resistance of cortex, phloem parenchyma cells and companion cells (CC) around the puncture site. (b) Ca²⁺-binding by aphid watery saliva may suppress the local defense responses in the SEs. (c) Signaling cascades triggered in CCs may lead to systemic resistance.Key words: aphid saliva, calcium binding, elicitor, oligogalacturonides, local plant defense, systemic plant defense, phloem translocation, aphid/plant-interactionAfter having penetrated the sieve-element (SE) plasma membrane, aphids encounter unspecific wound-induced occlusion reactions to prevent sap leakage.^1–4 Occlusion mechanisms by callose, structural P-proteins and forisomes are likely induced by a sudden calcium influx into the sieve-tube lumen.⁵ Calcium possibly enters the sieve-tube lumen through the stylet wounding-site in the plasma membrane and/or stretch-activated calcium-channels.^6–8 After SE penetration, aphids secrete watery saliva that contains calcium-binding proteins presumed to sabotage sieve-plate occlusion.^9,10We demonstrated that Megoura viciae (Buckton) is most likely able to prevent or reduce sieve-tube occlusion in Vicia faba by secretion of watery saliva. By in vitro confrontation of isolated forisomes, protein bodies responsible for sieve-tube occlusion in Fabaceaen,⁵ and watery saliva concentrate, we were able to show that salivary proteins convey forisomes from a dispersed (+Ca²⁺) into a condensed (−Ca²⁺) state.¹⁰ The dispersed forisome functions in vivo as a plug, leading to stoppage of mass flow.⁵This in vitro evidence was corroborated by aphid behavior in response to leaf tip burning, which triggers an electrical potential wave (EPW) along the sieve tubes. Such an EPW induces Ca²⁺-influx and corresponding SE occlusion along the pathway.¹¹ The passage of the EPW is associated with a prolonged secretion of watery saliva of aphids. This is interpreted as an attempt to unplug the SEs by calcium binding.¹⁰ Similar behavioral changes in response to leaf-tip burning were observed in an extended set of aphid/plant species combinations, indicating that attempted sabotage of sieve-tube occlusion by aphid saliva is a widespread phenomenon (unpublished).Aphid feeding was reported to induce local (on the same leaf) and systemic (in distant leaves) reactions of the host plant. The local response led to enhanced feeding,^12–14 while the systemic response showed reduced ingestion and extended periods of watery saliva secretion in sieve tubes distant from previous feeding sites.^12–14 These contrasting observations were described to be independent of the aphid species.¹³ The question arises how aphids induce these seemingly opposite plant responses?The aphid stylet pushing forward through cortical and vascular tissue is surrounded by a sheath of gel saliva, secreted into the apoplast.^15,16 Gel saliva contains cellulase and pectinase that amongst others produce oligogalacturonides (OGs) along the stylet sheath by digestion of cell wall material.^17,18 Usually, OGs act as elicitors, triggering a variety of plant responses against pathogens and insects in which the activation of calcium channels is involved.^19,20 This seems to conflict with a suppression of resistance as observed for the impact of watery saliva in SEs.¹⁰ We will make an attempt to explain this paradoxon.OG induced defense responses may be triggered in all cell types adjacent to the salivary sheath (Fig. 1). Because watery saliva is only secreted briefly into these cells, which are punctured for orientation purposes (Hewer et al., unpublished), it seems unlikely that OG induced defense is suppressed here by saliva-mediated calcium binding.¹⁵ The diffusion range of OGs may be restricted to the close vicinity of the stylet sheath leading to an enhanced regional defense with a limited sphere of action (Fig. 1). Because the settling distance of aphids is restricted by their body size (1–10 mm),²¹ aphids feeding on the same leaf are probably hardly confronted with the regional defense induced by another aphid (Fig. 1). Otherwise, they would show an increased number of test probes before first phloem activity, as described for volatile mediated plant defense in cortex cells.¹³ Circumstantial support in favor of our hypothesis is provided by production of hydrogen peroxide in the apoplast,²² which is most likely associated with the action of OGs.²² Observations of hydrogen peroxide production during aphid (Macrosiphum euphorbiae) infestation of tomato in a limited area along the leaf veins, the preferred feeding sites of this species, indicate a locally restricted defense response (Fig. 1 and ​and22).⁴ The question arises why the cell signals are not spread via plasmodesmata to adjacent cells to induce resistance in a more extended leaf area? Dissemination of the signals may be prevented by closure of plasmodesmata (Fig. 1) through callose deposition,^23,24 which is most likely directly coupled with calcium influx induced by OGs,²⁵ by apoplastic hydrogen peroxide and to a minor extent by stylet puncture (Fig. 2).^7,26Open in a separate windowFigure 1Hypothetical model on how stylet penetration induces and suppresses plant defense. Sheath saliva (light blue) that envelopes the stylet during propagation through the apoplast contains cellulase and pectinase,^17,18 enzymes producing elicitors (e.g., oligogalacturonides (oGs)) by local cell wall digestion.¹⁹ Parenchyma cells adjacent to the sheath may develop a defense response owing to signaling cascades triggered by oG-mediated Ca²⁺-influx.¹⁹ Together with a Ca²⁺-dependent transient closure of plasmodesmata by callose (black crosses),^23,24 the focused production of oGs may cause a defense response with a limited sphere of action (red—strong, brown—light, green—none). This restricted domain of defense may not be perceived by other aphids, since the settling distance is limited by the aphid body size. Nearby aphids do not show any sign of defense perception in their probing and feeding behavior.¹⁴ Signaling cascade compounds may be channeled from parenchyma cells to CCs (dashed yellow arrows), where they are subsequently released into the SEs. There they may act as long-distance systemic defense components (grey arrows). In contrast to the parenchyma domain (where only minor amounts of watery saliva are secreted), Ca²⁺-mediated reactions such as defense cascades and sieve-plate (SP) occlusion are suppressed in SEs by large amounts of watery saliva. The left aphid penetrates an SE and injects watery saliva (red cloud; ws) that inhibits local sieve-plate occlusion and,¹⁰ most likely, is transported by mass flow (black arrow) to adjacent SEs,²⁷ where occlusion is impeded as well. A short-distance systemic spread over a few centimeters may explain local suppression of plant defense resulting in a higher rate of colonization. Salivary proteins or their degradation products may serve as systemic defense signals as well (grey arrows), but may also diffuse via the PPUs into CCs where additional systemic signals are induced (yellow arrows).Open in a separate windowFigure 2Hypothetical involvement of Ca²⁺-channels in aphid-induced cell defense (detail). During probing with its stylet the aphid secretes gel saliva as a lubrication substance (light blue) into the apoplast.¹⁵ on the way to the sieve tubes, aphids briefly puncture most non-phloem cells (red) after which the puncturing sites are sealed with gel saliva.^7,16 Gel saliva also most likely prevents the influx of apoplastic calcium into pierced sieve elements (green) by sealing the penetration site.⁷ Watery saliva (red cloud), injected into the SE lumen,⁹ contains proteins which bind calcium ions (marked by X) that enter the SE via e.g., mechano sensitive Ca²⁺-channels activated by stylet penetration (blue tons).¹⁰ In this way, aphids suppress SE occlusion and activation of local defense cascades. In the parenchyma cells around the gel saliva sheath, a small cylindrical zone of defense may be induced by oligogalacturonides (oGs; brown triangles) produced by cell wall (grey) digestion.^17–19 Perceived by unknown receptor proteins (R; e.g., a receptor like protein kinase)³⁴ and kinase mediation (black dotted and dashed arrows), oGs lead to a Ca²⁺-influx through kinase activated calcium channels (orange tons).²⁵ Around the probing site, aphids apparently induce the production of superoxide by Ca²⁺-induced activation of the NADPH oxidase (violet box) and its following conversion to hydrogen peroxide (red spots) is mediated by superoxide dismutase (SoD).⁴ Hydrogen peroxide activates Ca²⁺-channels (violet tons) and diffuses through plasma membrane (curled arrows) therefore potentially acting as a intracellular signal.²⁶By contrast, Ca²⁺-influx into SEs, induced by presence of OGs or stylet insertion (Fig. 2), is not expected to trigger local defense given the abundant excretion of Ca²⁺-binding watery saliva.^7,10,25 Watery saliva may spread to down-stream and adjacent SEs through transverse and lateral sieve plates (Fig. 1).^7,27 Aphids puncturing nearby SEs may therefore encounter less severe sieve-plate occlusion which results in facilitated settling and thus in increased population growth. Aggregation of feeding aphids would self-amplify population growth until a certain density is attained. Farther from the colonization site, this effect may be lost due to dilution. Stimulation of aphid feeding by aphid infestation was observed locally on potato by Myzus persicae and M. euphorbiae, respectively, 96 h after infestation.¹³ However, a similar effect was not observed for M. persicae on Arabidopsis thaliana where aphids induced premature leaf senescence and resistance 12 h after infestation,²⁸ possibly induced by OGs.¹⁹As a speculation, OG induced Ca²⁺-influx into parenchyma cells adjacent to the salivary sheath activate Ca²⁺-induced signaling cascades via CaM,^26,29 CDPKs,^30,31 MAPKinases and reactive oxygen species (Fig. 2).³² Systemic resistance, induced by aphid infestation,^12–14 is mediated by unknown compounds such as, e.g., salivary proteins, their degradation products, signal cascade products or volatiles.¹³ Compounds produced in CCs first have to pass the PPUs, while SE signaling elements can be directly transported via mass flow (Fig. 1).The question arises if aphids profit from induced resistance on local (cortex and parenchyma cells) and systemic (distant plant organs) levels as holds for suppression of defense in SEs. Possibly settling and subsequent spread of competing pathogens/herbivores (e.g., fungi or other piercing-sucking insects) are suppressed by induced defense. In this context it is intriguing to understand how aphids cope with the self-induced systemic resistance, which probably lasts over weeks.³³     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.