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Visualizing phosphoinositide signalling in single neurons gets a green light
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Visualizing phosphoinositide signalling in single neurons gets a green light Stefan R. Nahorski, Kenneth W. Young, R.A. John Challiss and Mark S. Nash Department of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, UK
There is now substantial evidence, from single-cell imaging, that complex patterns of release from Ca21 stores play an important role in regulating synaptic efficacy and plasticity. Moreover, the major mechanism of store release depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] through the action of phospholipase(s) C on phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and several neurotransmitters can enhance receptor-mediated activation of this enzyme. The recent development of techniques to image real-time changes in PtdIns(4,5)P2 hydrolysis according to generation of Ins(1,4,5)P3 and diacylglycerol in single cells has significantly advanced our ability to investigate these signalling pathways, particularly in relation to single-cell Ca21 signals. This article reviews these new approaches and how they have provided novel insights into mechanisms underlying spatio-temporal Ca21 signals and phospholipase C activation in neurons. The past two decades have seen an extraordinary expansion in our understanding of the role of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], both as a direct regulator of membrane and cytoskeletal proteins and as the precursor of key signalling molecules [1– 5]. Classical receptor-activated phospholipase C (PLC)mediated hydrolysis of PtdIns(4,5)P2 yields both the diffusible messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which can regulate Ca2þ release from intracellular stores, and diacylglycerol (DAG), the activator of classical and novel forms of protein kinase C (PKC) [6]. This phosphoinositide cascade is present in virtually all cells and within the CNS a large number of G-proteincoupled receptors (GPCRs) use this signalling pathway to modulate neuronal activity. The recent recognition of spatio-temporal Ins(1,4,5)P3-mediated Ca2þ release from neuronal stores, in addition to PKC-mediated phosphorylation of other signalling proteins, strongly implicates phosphoinositide signalling in synaptic regulation and plasticity [7,8] With the development of Ca2þ-sensitive dyes, imaging of single-cell intracellular Ca2þ has revealed a rich diversity of patterns, including oscillations and waves, Corresponding author: Stefan R. Nahorski ([email protected]).
and there is much evidence that these complex spatiotemporal responses add enormously to the versatility of Ca2þ signalling [6]. However, until recently evaluation of upstream phosphoinositide signalling has been limited to biochemical assays on large numbers of cells that can provide only a summation of the population PLC response [9,10]. Such techniques cannot provide information on spatio-temporal changes (e.g. oscillations and waves), on the number of responding cells or on whether responses are graded; nor can they identify differences in synchronicity. Moreover, they crucially fail to yield accurate data on signalling in heterogeneous populations (e.g. primary neurons). Overall, this inability to visualize Ins(1,4,5)P3 and DAG levels in real time in individual cells has hindered the correlation of this signalling pathway with the complex patterns of Ca2þ signal observed at the singlecell level. Recent advances have now transformed this situation with the development of approaches to track in single cells signalling domains tagged with fluorescent proteins derived from the jellyfish Aequorea victoria. These imaging techniques have many advantages, as well as some limitations (Table 1), over traditional biochemical measurements and they provide the means to address key issues arising from earlier biochemical data. Currently, a variety of such ‘biosensors’ can detect most aspects of phosphoinositide signalling: Ins(1,4,5)P3 can be studied using the pleckstrin-homology domain of PLCd1 (PHPLCd) [11 – 20], Ca2þ and DAG can be studied using using C2 and C1 domains of PKCg, respectively [21,22]; and there are full-length enhanced green-fluorescent protein (eGFP)fusion isoforms of PKC [23] and probes to detect PtdIns(3,4,5)P3 [24] (Fig. 1). This provides us with the means to correlate the changes in each component of the pathway in real time, which is particularly significant when one considers the potential for differences in the rate of metabolism or clearance. This review highlights how such approaches have provided new insights into the mechanisms underlying spatio-temporal Ca2þ signals and the nature of PLC signalling within neurons. A window on phosphoinositide signalling in single cells Visualizing changes in Ins(1,4,5)P3 levels within individual cells relies upon harnessing the in vivo Ins(1,4,5)P3-sensing ability of PHPLCd. This domain
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binds with high affinity and selectivity to PtdIns(4,5)P2 [12] and a fusion construct of PHPLCd and eGFP enriches in the plasma membrane (Fig. 2a). Increases in levels of Ins(1,4,5)P3 compete for PHPLCd, leading to translocation of the construct to the cytosol over the physiological range (,0.1 – 10.0 mM [25]) produced following activation of GPCRs coupled to Gaq proteins (GqPCRs) [12,25]. Importantly, the subcellular distribution of the PHPLCd appears to be predominantly controlled by the amount of Ins(1,4,5)P3 produced rather than by the fall in PtdIns(4,5)P2 levels following hydrolysis by PLC (Table 2). The first use of this methodology revealed a transient translocation of eGFP – PHPLCd in plateletactivating factor (PAF)-activated basophilic leukaemic cells [11] and adrenal glomerulosa cells stimulated with angiotensin II [13]. More recent studies have started to reveal clear differences in the degree, kinetics and profile of Ins(1,4,5)P3 responses to different receptors and their association with single-cell Ca2þ responses (Fig. 3; Table 3). An attractive alternative to using PHPLCd to detect PLC activity is to use the cysteine-rich C1 domains of PKC. These bind to the plasma membrane through their interaction with DAG and can be exploited effectively as
Table 1. Advantages and limitations of biosensors for PLC signallinga Advantages 1. Provides real-time acquisition of data in single cells 2. Captures accurate data from cell populations responding asynchronously (i.e. oscillatory signals or differences in the percentage of responsive cells) 3. Facilitates the study of identifiable cell types within heterogeneous populations 4. Allows the visualization of regionally specific signalling or waves 5. Can be used in combination using GFP variants or in conjunction with Ca2þ-sensitive dyes to correlate changes in different elements of the PLC pathway 6. Genetic biosensors have the potential to be targeted to specific subcellular regions Limitations 1. Provides information on relative changes, not on absolute concentrations 2. Requires transfection of plasmid DNA, which can be difficult in neurons 3. At high concentrations, could interfere with signalling or promote long-term adaptive changes 4. Heterogeneous distribution or segregation of the biosensor within complex cells could compromise data a
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Abbreviations: GFP, green-fluorescent protein; PLC, phospholipase C.
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Fig. 1. Translocating fluorescent biosensors to visualize G-protein-coupled receptor (GPCR)-mediated phosphoinositide signalling in single cells. Several protein modules show specificity for individual components of the phospholipase C (PLC) signalling system. These can be tagged with enhanced green-fluorescent protein (eGFP) and visualized using microscopy to track fluctuations in levels of each messenger in this system. Activation of PLC induces hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane to produce two messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (1) and diacylglycerol (DAG) (2). These changes can be detected by the translocation of the pleckstrin-homology domain of PLCd1 (PHPLCd) away from the plasma membrane (a), and of C12 domains of PKCg to the plasma membrane (b), respectively. The interaction of Ins(1,4,5)P3 with its receptor on the endoplasmic reticulum (ER) (3) causes release of Ca2þ from these intracellular stores (4). This can be tracked by the increase in the affinity of C2 domains for the phospholipids upon Ca2þ binding (5), which results in its translocation of this domain to the plasma membrane (c). Activation of GPCRs can also lead to activation of phosphatidylinositol 3-kinase (PI3-K), which phosphorylates PtdIns(4,5)P2 to give phosphatidyl Ins(3,4,5)P3 [PtdIns(3,4,5)P3] (6). The high selectivity of the pleckstrin-homology domain of Bruton’s tyrosine kinase (PHBtk) for PtdIns(3,4,5)P3 results in its translocation to the plasma membrane (d). Red arrows indicate steps in physiological GPCR-mediated phosphoinositide signalling; green arrows indicate translocation of fluorescent biosensors that can be used to visualize these signalling events. Abbreviation: G, G protein. http://tins.trends.com
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Fig. 2. Imaging inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) signalling in single cells. Two biosensors are currently available to image the immediate products of phosphoinositide hydrolysis by G-protein-coupled receptor (GPCR) activation of phospholipase C (PLC) – that is, to image Ins(1,4,5)P3 and DAG production. The pleckstrin-homology domain of PLCd1 initially binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane (PM) (ai) but translocates to the cytosol (aii) as a consequence of increases in Ins(1,4,5)P3 levels, whereas the C12 domain of protein kinase Cg (PKCg) is cytosolic in unstimulated cells (bi) but binds to DAG produced from PtdIns(4,5)P2 hydrolysis at the membrane (bii). The images show Chinese-hamster ovary (CHO) cells expressing the metabotropic glutamate receptor mGluR1a that have been transiently transfected with the two biosensors tagged with the green-fluorescent protein. Clear translocations of the biosensors from or to the plasma membrane occur as Ins(1,4,5)P3 and DAG levels increase, respectively, following glutamate-induced activation of mGluR1a. This is clearly illustrated in the graphs, which show the redistribution of fluorescent intensity across a line drawn through the plasma membrane and cytosol of each cell. Fluorescence values are in arbitrary units.
sensors of the DAG released following PtdIns(4,5)P2 hydrolysis by PLC [22]. When expressed in cells, the eGFP-tagged tandem C1 domains (C12 domains) of PKCg distribute across the cytosol and nucleus (Fig. 1b) but GPCR activation of PLC results in the translocation of the Table 2. eGFP –PHPLCd detects Ins(1,4,5)P3 accumulation rather than PtdIns(4,5)P2 depletiona Evidence
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1.The affinity of the pleckstrin-homology domain for Ins(1,4,5)P3 is 20-fold higher than for PtdIns(4,5)P2 2. Microinjection or uncaging of Ins(1,4,5)P3 in the absence of changes in PtdIns(4,5)P2 levels elicits translocation of eGFP –PHPLCd 3. The temporal pattern of translocation more closely mirrors that of Ins(1,4,5)P3 mass than that of PtdIns(4,5)P2 in populations 4. Enhancing the metabolism of Ins(1,4,5)P3 following GqPCR activation with 5-phosphatase or 3-kinase inhibits translocation, despite falls in PtdIns(4,5)P2 levels
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Abbreviations: eGFP –PHPLCd, enhanced green-fluorescent protein attached to the pleckstrin-homology domain of PLCd1; GqPCR, receptor coupled to the Gq protein; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate. http://tins.trends.com
fusion protein to the plasma membrane. These eGFPtagged C12 domains have been used to follow activation of forms of PLC by tyrosine kinase receptors or GPCRs in a mast-cell line, consistent with the early generation of DAG from PLCb or PLCg action on PtdIns(4,5)P2 [22]. Moreover, tandem C1 domains from PKCd, which have a high affinity for DAG, have been successfully used to monitor DAG accumulation in glutamate-stimulated astrocytes [26]. This study also used total internal-reflection fluorescence (TIRF) evanescent-wave technology to visualize glutamate-induced waves of DAG synthesis. Ins(1,4,5)P3 and Ca21 oscillations In both excitable and non-excitable cell types, activation of the PLC –Ins(1,4,5)P3 signalling pathway often evokes repetitive Ca2þ oscillations that arise when successive waves of Ca2þ propagate through the cell [6,27 – 31]. Increasingly, it has been recognized that the pattern and the periodicity of Ca2þ oscillations provide a variety of effectors with a digitally encoded signal. Thus, regulation of Ca2þ/calmodulin-dependent kinase II (CaMKII) [32] and PKC [21], and selective activation of different gene
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1. Distinct temporal patterns of Ins(1,4,5)P3 production are stimulated by different GPCRs 2. Distinct phases of Ins(1,4,5)P3 production occur during sustained agonist challenge 3. Ins(1,4,5)P3 oscillations can underlie GPCRmediated Ca2þ oscillations 4. Intracellular and intercellular Ins(1,4,5)P3 waves accompany Ca2þ waves 5. Graded, rather than all-or-none, single-cell changes in Ins(1,4,5)P3concentrations occur following agonist stimulation 6. Ca2þ-dependent Ins(1,4,5)P3 production can be mediated by GPCRs and ligand-gated channels
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1. Regionally constricted, or compartmentalized, Ins(1,4,5)P3 production in complex cells (i.e. neurons) 2. Dual imaging of Ins(1,4,5)P3 with PtdIns(4,5)P2, PtdIns(3,4,5)P3, Ins(1,3,4,5)P4, various PKCs, DAG or cyclic nucleotides 3. Analysis of receptor regulation, desensitization and recovery in single cells 4. Examination of the activation of heterotrimeric G proteins in single cells 5. Use of new imaging techniques to detect phosphorylated proteins and transcriptional activity 6. In vivo imaging of phosphoinositide signalling using multiphoton confocal microscopy
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a Abbreviations: DAG, diacylglycerol; GPCRs, G-protein-coupled receptors; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; PLC, phospholipase C; PKCs, forms of protein kinase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.
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Fig. 3. New information is revealed by imaging single cell inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production in real time. A fundamental question in signalling is whether the detection of agonist-concentration-related increases in second messenger using a population-based assay reflects graded changes in messenger production within each cell or a change in the proportion of cells showing an all-ornone type response. For G-protein-coupled receptor (GPCR)-induced phospholipase C (PLC) activation, this was addressed directly using enhanced green-fluorescent protein attached to the pleckstrin-homology domain of PLCd1 (eGFP– PHPLCd), to visualize Ins(1,4,5)P3 production in Chinese-hamster ovary (CHO) cells expressing the metabotropic glutamate receptor mGluR1a during application of increasing concentrations of glutamate (a). Reproduced with permission, from Ref. [14], q (2001) the Biochemical Society. Similarly, without single-cell measurements it is impossible to interpret correctly temporal information as a reaction in individuals cells rather than a change in the number of responsive cells. So the temporal profile of Ins(1,4,5)P3 production can be shown to follow a peak and reduced plateau pattern, and for mGluR1a these two distinct phases have been shown to have different requirements for the presence of extracellular Ca2þ (Ca2þ o ) (b). Reproduced, with permission, from Ref. [18]. Perhaps the most important advance to date from the use of eGFP –PHPLCd to image Ins(1,4,5)P3 has been the http://tins.trends.com
transcription factors [33,34], are influenced by the pattern and frequency of Ca2þ transients. Models to explain these oscillations are currently based either on the regulation of Ins(1,4,5)P3 generation by negative feedback via PKC [28,35] and regulator of G-protein signalling (RGS) proteins [36], or on regenerative Ca2þ-induced Ca2þ release (CICR), which is an integral property of the Ins(1,4,5)P3 receptor [6,27,28]. Clearly, a distinction between the two models relies on whether levels of Ins(1,4,5)P3 oscillate following the repetitive activation and deactivation of PLC or whether Ca2þ alone controls its store release at a steady-state level of Ins(1,4,5)P3 (Fig. 4). The use of eGFP – PHPLCd has recently allowed these alternatives to be directly tested and there is now substantial evidence that Ins(1,4,5)P3 can, indeed, oscillate in synchrony with cytosolic Ca2þ increases following stimulation of Madin-Darby canine kidney (MDCK) cells observation that levels can fluctuate, which is exemplified by the cellular response of CHO cells expressing the metabotropic glutamate receptor mGluR5a during glutamate challenge (c). Reproduced, with permission, from Ref. [19], q 2001 Macmillan Magazines Limited. For this receptor, these oscillations arise through cyclic inhibition and activation involving classical protein kinase Cs and they underlie global Ca2þ oscillations [17,19]. Data in each trace are expressed as the relative change in fluorescence within a region of interest in the cytoplasm, which provides an index of cellular Ins(1,4,5)P3 levels.
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Fig. 4. Models for the generation of intracellular Ca2þ oscillations. The mechanisms proposed to underlie oscillatory Ca2þ responses can broadly be categorized into two classes. The first, illustrated on the left, is based upon the regenerative properties of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor whereby low concentrations of intracellular Ca2þ ([Ca2þ]i) increase the open probability of the channels but higher concentrations close them. This leads to Ca2þ-induced Ca2þ release (CICR) waves and, hence, Ca2þ oscillations, which arise against a background of low and stable Ins(1,4,5)P3 concentration. By contrast, the second broad category relies upon the activation of an inhibitor, for example protein kinase C (PKC) or regulator of G-protein signalling (RGS) proteins, following Ca2þ release. These then exert negative feedback upon the phospholipase C (PLC) signalling pathway, to switch Ins(1,4,5)P3 production off until [Ca2þ]I falls. Clearly, for these dynamic uncoupling models, Ins(1,4,5)P3 concentration must oscillate in synchrony with the Ca2þ oscillations. Superficially, therefore, determining whether Ins(1,4,5)P3 levels fluctuate provides the answer to which mechanism is involved. However, Ca2þ oscillations could, theoretically, secondarily influence Ins(1,4,5)P3 synthesis or catabolism through stimulation of PLC activity or Ins(1,4,5)P3 3kinase, respectively. Although the latter possibility has recently been shown not to occur in hepatocytes [66], Young et al. have shown that a1B-adrenoceptors stimulate Ca2þ oscillations in Chinese-hamster ovary cells by regenerative CICR, and that these enhance PLC activity to give concurrent Ins(1,4,5)P3 oscillations [63]. Abbreviation: Gq, Gq protein.
with ATP [12] or stimulation of metabotropic-glutamatereceptor 5 (mGluR5)-expressing Chinese-hamster ovary (CHO) cells with glutamate [17,19] (Fig. 2c). In the latter case, a PKC-dependent dynamic uncoupling of the receptor and G protein by phosphorylation is almost certainly responsible for such periodicity. Similar mechanisms have been proposed for DAG oscillations following glutamate activation of astrocytes [26] and Ca2þ oscillations following activation of another family C GPCR, the Ca2þ-sensing receptor [37]. By contrast, muscarinic M3-ACh-receptor-driven sinusoidal Ca2þ oscillations in the same CHO-cell background is associated with a modest steady-state increase in single cell Ins(1,4,5)P3 levels, consistent with CICR [19]. These mechanisms of Ca2þ oscillation are not mutually exclusive [29] and a ‘signalling slide rule’ model that can accommodate these two mechanisms according to the stimulus strength of the receptor signals has recently been proposed [17]. The Ca2þ oscillation frequency in mGluR5 expressing cells is independent of the concentration of agonist but is dependent on the expression level of cell surface receptors [17], which would be wholly consistent with a substratedependent effect on the rates of activation and deactivation by phosphorylation and dephosphorylation [38]. The physiological significance of this mGluR5-density-determined Ca2þ output remains to be investigated, particularly in a neuronal setting. However, there is now increasing evidence that the cellular distribution of group 1 mGluRs can be influenced in neurons by the members of the Homer adaptor protein family, which can, in turn, be dynamically regulated by neuronal http://tins.trends.com
activity [39]. Such regulation could provide sophisticated control of Ca2þ wave and oscillation frequency and their propagation to the nucleus, to regulate gene transcription that might underlie some forms of synaptic plasticity [34,39]. PLC signalling in single neurons It has been known for many years that PtdIns(4,5)P2 – PLC signalling is very prominent in the CNS and that a wide range of GPCRs can activate this system within the brain [40]. More recently, it has been increasingly appreciated that the subtleties of Ins(1,4,5)P3-mediated Ca2þ release from neuronal stores might have an impact on the regulation of neuronal excitability, neurotransmitter release, gene transcription and synaptic plasticity [7,34, 41 – 45]. However, any progress in understanding this important signalling system in such a complex tissue as the CNS will depend upon imaging and manipulating the spatial and temporal dynamics of both extracellular transmitters and intracellular messengers. The ability to image Ca2þ with high resolution has recently been associated with the translocation of eGFP – aCAMKII to postsynaptic densities in hippocampal neurons [45]. By contrast, until very recently, studies on the activation of PLC have been limited to biochemical studies of cell populations in slices, or of heterogeneous neuronal and/or glial cells in culture. The Ins(1,4,5)P3 biosensor now makes it possible to image PLC activity in single neurons (Box 1) and the promise afforded by this development and the use of C1 domains to detect DAG can be gauged from recent studies.
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Box 1. Ins(1,4,5)P3 signalling in individual neurons The inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] biosensor has clearly provided a wealth of information on G-protein-coupled receptor (GPCR)-induced phospholipase C (PLC) signalling in simple cells beyond what could previously be obtained using traditional biochemical techniques, such as the detection of Ins(1,4,5)P3 oscillations and graded responses. However, there are several additional levels at which this new technology is crucial for the study of phosphoinositide signalling in a complex cellular system such as the CNS. First, even within a defined structure such as the hippocampus or cerebellum, there are diverse neuronal cell types with different roles, in addition to many non-neuronal cells, including astrocytes and microglia. This rather obvious point highlights the inadequacies of data on phosphoinositide turnover accrued from population-based analyses because it is likely to be the summation of a very complex response. Only by visualizing changes in individual defined cells will it be possible to get a true picture of regulation of the phosphoinositide pathway within the CNS. Second, neurons are extremely structured (Fig. I), with different elements performing distinct functions (i.e. axon, dendrites, soma and nucleus) or similar structures that can act as independent functional units (i.e. individual boutons, dendritic spines or specific arborizations). Most neuronal functions (e.g. neurotransmission, synaptic plasticity and gene transcription) are regulated to some extent by the phosphoi-
nositide pathway and/or by Ca2þ release from intracellular stores [4,7,40,41,43]. However, the many elements of this signalling system are regionally constrained through, for example, buffering and extrusion of intracellular Ca2þ [7,67] or metabolism of Ins(1,4,5)P3 [68]. The sites of Ins(1,4,5)P3 production can also be distinct from sites of action because Ins(1,4,5)P3 receptors are not present in dendritic spines of CA1 pyramidal neurons [43]. Moreover, despite the close proximity of GPCR –PLC signalling and Ins(1,4,5)P3 receptors in Purkinje cells, the apparent affinity of the receptors is lower than predicted [43]. Factors including the different diffusion rates for Ins(1,4,5)P3 and Ca2þ through the cellular milieu [69] and the degree to which these pathways are reinforced through either Ca2þ-induced Ca2þ release or Ca2þ-induced Ins(1,4,5)P3 production must also be considered. The existence of such spatial constraints means that, if we are to understand how regionally restricted phosphoinositide and intracellular Ca2þ signals are integrated to influence function of an individual neuron or its response to stimuli, it is necessary to be able to visualize the whole cell rather than to obtain a global summary. Indeed, studies using Ca2þ-sensitive fluorescent dyes revealed that there is a huge diversity of regional, elementary and global Ca2þ signals in neurons [7] and imaging Ins(1,4,5)P3 (and diacylglycerol) now means we have the potential to incorporate these data into the developing picture of the role and regulation of the PLC signalling pathway in neurons.
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Fig. I. Visualizing phospholipase C (PLC) activation in neurons. A cultured rat hippocampal neuron (at 22 days in vitro) that had been transfected with enhanced greenfluorescent protein attached to the pleckstrin-homology domain of PLCd1 (eGFP –PHPLCd) by lipofection at five days in vitro (centre). Importantly, at this level of expression no significant toxicity is apparent [16]. Moreover, although transfection efficiency is invariably low, this actually makes visualizing the extensive dendritic tree of an individual neuron easier. The left panel and inset are higher magnification confocal images, showing the membrane localization of the pleckstrin-homology domain within a dendritic arborization and the presence of dendritic spines. The right panel shows a confocal section through the soma of the same neuron before (top) and after (bottom) challenge with methacholine (MCh; 100 mM ) in the presence of tetrodotoxin (500 nM ) (to block synaptic activity)]. This agonist stimulates the Gq-coupled muscarinic ACh receptors present in these neurons to activate PLC [61] and clearly causes translocation of the inositol 1,4,5-trisphosphate biosensor from the plasma membrane to the cytosol in a reversible manner. This experiment illustrates a global response to PLC activation but future research will also be aimed at determining how regional activation of phosphoinositide signalling within neurons (i.e. within individual spines or branches of the dendritic tree) integrates with spatially restricted Ca2þ signalling to influence local and global cell function.
Okubo et al. [15] used a Sindbis virus vector to infect Purkinje cells of the cerebellum with this construct and provided strong evidence using overexpressed Ins(1,4,5)P3 5-phosphatase that translocation of this biosensor to the cytoplasm detects Ins(1,4,5)P3 accumulation rather than PtdIns(4,5)P2 depletion. Their studies also reveal that, in addition to metabotropic-glutamate-receptor activation, there is a major AMPA receptor-mediated Ins(1,4,5)P3 accumulation in Purkinje cells. Although at first sight these data obtained using an agonist of ligand-gated channels might appear surprising, it should be recalled http://tins.trends.com
that activation of PLC in cerebral cortex slices with AMPA, NMDA or indeed depolarizing stimuli has long been known to be secondary to Ca2þ entry [46 –48]. This has now been confirmed at a single cell level in Purkinje cells in which the response to AMPA could be blocked by inhibitors of P-type Ca2þ channels [15]. Although this action remains to be unequivocally associated with a direct rather than an indirect (release of neurotransmitter) effect of Ca2þ, these data highlight that in CNS neurons, Ca2þ-driven PLC(s) [49] might play an important coincidence-detection role, possibly in synergy with GPCR-mediated PLC signalling.
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In this respect, it is of considerable interest that the C terminus of P/Q-type voltage-sensitive Ca2þ channels possesses a Homer-protein-binding consensus sequence [50] and that Ca2þ entry through voltage-dependent channels promotes formation of Homer 1c clusters in hippocampal neurons [51]. Because Homer can couple metabotropic glutamate receptors to the Ins(1,4,5)P3 receptor [39], perhaps specific Ca2þ entry can initiate local CICR at this endoplasmic-reticulum-associated receptor. Signalling microdomains in neurons It is clear that within complex neuronal settings, signalling microdomains are likely to be crucial to the specificity of PLC – Ca2þsignalling [44,52,53]. The recent report [54] that phosphatidylinositol 4-phosphate 5-kinase 1g can be recruited by the cytoskeletal protein talin locally to generate PtdIns(4,5)P2 at synapses and that PLCb1 is a component of multiprotein postsynaptic complexes [55] again emphasizes that signalling microdomains are crucial to the specificity of PLC – Ca2þ signalling in neurons. An added dimension to this new information is the evidence that PtdIns(4,5)P2 can directly inhibit the opening of P/Q-type Ca2þ channels [3], perhaps indicating a complex regulation of synaptic activity by both the substrate and the products of PLC activity. The importance of signalling microdomains in neurons is further emphasized by a recent study using single-cell biosensors prompted by the paradoxical regulation of the M-current by different GqPCRs [52]. In superior cervical ganglion neurons, bradykinin and muscarinic receptors both deactivate M-currents but, despite coupling to the same Gq protein, the mechanism for inactivation is different. Delmas et al. [52] used eGFP-tagged C12 domains to show that both bradykinin and muscarinic receptors induced DAG production; however, using C2 domains to detect changes in Ca2þ levels revealed that only bradykinin could elicit Ca2þ release. As an elegant alternative to investigate this phenomenon, the authors also used a channel-based assay of PtdIns(4,5)P2 hydrolysis. They expressed, in the sympathetic neurons, different transient receptor potential (Trp) channels that are selectively activated by Ins(1,4,5)P3-mediated Ca2þ signalling (hTrpC1) or by DAG (mTrpC6). Using these specific biosensors not only allowed a real-time assessment of PLC activity but also confirmed the striking differences between M1-muscarinic-receptor and B2-bradykininreceptor signalling. Whereas both GPCRs activate PLC efficiently in these neurons, exemplified by DAG-activated mTrpC6 and C12 translocation, only bradykinin was able to stimulate significantly Ins(1,4,5)P3-induced Ca2þ release. This work further highlighted a possible role of the actin cytoskeleton in controlling the proximity of different signalling components and an apparent uncoupling of PLC activity from Ca2þ mobilization. Micheva et al. [16] transiently transfected eGFP – PHPLCd into hippocampal neurons and by loading synapses with the dye FM 4-64, imaged local PtdIns(4,5)P2 within presynaptic boutons. Electrical stimulation of these neurons led to a translocation of this biosensor to the cytoplasm, dependent on NMDA activation and a possible http://tins.trends.com
retrograde action of nitric oxide. These studies emphasize the potential utility of biosensors to detect local changes in plasma membrane PtdIns(4,5)P2 and, with increasing evidence that this lipid is a key regulator of many ion channels [1 –3,56], the future use of biosensors specific for PtdIns(4,5)P2, might elucidate the dynamic nature and mechanisms of this regulation. One way to assess PtdIns(4,5)P2 rather than Ins(1,4,5)P3 concentrations might be the use of channel-based biosensors. Hardie et al. [57] exploited the properties of inward-rectifier channels (particularly Kir2.1) for their ability to be activated by PtdIns(4,5)P2. By expressing the human Kir2.1 channel in Drosophila photoreceptors, this group was able to associate casually a suppression of Kþ currents with the loss of PtdIns(4,5)P2 that is required for phototransduction. Ins(1,4,5)P3-induced Ca2þ release and synaptic activity There are now several studies suggesting that synaptically evoked Ca2þ release from stores can be initiated by activation of Ins(1,4,5)P3 receptors within dendrites and spines. Thus, synergistic (also termed ‘supralinear’) Ca2þ responses supported by Ca2þ influx through voltage-gated or ligand-gated channels following GPCR activation almost certainly results from Ins(1,4,5)P3-dependent CICR from stores [7,42,43,58– 60]. That this might be initiated by pairing synaptic activity with single or multiple action potentials to achieve the crucial spatial and temporal characteristics [58] implies that there must be compartments with the dendritic tree and a discrete localization of Ins(1,4,5)P3 and ryanodine receptors, GPCRs and channels. Couple this to the complexity of neuronal endoplasmic reticulum in the dendrites, axons and soma of different neurons and it is clear that the mechanisms of Ca2þ release are very diverse. Recent studies suggest that the local production and diffusion of Ins(1,4,5)P3, rather than that of Ca2þ, to distant targets could be operative in Ca2þ wave propagation in hippocampal CA1 pyramidal cells [59]. Therefore, superimposed upon CICR at Ins(1,4,5)P3 receptors, there could be Ca2þentry-mediated activation of Ins(1,4,5)P3 production further enhancing the probability of regenerative Ca2þ release. Indeed, a recent report [61] has described how activation of muscarinic receptors by synaptic stimulation of cholinergic afferents in hippocampal CA1 neurons led to a focal rise in apical dendritic Ca2þ concentration that then propagated to the soma and invaded the nucleus. Concluding remarks The ability to detect the activity of PLC in single neurons in real time with sufficient resolution alongside Ca2þ imaging will provide new insights into synaptic modulation and plasticity. Overall, to understand how information from transmitter receptors at dendritic spines is conveyed over potentially long distances to the nucleus in the soma will be crucial in linking synaptic activity to gene transcription and long-term neuronal adaptation. Clearly, the advances in imaging technology can provide significant information about the spatio-temporal aspects of PLC signalling (Table 3) and the field is now in the exciting position of being able to address
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fundamental issues associated with this important pathway in neurons. Acknowledgements We thank the Wellcome Trust for support (Programme Grant; 062495), Tobias Meyer for several constructs, Lyn McCarthy for manuscript preparation and the University of Leicester for study leave (S.R.N.).
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activity-dependent transcription of syntaxin-1A through coupling to Homer. Soc. Neurosci. Abstr., 14.4 Okabe, S. et al. (2001) Rapid redistribution of PSD-ZIP-4S (Homer 1c) and its differential regulation by NMDA receptors and calcium channels. J. Neurosci. 21, 9561– 9571 Delmas, P. et al. (2002) Signalling microdomains define the specificity of receptor-mediated InsP3 pathways in neurones. Neuron 34, 209–220 Delmas, P. and Brown, D.A. (2002) Junctional signalling microdomains: bridging the gap between the neuronal cell surface and Ca2þ stores. Neuron 36, 787– 790 Di Paulo, G. et al. (2002) Recruitment and regulation of phosphatidylinositol phosphate kinase type 1y by the FERM domain of talin. Nature 420, 85 – 89 Erzurumlu, R.S. and Kind, P.C. (2001) Neural activity: sculptor of ‘barrels’ in the neocortex. Trends Neurosci. 24, 589– 595 Zhang, H. et al. (2003) PIP2 activates KCNQ channels and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37, 963 – 975 Hardie, R.C. et al. (2001) Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors. Neuron 30, 149 – 159 Nakamura, T. et al. (2000) Inositol 1,4,5-trisphosphate mediated Ca2þ release evoked by metabotropic agonists and back propagating action potentials. J. Neurosci. 20, 8365– 8376 Nakamura, T. et al. (2002) Spatial segregation and interaction of calcium signalling mechanisms in rat hippocampal CA1 pyramidal neurons. J. Physiol. 543, 465 – 480
60 Finch, E.A. and Augustine, G.J. (1998) Local calcium signalling by inositol 1,4,5-trisphosphate in Purkinje cells dendrites. Nature 396, 753– 756 61 Power, J.M. and Sah, P. (2002) Nuclear calcium signalling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons. J. Neurosci. 22, 3454– 3462 62 Van der Wal, J. et al. (2001) Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J. Biol. Chem. 276, 15337 – 15344 63 Young, K.W. et al. (2003) Role of Ca2þ feedback on single cell Ins(1,4, 5)P3 oscillations mediated by G-protein coupled receptors. J. Biol. Chem. 278, 20753 – 20760 64 Janetopoulos, C. et al. (2001) Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science 291, 2408 – 2411 65 Sato, M. et al. (2002) Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat. Biotechnol. 20, 287– 294 66 Dupont, G. et al. (2003) Ca2þ oscillations in hepatocytes do not require the modulation of InsP3 3-kinase activity by Ca2þ. FEBS Lett. 534, 101– 105 67 Sabatini, B.L. et al. (2001) Ca2þ signalling in dendritic spines. Curr. Opin. Neurobiol. 11, 349 – 356 68 Schell, M.J. et al. (2001) Inositol 1,4,5-trisphosphate 3-kinase A associates with F-actin and dendritic spines via its N terminus. J. Biol. Chem. 276, 37537 – 37546 69 Allbritton, N.L. et al. (1992) Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258, 1812– 1815
Articles of interest in other Trends and Current Opinion journals Prolactin-induced neurogenesis in the maternal brain Robert S. Bridges and David R. Grattan Trends in Endocrinology and Metabolism 10.1016/S1043-2760(03)00082-1 Mechanisms of sodium channel inactivation Alan L. Goldin Current Opinion in Neurobiology 10.1016/S0959-4388(03)00065-5 Is frequent dosing with ecstasy a risky business for dopamine-containing neurons? Esther O’Shea and M. Isabel Colado Trends in Pharmacological Sciences 10.1016/S0165-6147(03)00133-0 The role of similarity in the development of categorization Vladimir M. Sloutsky Trends in Cognitive Sciences 7, 246–251 (June 2003) Nonsyndromic X-linked mental retardation: where are the missing mutations? Hans-Hilger Ropers et al. Trends in Genetics 19, 316–320 (June 2003) Is loss of function of the prion protein the cause of prion disorders? Claudio Hetz, Kinsey Maundrell and Claudio Soto Trends in Molecular Medicine 10.1016/S1471-4914(03)00069-8 Prion proteins meet protein quality control Derek E. Dimcheff, John L. Portis and Byron Caughey Trends in Cell Biology 10.1016/S0962-8924(03)00125-9
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Visualizing phosphoinositide signalling in single neurons gets a green light Stefan R. Nahorski, Kenneth W. Young, R.A. John Challiss and Mark S. Nash Department of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, UK
There is now substantial evidence, from single-cell imaging, that complex patterns of release from Ca21 stores play an important role in regulating synaptic efficacy and plasticity. Moreover, the major mechanism of store release depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] through the action of phospholipase(s) C on phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and several neurotransmitters can enhance receptor-mediated activation of this enzyme. The recent development of techniques to image real-time changes in PtdIns(4,5)P2 hydrolysis according to generation of Ins(1,4,5)P3 and diacylglycerol in single cells has significantly advanced our ability to investigate these signalling pathways, particularly in relation to single-cell Ca21 signals. This article reviews these new approaches and how they have provided novel insights into mechanisms underlying spatio-temporal Ca21 signals and phospholipase C activation in neurons. The past two decades have seen an extraordinary expansion in our understanding of the role of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], both as a direct regulator of membrane and cytoskeletal proteins and as the precursor of key signalling molecules [1– 5]. Classical receptor-activated phospholipase C (PLC)mediated hydrolysis of PtdIns(4,5)P2 yields both the diffusible messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which can regulate Ca2þ release from intracellular stores, and diacylglycerol (DAG), the activator of classical and novel forms of protein kinase C (PKC) [6]. This phosphoinositide cascade is present in virtually all cells and within the CNS a large number of G-proteincoupled receptors (GPCRs) use this signalling pathway to modulate neuronal activity. The recent recognition of spatio-temporal Ins(1,4,5)P3-mediated Ca2þ release from neuronal stores, in addition to PKC-mediated phosphorylation of other signalling proteins, strongly implicates phosphoinositide signalling in synaptic regulation and plasticity [7,8] With the development of Ca2þ-sensitive dyes, imaging of single-cell intracellular Ca2þ has revealed a rich diversity of patterns, including oscillations and waves, Corresponding author: Stefan R. Nahorski ([email protected]).
and there is much evidence that these complex spatiotemporal responses add enormously to the versatility of Ca2þ signalling [6]. However, until recently evaluation of upstream phosphoinositide signalling has been limited to biochemical assays on large numbers of cells that can provide only a summation of the population PLC response [9,10]. Such techniques cannot provide information on spatio-temporal changes (e.g. oscillations and waves), on the number of responding cells or on whether responses are graded; nor can they identify differences in synchronicity. Moreover, they crucially fail to yield accurate data on signalling in heterogeneous populations (e.g. primary neurons). Overall, this inability to visualize Ins(1,4,5)P3 and DAG levels in real time in individual cells has hindered the correlation of this signalling pathway with the complex patterns of Ca2þ signal observed at the singlecell level. Recent advances have now transformed this situation with the development of approaches to track in single cells signalling domains tagged with fluorescent proteins derived from the jellyfish Aequorea victoria. These imaging techniques have many advantages, as well as some limitations (Table 1), over traditional biochemical measurements and they provide the means to address key issues arising from earlier biochemical data. Currently, a variety of such ‘biosensors’ can detect most aspects of phosphoinositide signalling: Ins(1,4,5)P3 can be studied using the pleckstrin-homology domain of PLCd1 (PHPLCd) [11 – 20], Ca2þ and DAG can be studied using using C2 and C1 domains of PKCg, respectively [21,22]; and there are full-length enhanced green-fluorescent protein (eGFP)fusion isoforms of PKC [23] and probes to detect PtdIns(3,4,5)P3 [24] (Fig. 1). This provides us with the means to correlate the changes in each component of the pathway in real time, which is particularly significant when one considers the potential for differences in the rate of metabolism or clearance. This review highlights how such approaches have provided new insights into the mechanisms underlying spatio-temporal Ca2þ signals and the nature of PLC signalling within neurons. A window on phosphoinositide signalling in single cells Visualizing changes in Ins(1,4,5)P3 levels within individual cells relies upon harnessing the in vivo Ins(1,4,5)P3-sensing ability of PHPLCd. This domain
http://tins.trends.com 0166-2236/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0166-2236(03)00178-4
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binds with high affinity and selectivity to PtdIns(4,5)P2 [12] and a fusion construct of PHPLCd and eGFP enriches in the plasma membrane (Fig. 2a). Increases in levels of Ins(1,4,5)P3 compete for PHPLCd, leading to translocation of the construct to the cytosol over the physiological range (,0.1 – 10.0 mM [25]) produced following activation of GPCRs coupled to Gaq proteins (GqPCRs) [12,25]. Importantly, the subcellular distribution of the PHPLCd appears to be predominantly controlled by the amount of Ins(1,4,5)P3 produced rather than by the fall in PtdIns(4,5)P2 levels following hydrolysis by PLC (Table 2). The first use of this methodology revealed a transient translocation of eGFP – PHPLCd in plateletactivating factor (PAF)-activated basophilic leukaemic cells [11] and adrenal glomerulosa cells stimulated with angiotensin II [13]. More recent studies have started to reveal clear differences in the degree, kinetics and profile of Ins(1,4,5)P3 responses to different receptors and their association with single-cell Ca2þ responses (Fig. 3; Table 3). An attractive alternative to using PHPLCd to detect PLC activity is to use the cysteine-rich C1 domains of PKC. These bind to the plasma membrane through their interaction with DAG and can be exploited effectively as
Table 1. Advantages and limitations of biosensors for PLC signallinga Advantages 1. Provides real-time acquisition of data in single cells 2. Captures accurate data from cell populations responding asynchronously (i.e. oscillatory signals or differences in the percentage of responsive cells) 3. Facilitates the study of identifiable cell types within heterogeneous populations 4. Allows the visualization of regionally specific signalling or waves 5. Can be used in combination using GFP variants or in conjunction with Ca2þ-sensitive dyes to correlate changes in different elements of the PLC pathway 6. Genetic biosensors have the potential to be targeted to specific subcellular regions Limitations 1. Provides information on relative changes, not on absolute concentrations 2. Requires transfection of plasmid DNA, which can be difficult in neurons 3. At high concentrations, could interfere with signalling or promote long-term adaptive changes 4. Heterogeneous distribution or segregation of the biosensor within complex cells could compromise data a
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Abbreviations: GFP, green-fluorescent protein; PLC, phospholipase C.
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Fig. 1. Translocating fluorescent biosensors to visualize G-protein-coupled receptor (GPCR)-mediated phosphoinositide signalling in single cells. Several protein modules show specificity for individual components of the phospholipase C (PLC) signalling system. These can be tagged with enhanced green-fluorescent protein (eGFP) and visualized using microscopy to track fluctuations in levels of each messenger in this system. Activation of PLC induces hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane to produce two messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (1) and diacylglycerol (DAG) (2). These changes can be detected by the translocation of the pleckstrin-homology domain of PLCd1 (PHPLCd) away from the plasma membrane (a), and of C12 domains of PKCg to the plasma membrane (b), respectively. The interaction of Ins(1,4,5)P3 with its receptor on the endoplasmic reticulum (ER) (3) causes release of Ca2þ from these intracellular stores (4). This can be tracked by the increase in the affinity of C2 domains for the phospholipids upon Ca2þ binding (5), which results in its translocation of this domain to the plasma membrane (c). Activation of GPCRs can also lead to activation of phosphatidylinositol 3-kinase (PI3-K), which phosphorylates PtdIns(4,5)P2 to give phosphatidyl Ins(3,4,5)P3 [PtdIns(3,4,5)P3] (6). The high selectivity of the pleckstrin-homology domain of Bruton’s tyrosine kinase (PHBtk) for PtdIns(3,4,5)P3 results in its translocation to the plasma membrane (d). Red arrows indicate steps in physiological GPCR-mediated phosphoinositide signalling; green arrows indicate translocation of fluorescent biosensors that can be used to visualize these signalling events. Abbreviation: G, G protein. http://tins.trends.com
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Fig. 2. Imaging inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) signalling in single cells. Two biosensors are currently available to image the immediate products of phosphoinositide hydrolysis by G-protein-coupled receptor (GPCR) activation of phospholipase C (PLC) – that is, to image Ins(1,4,5)P3 and DAG production. The pleckstrin-homology domain of PLCd1 initially binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane (PM) (ai) but translocates to the cytosol (aii) as a consequence of increases in Ins(1,4,5)P3 levels, whereas the C12 domain of protein kinase Cg (PKCg) is cytosolic in unstimulated cells (bi) but binds to DAG produced from PtdIns(4,5)P2 hydrolysis at the membrane (bii). The images show Chinese-hamster ovary (CHO) cells expressing the metabotropic glutamate receptor mGluR1a that have been transiently transfected with the two biosensors tagged with the green-fluorescent protein. Clear translocations of the biosensors from or to the plasma membrane occur as Ins(1,4,5)P3 and DAG levels increase, respectively, following glutamate-induced activation of mGluR1a. This is clearly illustrated in the graphs, which show the redistribution of fluorescent intensity across a line drawn through the plasma membrane and cytosol of each cell. Fluorescence values are in arbitrary units.
sensors of the DAG released following PtdIns(4,5)P2 hydrolysis by PLC [22]. When expressed in cells, the eGFP-tagged tandem C1 domains (C12 domains) of PKCg distribute across the cytosol and nucleus (Fig. 1b) but GPCR activation of PLC results in the translocation of the Table 2. eGFP –PHPLCd detects Ins(1,4,5)P3 accumulation rather than PtdIns(4,5)P2 depletiona Evidence
Refs
1.The affinity of the pleckstrin-homology domain for Ins(1,4,5)P3 is 20-fold higher than for PtdIns(4,5)P2 2. Microinjection or uncaging of Ins(1,4,5)P3 in the absence of changes in PtdIns(4,5)P2 levels elicits translocation of eGFP –PHPLCd 3. The temporal pattern of translocation more closely mirrors that of Ins(1,4,5)P3 mass than that of PtdIns(4,5)P2 in populations 4. Enhancing the metabolism of Ins(1,4,5)P3 following GqPCR activation with 5-phosphatase or 3-kinase inhibits translocation, despite falls in PtdIns(4,5)P2 levels
[12]
a
[12,62]
[14]
[12,15,17]
Abbreviations: eGFP –PHPLCd, enhanced green-fluorescent protein attached to the pleckstrin-homology domain of PLCd1; GqPCR, receptor coupled to the Gq protein; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate. http://tins.trends.com
fusion protein to the plasma membrane. These eGFPtagged C12 domains have been used to follow activation of forms of PLC by tyrosine kinase receptors or GPCRs in a mast-cell line, consistent with the early generation of DAG from PLCb or PLCg action on PtdIns(4,5)P2 [22]. Moreover, tandem C1 domains from PKCd, which have a high affinity for DAG, have been successfully used to monitor DAG accumulation in glutamate-stimulated astrocytes [26]. This study also used total internal-reflection fluorescence (TIRF) evanescent-wave technology to visualize glutamate-induced waves of DAG synthesis. Ins(1,4,5)P3 and Ca21 oscillations In both excitable and non-excitable cell types, activation of the PLC –Ins(1,4,5)P3 signalling pathway often evokes repetitive Ca2þ oscillations that arise when successive waves of Ca2þ propagate through the cell [6,27 – 31]. Increasingly, it has been recognized that the pattern and the periodicity of Ca2þ oscillations provide a variety of effectors with a digitally encoded signal. Thus, regulation of Ca2þ/calmodulin-dependent kinase II (CaMKII) [32] and PKC [21], and selective activation of different gene
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(a)
Table 3. Key issues addressed by the imaging of Ins(1,4,5)P3 in individual cellsa
7
Ins(1,4,5)P3
mGluR1α 6
Current Advances
Refs
5
1. Distinct temporal patterns of Ins(1,4,5)P3 production are stimulated by different GPCRs 2. Distinct phases of Ins(1,4,5)P3 production occur during sustained agonist challenge 3. Ins(1,4,5)P3 oscillations can underlie GPCRmediated Ca2þ oscillations 4. Intracellular and intercellular Ins(1,4,5)P3 waves accompany Ca2þ waves 5. Graded, rather than all-or-none, single-cell changes in Ins(1,4,5)P3concentrations occur following agonist stimulation 6. Ca2þ-dependent Ins(1,4,5)P3 production can be mediated by GPCRs and ligand-gated channels
[12,14,17,18]
The Future
Refs
1. Regionally constricted, or compartmentalized, Ins(1,4,5)P3 production in complex cells (i.e. neurons) 2. Dual imaging of Ins(1,4,5)P3 with PtdIns(4,5)P2, PtdIns(3,4,5)P3, Ins(1,3,4,5)P4, various PKCs, DAG or cyclic nucleotides 3. Analysis of receptor regulation, desensitization and recovery in single cells 4. Examination of the activation of heterotrimeric G proteins in single cells 5. Use of new imaging techniques to detect phosphorylated proteins and transcriptional activity 6. In vivo imaging of phosphoinositide signalling using multiphoton confocal microscopy
[52]
4 3 2 1 1
3
0
5 8 10 [Glutamate] (µM)
100
–1 0 (b)
4
200
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600 800 Time (s)
1000
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Control Nominal Ca2+0
2
1
0 0
2.0
100
200 Time (s)
300
400
[12,17,19,20] [12] [14]
[13,15,63]
[64] [65]
a Abbreviations: DAG, diacylglycerol; GPCRs, G-protein-coupled receptors; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; PLC, phospholipase C; PKCs, forms of protein kinase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.
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Fig. 3. New information is revealed by imaging single cell inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production in real time. A fundamental question in signalling is whether the detection of agonist-concentration-related increases in second messenger using a population-based assay reflects graded changes in messenger production within each cell or a change in the proportion of cells showing an all-ornone type response. For G-protein-coupled receptor (GPCR)-induced phospholipase C (PLC) activation, this was addressed directly using enhanced green-fluorescent protein attached to the pleckstrin-homology domain of PLCd1 (eGFP– PHPLCd), to visualize Ins(1,4,5)P3 production in Chinese-hamster ovary (CHO) cells expressing the metabotropic glutamate receptor mGluR1a during application of increasing concentrations of glutamate (a). Reproduced with permission, from Ref. [14], q (2001) the Biochemical Society. Similarly, without single-cell measurements it is impossible to interpret correctly temporal information as a reaction in individuals cells rather than a change in the number of responsive cells. So the temporal profile of Ins(1,4,5)P3 production can be shown to follow a peak and reduced plateau pattern, and for mGluR1a these two distinct phases have been shown to have different requirements for the presence of extracellular Ca2þ (Ca2þ o ) (b). Reproduced, with permission, from Ref. [18]. Perhaps the most important advance to date from the use of eGFP –PHPLCd to image Ins(1,4,5)P3 has been the http://tins.trends.com
transcription factors [33,34], are influenced by the pattern and frequency of Ca2þ transients. Models to explain these oscillations are currently based either on the regulation of Ins(1,4,5)P3 generation by negative feedback via PKC [28,35] and regulator of G-protein signalling (RGS) proteins [36], or on regenerative Ca2þ-induced Ca2þ release (CICR), which is an integral property of the Ins(1,4,5)P3 receptor [6,27,28]. Clearly, a distinction between the two models relies on whether levels of Ins(1,4,5)P3 oscillate following the repetitive activation and deactivation of PLC or whether Ca2þ alone controls its store release at a steady-state level of Ins(1,4,5)P3 (Fig. 4). The use of eGFP – PHPLCd has recently allowed these alternatives to be directly tested and there is now substantial evidence that Ins(1,4,5)P3 can, indeed, oscillate in synchrony with cytosolic Ca2þ increases following stimulation of Madin-Darby canine kidney (MDCK) cells observation that levels can fluctuate, which is exemplified by the cellular response of CHO cells expressing the metabotropic glutamate receptor mGluR5a during glutamate challenge (c). Reproduced, with permission, from Ref. [19], q 2001 Macmillan Magazines Limited. For this receptor, these oscillations arise through cyclic inhibition and activation involving classical protein kinase Cs and they underlie global Ca2þ oscillations [17,19]. Data in each trace are expressed as the relative change in fluorescence within a region of interest in the cytoplasm, which provides an index of cellular Ins(1,4,5)P3 levels.
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Agonist Dynamic uncoupling
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Fig. 4. Models for the generation of intracellular Ca2þ oscillations. The mechanisms proposed to underlie oscillatory Ca2þ responses can broadly be categorized into two classes. The first, illustrated on the left, is based upon the regenerative properties of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor whereby low concentrations of intracellular Ca2þ ([Ca2þ]i) increase the open probability of the channels but higher concentrations close them. This leads to Ca2þ-induced Ca2þ release (CICR) waves and, hence, Ca2þ oscillations, which arise against a background of low and stable Ins(1,4,5)P3 concentration. By contrast, the second broad category relies upon the activation of an inhibitor, for example protein kinase C (PKC) or regulator of G-protein signalling (RGS) proteins, following Ca2þ release. These then exert negative feedback upon the phospholipase C (PLC) signalling pathway, to switch Ins(1,4,5)P3 production off until [Ca2þ]I falls. Clearly, for these dynamic uncoupling models, Ins(1,4,5)P3 concentration must oscillate in synchrony with the Ca2þ oscillations. Superficially, therefore, determining whether Ins(1,4,5)P3 levels fluctuate provides the answer to which mechanism is involved. However, Ca2þ oscillations could, theoretically, secondarily influence Ins(1,4,5)P3 synthesis or catabolism through stimulation of PLC activity or Ins(1,4,5)P3 3kinase, respectively. Although the latter possibility has recently been shown not to occur in hepatocytes [66], Young et al. have shown that a1B-adrenoceptors stimulate Ca2þ oscillations in Chinese-hamster ovary cells by regenerative CICR, and that these enhance PLC activity to give concurrent Ins(1,4,5)P3 oscillations [63]. Abbreviation: Gq, Gq protein.
with ATP [12] or stimulation of metabotropic-glutamatereceptor 5 (mGluR5)-expressing Chinese-hamster ovary (CHO) cells with glutamate [17,19] (Fig. 2c). In the latter case, a PKC-dependent dynamic uncoupling of the receptor and G protein by phosphorylation is almost certainly responsible for such periodicity. Similar mechanisms have been proposed for DAG oscillations following glutamate activation of astrocytes [26] and Ca2þ oscillations following activation of another family C GPCR, the Ca2þ-sensing receptor [37]. By contrast, muscarinic M3-ACh-receptor-driven sinusoidal Ca2þ oscillations in the same CHO-cell background is associated with a modest steady-state increase in single cell Ins(1,4,5)P3 levels, consistent with CICR [19]. These mechanisms of Ca2þ oscillation are not mutually exclusive [29] and a ‘signalling slide rule’ model that can accommodate these two mechanisms according to the stimulus strength of the receptor signals has recently been proposed [17]. The Ca2þ oscillation frequency in mGluR5 expressing cells is independent of the concentration of agonist but is dependent on the expression level of cell surface receptors [17], which would be wholly consistent with a substratedependent effect on the rates of activation and deactivation by phosphorylation and dephosphorylation [38]. The physiological significance of this mGluR5-density-determined Ca2þ output remains to be investigated, particularly in a neuronal setting. However, there is now increasing evidence that the cellular distribution of group 1 mGluRs can be influenced in neurons by the members of the Homer adaptor protein family, which can, in turn, be dynamically regulated by neuronal http://tins.trends.com
activity [39]. Such regulation could provide sophisticated control of Ca2þ wave and oscillation frequency and their propagation to the nucleus, to regulate gene transcription that might underlie some forms of synaptic plasticity [34,39]. PLC signalling in single neurons It has been known for many years that PtdIns(4,5)P2 – PLC signalling is very prominent in the CNS and that a wide range of GPCRs can activate this system within the brain [40]. More recently, it has been increasingly appreciated that the subtleties of Ins(1,4,5)P3-mediated Ca2þ release from neuronal stores might have an impact on the regulation of neuronal excitability, neurotransmitter release, gene transcription and synaptic plasticity [7,34, 41 – 45]. However, any progress in understanding this important signalling system in such a complex tissue as the CNS will depend upon imaging and manipulating the spatial and temporal dynamics of both extracellular transmitters and intracellular messengers. The ability to image Ca2þ with high resolution has recently been associated with the translocation of eGFP – aCAMKII to postsynaptic densities in hippocampal neurons [45]. By contrast, until very recently, studies on the activation of PLC have been limited to biochemical studies of cell populations in slices, or of heterogeneous neuronal and/or glial cells in culture. The Ins(1,4,5)P3 biosensor now makes it possible to image PLC activity in single neurons (Box 1) and the promise afforded by this development and the use of C1 domains to detect DAG can be gauged from recent studies.
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Box 1. Ins(1,4,5)P3 signalling in individual neurons The inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] biosensor has clearly provided a wealth of information on G-protein-coupled receptor (GPCR)-induced phospholipase C (PLC) signalling in simple cells beyond what could previously be obtained using traditional biochemical techniques, such as the detection of Ins(1,4,5)P3 oscillations and graded responses. However, there are several additional levels at which this new technology is crucial for the study of phosphoinositide signalling in a complex cellular system such as the CNS. First, even within a defined structure such as the hippocampus or cerebellum, there are diverse neuronal cell types with different roles, in addition to many non-neuronal cells, including astrocytes and microglia. This rather obvious point highlights the inadequacies of data on phosphoinositide turnover accrued from population-based analyses because it is likely to be the summation of a very complex response. Only by visualizing changes in individual defined cells will it be possible to get a true picture of regulation of the phosphoinositide pathway within the CNS. Second, neurons are extremely structured (Fig. I), with different elements performing distinct functions (i.e. axon, dendrites, soma and nucleus) or similar structures that can act as independent functional units (i.e. individual boutons, dendritic spines or specific arborizations). Most neuronal functions (e.g. neurotransmission, synaptic plasticity and gene transcription) are regulated to some extent by the phosphoi-
nositide pathway and/or by Ca2þ release from intracellular stores [4,7,40,41,43]. However, the many elements of this signalling system are regionally constrained through, for example, buffering and extrusion of intracellular Ca2þ [7,67] or metabolism of Ins(1,4,5)P3 [68]. The sites of Ins(1,4,5)P3 production can also be distinct from sites of action because Ins(1,4,5)P3 receptors are not present in dendritic spines of CA1 pyramidal neurons [43]. Moreover, despite the close proximity of GPCR –PLC signalling and Ins(1,4,5)P3 receptors in Purkinje cells, the apparent affinity of the receptors is lower than predicted [43]. Factors including the different diffusion rates for Ins(1,4,5)P3 and Ca2þ through the cellular milieu [69] and the degree to which these pathways are reinforced through either Ca2þ-induced Ca2þ release or Ca2þ-induced Ins(1,4,5)P3 production must also be considered. The existence of such spatial constraints means that, if we are to understand how regionally restricted phosphoinositide and intracellular Ca2þ signals are integrated to influence function of an individual neuron or its response to stimuli, it is necessary to be able to visualize the whole cell rather than to obtain a global summary. Indeed, studies using Ca2þ-sensitive fluorescent dyes revealed that there is a huge diversity of regional, elementary and global Ca2þ signals in neurons [7] and imaging Ins(1,4,5)P3 (and diacylglycerol) now means we have the potential to incorporate these data into the developing picture of the role and regulation of the PLC signalling pathway in neurons.
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Fig. I. Visualizing phospholipase C (PLC) activation in neurons. A cultured rat hippocampal neuron (at 22 days in vitro) that had been transfected with enhanced greenfluorescent protein attached to the pleckstrin-homology domain of PLCd1 (eGFP –PHPLCd) by lipofection at five days in vitro (centre). Importantly, at this level of expression no significant toxicity is apparent [16]. Moreover, although transfection efficiency is invariably low, this actually makes visualizing the extensive dendritic tree of an individual neuron easier. The left panel and inset are higher magnification confocal images, showing the membrane localization of the pleckstrin-homology domain within a dendritic arborization and the presence of dendritic spines. The right panel shows a confocal section through the soma of the same neuron before (top) and after (bottom) challenge with methacholine (MCh; 100 mM ) in the presence of tetrodotoxin (500 nM ) (to block synaptic activity)]. This agonist stimulates the Gq-coupled muscarinic ACh receptors present in these neurons to activate PLC [61] and clearly causes translocation of the inositol 1,4,5-trisphosphate biosensor from the plasma membrane to the cytosol in a reversible manner. This experiment illustrates a global response to PLC activation but future research will also be aimed at determining how regional activation of phosphoinositide signalling within neurons (i.e. within individual spines or branches of the dendritic tree) integrates with spatially restricted Ca2þ signalling to influence local and global cell function.
Okubo et al. [15] used a Sindbis virus vector to infect Purkinje cells of the cerebellum with this construct and provided strong evidence using overexpressed Ins(1,4,5)P3 5-phosphatase that translocation of this biosensor to the cytoplasm detects Ins(1,4,5)P3 accumulation rather than PtdIns(4,5)P2 depletion. Their studies also reveal that, in addition to metabotropic-glutamate-receptor activation, there is a major AMPA receptor-mediated Ins(1,4,5)P3 accumulation in Purkinje cells. Although at first sight these data obtained using an agonist of ligand-gated channels might appear surprising, it should be recalled http://tins.trends.com
that activation of PLC in cerebral cortex slices with AMPA, NMDA or indeed depolarizing stimuli has long been known to be secondary to Ca2þ entry [46 –48]. This has now been confirmed at a single cell level in Purkinje cells in which the response to AMPA could be blocked by inhibitors of P-type Ca2þ channels [15]. Although this action remains to be unequivocally associated with a direct rather than an indirect (release of neurotransmitter) effect of Ca2þ, these data highlight that in CNS neurons, Ca2þ-driven PLC(s) [49] might play an important coincidence-detection role, possibly in synergy with GPCR-mediated PLC signalling.
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In this respect, it is of considerable interest that the C terminus of P/Q-type voltage-sensitive Ca2þ channels possesses a Homer-protein-binding consensus sequence [50] and that Ca2þ entry through voltage-dependent channels promotes formation of Homer 1c clusters in hippocampal neurons [51]. Because Homer can couple metabotropic glutamate receptors to the Ins(1,4,5)P3 receptor [39], perhaps specific Ca2þ entry can initiate local CICR at this endoplasmic-reticulum-associated receptor. Signalling microdomains in neurons It is clear that within complex neuronal settings, signalling microdomains are likely to be crucial to the specificity of PLC – Ca2þsignalling [44,52,53]. The recent report [54] that phosphatidylinositol 4-phosphate 5-kinase 1g can be recruited by the cytoskeletal protein talin locally to generate PtdIns(4,5)P2 at synapses and that PLCb1 is a component of multiprotein postsynaptic complexes [55] again emphasizes that signalling microdomains are crucial to the specificity of PLC – Ca2þ signalling in neurons. An added dimension to this new information is the evidence that PtdIns(4,5)P2 can directly inhibit the opening of P/Q-type Ca2þ channels [3], perhaps indicating a complex regulation of synaptic activity by both the substrate and the products of PLC activity. The importance of signalling microdomains in neurons is further emphasized by a recent study using single-cell biosensors prompted by the paradoxical regulation of the M-current by different GqPCRs [52]. In superior cervical ganglion neurons, bradykinin and muscarinic receptors both deactivate M-currents but, despite coupling to the same Gq protein, the mechanism for inactivation is different. Delmas et al. [52] used eGFP-tagged C12 domains to show that both bradykinin and muscarinic receptors induced DAG production; however, using C2 domains to detect changes in Ca2þ levels revealed that only bradykinin could elicit Ca2þ release. As an elegant alternative to investigate this phenomenon, the authors also used a channel-based assay of PtdIns(4,5)P2 hydrolysis. They expressed, in the sympathetic neurons, different transient receptor potential (Trp) channels that are selectively activated by Ins(1,4,5)P3-mediated Ca2þ signalling (hTrpC1) or by DAG (mTrpC6). Using these specific biosensors not only allowed a real-time assessment of PLC activity but also confirmed the striking differences between M1-muscarinic-receptor and B2-bradykininreceptor signalling. Whereas both GPCRs activate PLC efficiently in these neurons, exemplified by DAG-activated mTrpC6 and C12 translocation, only bradykinin was able to stimulate significantly Ins(1,4,5)P3-induced Ca2þ release. This work further highlighted a possible role of the actin cytoskeleton in controlling the proximity of different signalling components and an apparent uncoupling of PLC activity from Ca2þ mobilization. Micheva et al. [16] transiently transfected eGFP – PHPLCd into hippocampal neurons and by loading synapses with the dye FM 4-64, imaged local PtdIns(4,5)P2 within presynaptic boutons. Electrical stimulation of these neurons led to a translocation of this biosensor to the cytoplasm, dependent on NMDA activation and a possible http://tins.trends.com
retrograde action of nitric oxide. These studies emphasize the potential utility of biosensors to detect local changes in plasma membrane PtdIns(4,5)P2 and, with increasing evidence that this lipid is a key regulator of many ion channels [1 –3,56], the future use of biosensors specific for PtdIns(4,5)P2, might elucidate the dynamic nature and mechanisms of this regulation. One way to assess PtdIns(4,5)P2 rather than Ins(1,4,5)P3 concentrations might be the use of channel-based biosensors. Hardie et al. [57] exploited the properties of inward-rectifier channels (particularly Kir2.1) for their ability to be activated by PtdIns(4,5)P2. By expressing the human Kir2.1 channel in Drosophila photoreceptors, this group was able to associate casually a suppression of Kþ currents with the loss of PtdIns(4,5)P2 that is required for phototransduction. Ins(1,4,5)P3-induced Ca2þ release and synaptic activity There are now several studies suggesting that synaptically evoked Ca2þ release from stores can be initiated by activation of Ins(1,4,5)P3 receptors within dendrites and spines. Thus, synergistic (also termed ‘supralinear’) Ca2þ responses supported by Ca2þ influx through voltage-gated or ligand-gated channels following GPCR activation almost certainly results from Ins(1,4,5)P3-dependent CICR from stores [7,42,43,58– 60]. That this might be initiated by pairing synaptic activity with single or multiple action potentials to achieve the crucial spatial and temporal characteristics [58] implies that there must be compartments with the dendritic tree and a discrete localization of Ins(1,4,5)P3 and ryanodine receptors, GPCRs and channels. Couple this to the complexity of neuronal endoplasmic reticulum in the dendrites, axons and soma of different neurons and it is clear that the mechanisms of Ca2þ release are very diverse. Recent studies suggest that the local production and diffusion of Ins(1,4,5)P3, rather than that of Ca2þ, to distant targets could be operative in Ca2þ wave propagation in hippocampal CA1 pyramidal cells [59]. Therefore, superimposed upon CICR at Ins(1,4,5)P3 receptors, there could be Ca2þentry-mediated activation of Ins(1,4,5)P3 production further enhancing the probability of regenerative Ca2þ release. Indeed, a recent report [61] has described how activation of muscarinic receptors by synaptic stimulation of cholinergic afferents in hippocampal CA1 neurons led to a focal rise in apical dendritic Ca2þ concentration that then propagated to the soma and invaded the nucleus. Concluding remarks The ability to detect the activity of PLC in single neurons in real time with sufficient resolution alongside Ca2þ imaging will provide new insights into synaptic modulation and plasticity. Overall, to understand how information from transmitter receptors at dendritic spines is conveyed over potentially long distances to the nucleus in the soma will be crucial in linking synaptic activity to gene transcription and long-term neuronal adaptation. Clearly, the advances in imaging technology can provide significant information about the spatio-temporal aspects of PLC signalling (Table 3) and the field is now in the exciting position of being able to address
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fundamental issues associated with this important pathway in neurons. Acknowledgements We thank the Wellcome Trust for support (Programme Grant; 062495), Tobias Meyer for several constructs, Lyn McCarthy for manuscript preparation and the University of Leicester for study leave (S.R.N.).
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