Over the past 15 years or so, numerous studies have sought to characterise how nuclear calcium (Ca2+) signals are generated and reversed, and to understand how events that occur in the nucleoplasm influence cellular Ca2+ activity, and vice versa. In this Commentary, we describe mechanisms of nuclear Ca2+ signalling and discuss what is known about the origin and physiological significance of nuclear Ca2+ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca2+ signalling system that can generate its own Ca2+ transients that modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm.

Introduction

Calcium (Ca2+) is a key player in signal transduction. It modulates diverse cellular activities ranging from fertilisation to cell death (Berridge et al., 2000). It is well known that different cell types use various elements from a broad Ca2+ signalling `toolkit' (Box 1); consequently, the characteristics of global Ca2+ signals, and their physiological effects, can vary considerably (Berridge et al., 2000). This might also be the case for nuclear Ca2+ signalling, which is essentially a complex form of local Ca2+ signalling. As described below, the mechanisms of nuclear Ca2+ signalling are extensive, and it is likely that there is considerable flexibility in how Ca2+ signals are triggered in the nucleus and in the downstream targets that are affected. In this Commentary, we discuss what is known about the origin and physiological significance of nuclear Ca2+ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca2+ signalling system that can generate its own Ca2+ transients, which modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm. It should be noted that nuclear Ca2+ signalling has been the subject of previous reviews, some of which have presented different points of view (Bootman et al., 2000; Gerasimenko and Gerasimenko, 2004; Gomes et al., 2006; Lee et al., 1998; Santella and Carafoli, 1997).

The physiological significance of nuclear Ca2+ signalling

It has been well established that Ca2+ signals occurring inside cells affect activities within the nucleus. For example, the amplitude and frequency of global cellular Ca2+ signals can regulate the transcription of various genes (Dolmetsch et al., 1998). But what is the significance of the nuclear component of these Ca2+ transients? Do patterns of nuclear Ca2+ signalling simply track cytosolic Ca2+ signalling, or are there independent nuclear Ca2+ signalling mechanisms that are necessary for proper cell function?

A precise method for analysing the function of nuclear Ca2+ is to specifically localise a buffering molecule within the nucleoplasm, which prevents changes in nuclear Ca2+ levels but does not affect cytosolic Ca2+ signals. Nathanson and colleagues employed this approach to examine the involvement of nuclear Ca2+ in regulating the proliferation of hepatocyte cell lines (Rodrigues et al., 2007). They observed that targeting the Ca2+-binding protein parvalbumin to the nucleus significantly reduced cell proliferation and altered the proportions of cells that were in the S and G2-M phases of the cell cycle. By contrast, the expression of parvalbumin in the cytosol did not alter cell proliferation or cell cycle distribution. Most interestingly, the expression of the parvalbumin in the nucleus, but not in the cytosol, reduced the development of tumours when HepG2 cells were subcutaneously implanted into nude mice. Using a similar approach, the nuclear expression of parvalbumin was found to inhibit activation of the Elk1 transcription factor following stimulation of hepatoma cells with epidermial growth factor (EGF), whereas buffering cytosolic Ca2+ did not have the same effect (Pusl et al., 2002). Collectively, these data suggest that nuclear Ca2+signals are important for regulating cell proliferation in some contexts.

Ca2+ is a central player in many cellular signal-transduction cascades that modulate gene transcription. Signalling cascades mediate changes in gene expression by stimulating the translocation of transcription factors from the cytosol to the nucleus, or by causing the translocation and/or activation of enzymes that regulate the activity of nuclear transcription factors or the structure of chromatin. Although an increase in the concentration of nuclear Ca2+ might not be the initiating signal for such events, it can be necessary for gene transcription to occur or to be sustained. For example, nuclear Ca2+ can activate gene transcription via the nuclear factor of activated T cells (NFAT) family of proteins. In quiescent cells, NFAT is mainly found in the cytosol, but in response to an increase in cytosolic Ca2+, it rapidly (∼10 minutes) translocates to the nucleus (Tomida et al., 2003). The translocation of NFAT occurs following its dephosphorylation by the Ca2+-calmodulin-sensitive phosphatase calcineurin (Mellstrom et al., 2008), which exposes an intrinsic nuclear localisation sequence (NLS) in NFAT. NFAT and calcineurin associate in a Ca2+-dependent manner, and it has been reported that they translocate into the nucleus as a complex (Shibasaki et al., 1996). When Ca2+ is elevated in the nucleus, calcineurin remains active and associated with NFAT, and thereby sustains its transcriptional activity. Calcineurin mediates this effect by outcompeting kinases that promote NFAT re-phosphorylation (which would cause its nuclear export) and through a physical interaction that occludes a nuclear-export sequence (NES) in the NFAT protein.

Box 1. Cellular Ca2+-signalling machinery

Cytosolic Ca2+ signals can be evoked by chemical stimuli (such as hormones, growth factors and toxins), environmental changes (such as changes in pH or temperature shifts), mechanical deformation or depolarisation. Depending on the cell type, the nature of the stimulus and the extent of stimulation, Ca2+ signals can be transient, oscillatory or sustained (Thomas et al., 1997), and can occur globally or as subcellular events (Bootman et al., 2001). To generate such a diverse spectrum of signals, cells employ a range of messengers and mechanisms to evoke Ca2+ fluxes between various cellular compartments. For example, cells can access Ca2+ from a variety of intracellular stores (including the sarcoplasmic or endoplasmic reticulum, the Golgi, acidic vesicles and the NE) and via influx from the extracellular space. The figure depicts the basic Ca2+ cycle present in all mammalian cell types. The pathways that increase cytosolic Ca2+ levels are shown with red arrows and the mechanisms that reduce Ca2+ levels are depicted with blue arrows.

Ca2+ influx can occur via several different types of channels that have diverse activation mechanisms. The characteristics of the Ca2+ signals evoked by these channels depends on their biophysical properties, their expression levels and their location within the plasma membrane of the cells. Release of Ca2+ from intracellular stores is also mediated by several different types of Ca2+ channel, of which InsP3Rs (see Box 2) and RyRs are the best characterised. The magnitude of Ca2+ signals can be limited by various buffers inside cells, such as Ca2+-binding proteins and mitochondria. These buffers shape the spatial domain, time-course and amplitude of Ca2+ rises. Elevation of cytosolic Ca2+ concentration is transduced into functional changes in cellular activity by numerous sensors that bind Ca2+. Cellular Ca2+ sensors have varying affinities and locations, allowing them to specifically respond to different Ca2+ patterns.

An example of a nuclear-localised transcription factor that is regulated by both nuclear and cytosolic Ca2+ signals is cyclic AMP response element-binding protein (CREB). The signalling mechanisms that underlie CREB activation have been worked out particularly well in neurons, in which it has been established that depolarisation leads to the rapid phosphorylation of CREB on Ser133 by Ca2+-calmodulin-dependent kinase IV, which provides a necessary, but not sufficient, signal for CREB-mediated gene transcription (Dolmetsch et al., 2001). The triggering event is an increase in cytosolic Ca2+ levels in the immediate vicinity of L-type Ca2+ channels or N-methyl-D-aspartate receptors that are found in the synapse, at a site that is distal to the nucleus (Shaywitz and Greenberg, 1999). As CREB is only found in the nucleus, the phosphorylation of Ser133 is mediated by one of several Ca2+-sensitive signal transduction cascades that convey information from the remote synapses into the nucleus. In the nucleus, phosphorylated CREB binds additional proteins to form a transcriptionally active complex (Shaywitz and Greenberg, 1999). Although the synaptic Ca2+ signal does not need to propagate to the nucleus to induce phosphorylation of Ser133 on CREB, an increase in the level of nuclear Ca2+ is required for CREB-mediated gene transcription to occur. This is because additional Ca2+-dependent phosphorylation of CREB and its co-activators is required (Chawla et al., 1998; Kornhauser et al., 2002). Indeed, nuclear injection of an artificial Ca2+ buffer prevents CREB-mediated gene transcription, but not transcription induced by the serum-response element, which is sensitive to cytosolic Ca2+ buffering (Hardingham et al., 1997).

Another well-known target of nuclear Ca2+ signalling is the transcriptional regulator downstream regulatory element antagonist modulator (DREAM). It is well established that DREAM represses the expression of prodynorphin, an opiate-receptor precursor, and that mice that lack DREAM have a constitutive analgesic condition (Cheng et al., 2002). DREAM contains four Ca2+-binding EF hands (a widely expressed structural motif in proteins that bind Ca2+), and is directly regulated by an increase in nuclear Ca2+ levels (Mellstrom et al., 2008). When the concentration of Ca2+ in the nucleus is low, DREAM represses gene transcription by binding to DNA and displacing other transcriptional activators such as CREB (Ledo et al., 2002). An increase in the concentration of nuclear Ca2+ causes DREAM to dissociate from its DNA binding sites and thereby allows the transcription of its target genes.

Space limitations prevent a comprehensive discussion of the downstream targets of Ca2+ signalling pathways in the nucleus. However, it is evident from the in vitro and in vivo experiments described above that nucleoplasmic and cytosolic Ca2+ signals can synergistically control important cellular events. Furthermore, the nuclear component of global Ca2+ signals – or an independent increase in nuclear Ca2+ alone – can regulate specific nuclear processes.

Mechanisms that regulate the level or function of Ca2+ in the nucleus

Changes in nuclear Ca2+ concentration clearly impact on many cellular functions, but what are the mechanisms involved in regulating nuclear Ca2+ levels, and how are they related to those that operate in the cytosol? In the following section, we describe the mechanisms by which Ca2+ moves into and out of the nucleus and how intranuclear Ca2+ stores are triggered. Together, these mechanisms contribute to the regulation of Ca2+-dependent nuclear signalling pathways (Fig. 1).

InsP3Rs, RyRs and NAADPRs

To date, the most widely implicated mechanism for nuclear Ca2+ release is via the activation of inositol(1,4,5)-trisphosphate receptors (InsP3Rs) (see Box 2). Evidence in support of a role for nuclear InsP3Rs in regulating nuclear Ca2+ release has been obtained by using InsP3-binding assays (Humbert et al., 1996), InsP3R-specific antibodies (Leite et al., 2003; Malviya, 1994; Stehno-Bittel et al., 1995), electrophysiological recordings from nuclear envelope (NE) patches or from whole nuclei (Bustamante et al., 2000; Stehno-Bittel et al., 1995), nuclear microinjection of InsP3 (Hennager et al., 1995; Huh et al., 2007; Santella et al., 2003), direct visualisation of InsP3-evoked nuclear Ca2+ signals (Higazi et al., 2009), reconstitution of nuclear InsP3Rs into lipid bilayers (Leite et al., 2003) and prevention of nuclear Ca2+ signals by using InsP3R antagonists (Kumar et al., 2008). Furthermore, the addition of InsP3 to nuclei that were isolated from Xenopus oocytes, Aplysia neurons, mammalian epithelial cells or pancreatic acinar cells (Bezin et al., 2008a; Gerasimenko et al., 1995; Quesada and Verdugo, 2005; Stehno-Bittel et al., 1995) has been shown to induce nucleoplasmic Ca2+ transients. Not all of these studies differentiated between expression of InsP3Rs on the inner or outer NE. However, it is clear that InsP3Rs are present on both the inner and outer NE, and that those channels expressed on the inner NE can trigger the release of Ca2+ from the NE lumen directly into the nucleoplasm. The actual proportion of the total cellular InsP3Rs that are found on the inner NE (or other putative nuclear stores; see below) is probably very small (<1%) (Laflamme et al., 2002). However, their privileged access to the nucleoplasm might confer them with an essential local signalling function.

Fig. 1.

Summary of the pathways that regulate nuclear Ca2+ levels. Key aspects of this schematic are the separate Ca2+-releasing mechanisms in the cytosol and nucleus. In particular, the independent cytosolic and nuclear phosphoinositide cycles are depicted. Although Ca2+ release and the production of Ca2+-releasing messengers can be localised in the individual compartments, nuclear and cytosolic Ca2+ levels can also be coordinated via the diffusion of messengers through NPCs. Some of the putative pathways described in this figure are controversial. In particular, the translocation of growth-factor receptors into the nucleus has not been observed in all studies (marked by `?'). + and – indicate a positive or negative effect on a downstream process, respectively.

Fig. 1.

Summary of the pathways that regulate nuclear Ca2+ levels. Key aspects of this schematic are the separate Ca2+-releasing mechanisms in the cytosol and nucleus. In particular, the independent cytosolic and nuclear phosphoinositide cycles are depicted. Although Ca2+ release and the production of Ca2+-releasing messengers can be localised in the individual compartments, nuclear and cytosolic Ca2+ levels can also be coordinated via the diffusion of messengers through NPCs. Some of the putative pathways described in this figure are controversial. In particular, the translocation of growth-factor receptors into the nucleus has not been observed in all studies (marked by `?'). + and – indicate a positive or negative effect on a downstream process, respectively.

It has also been proposed that InsP3Rs are present within heterochromatin and euchromatin, where they are expressed on small vesicular Ca2+ stores that also contain high-capacity Ca2+-binding chromogranin proteins (Yoo et al., 2007). The presence of small InsP3-sensitive vesicular stores could help to explain the curious spotted distribution of nuclear InsP3R staining that is sometimes observed when using InsP3R-specific antibodies (particularly type-2-InsP3R-specific antibodies) (Garcia et al., 2004; Laflamme et al., 2002; Leite et al., 2003). If this is the case, these vesicular structures could represent nuclear InsP3-sensitive Ca2+ stores that might operate independently of cytosolic Ca2+ channels. Exactly how these vesicles are formed and how chromogranins and InsP3Rs are trafficked to them is unclear. It is known that chromogranins can interact with InsP3Rs and potentiate their opening (Thrower et al., 2003), and it has been proposed that the colocalisation of chromogranins with InsP3Rs on nuclear vesicles underlies the increased sensitivity of nuclei to InsP3-evoked Ca2+ release (Huh et al., 2007). However, more work is needed to verify the existence and function of these nucleoplasmic Ca2+ stores.

In addition to InsP3Rs, it has been demonstrated that ryanodine receptors (RyRs) and nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs) are expressed inside, or on the NE, of nuclei in various cell types, and that they regulate nuclear Ca2+ signalling. For example, cyclic ADP ribose or NAADP have been shown to cause the release of Ca2+ from the NE and to evoke Ca2+ transients in nuclei isolated from Aplysia neurons (Bezin et al., 2008b), osteoblasts (Adebanjo et al., 2000) and pancreatic acinar cells (Gerasimenko et al., 2003).

The nucleoplasmic reticulum

Ca2+-release channels have been found on nucleoplasm-facing invaginations of the NE, which are known as the nucleoplasmic reticulum (or nuclear tubules) (Echevarria et al., 2003; Lee et al., 2006; Lui et al., 2003; Schermelleh et al., 2008). Although such structures have been observed in a wide range of cell types (Fricker et al., 1997), they might not be universal (Bezin et al., 2008b). For reasons that are unknown, the structure of the nucleoplasmic reticulum seems to vary from being made up of simple solitary projections (Fig. 2) to a complex branched network, and it is interesting that the tubules often colocalise with nucleoli (Fricker et al., 1997). The lumen in the centre of the nucleoplasmic reticulum tubules is contiguous with the cytosol and presumably allows cytosolic messengers to gain increased access to the deeper parts of the nucleus. There is evidence that functional Ca2+-release channels localise on the nucleoplasmic reticulum (Echevarria et al., 2003; Marius et al., 2006). For example, photorelease of caged InsP3 (Echevarria et al., 2003) or caged Ca2+ (Marius et al., 2006) in the vicinity of the nucleoplasmic reticulum initiated regenerative nuclear Ca2+ signals. These data indicate that InsP3Rs and RyRs are present on the nucleoplasmic reticulum. It has been suggested that the presence of a nucleoplasmic reticulum increases the surface-to-volume ratio within the nucleus, thereby facilitating both the entry and exit of Ca2+.

Box 2. InsP3Rs are a generic pathway for Ca2+ release

With respect to Ca2+ release from intracellular stores, the best-characterised mechanisms involve the activation of InsP3Rs, RyRs and NAADPRs. InsP3 is a highly diffusible hydrophilic messenger that is produced by the hydrolysis of PtdIns(4,5)P2 (Irvine, 2006). This is catalysed by a family of PLC isozymes, which, depending on the isozyme, can be activated by G-proteins (Gq), phosphorylation or Ca2+ itself, or are introduced into egg cytosol during fertilisation (see figure). The other product of PtdIns(4,5)P2 hydrolysis is DAG, which stays within the plasma membrane where it promotes the activation of protein kinase C or is metabolised further (some of the metabolites promote Ca2+ signals). InsP3-induced Ca2+ release is probably the most widespread mechanism for Ca2+ mobilisation in mammalian cells, and is a focal point for the convergence of multiple signalling inputs (Roderick and Bootman, 2003).

RyRs are structurally similar to InsP3Rs in that they comprise a tetrameric pinwheel-like arrangement, but have approximately twice the molecular mass and conductance as InsP3Rs. They have a more restricted expression profile than InsP3Rs (Bennett et al., 1996), and are best characterised in their role as Ca2+-induced Ca2+-release channels in muscle and neurons. The opening of RyRs can be modulated by cADPR, which is produced from NAD+ by the action of ribosyl cyclase enzymes such as CD38 (Bezin et al., 2008a). The same enzymes may also catalyse an alternative reaction that culminates in the production of a different Ca2+-releasing messenger, NAADP (Soares et al., 2007). In addition to InsP3, cADPR and NAADP, Ca2+ mobilisation can also be evoked by metabolites including nitric oxide, DAG, arachidonic acid, sphingosine and sphingosine-1-phosphate and Ca2+ itself (Bootman et al., 2002).

Fig. 2.

An example of nucleoplasmic reticulum. (A) Single HeLa cell expressing ER-targeted EGFP. The net-like structure of the ER can be seen, especially at the periphery of the cell where the tubules of the organelle are less dense. The position of the nucleus is marked by N. (B) Confocal sections of the cell were volume-rendered to provide a 3D image of the fluorescence labelling. This 3D image was then serially sectioned in the vertical plane to produce the images in Bi-v. The images in Bi-v therefore represent optical sections running from the top to the bottom of the cell. An example of a position from which a vertical section was obtained is indicated by the blue bar in A. The nucleus is evident as an unlabelled region in the middle of the cell. Biii-v indicate a membranous protrusion that spans across the nucleus from one side to the other, which represents the nucleoplasmic reticulum. Images were obtained using using a Zeiss LSM 510 laser-scanning confocal microscope. The optical depth of each image is approximately 1 μm.

Fig. 2.

An example of nucleoplasmic reticulum. (A) Single HeLa cell expressing ER-targeted EGFP. The net-like structure of the ER can be seen, especially at the periphery of the cell where the tubules of the organelle are less dense. The position of the nucleus is marked by N. (B) Confocal sections of the cell were volume-rendered to provide a 3D image of the fluorescence labelling. This 3D image was then serially sectioned in the vertical plane to produce the images in Bi-v. The images in Bi-v therefore represent optical sections running from the top to the bottom of the cell. An example of a position from which a vertical section was obtained is indicated by the blue bar in A. The nucleus is evident as an unlabelled region in the middle of the cell. Biii-v indicate a membranous protrusion that spans across the nucleus from one side to the other, which represents the nucleoplasmic reticulum. Images were obtained using using a Zeiss LSM 510 laser-scanning confocal microscope. The optical depth of each image is approximately 1 μm.

Calmodulin

As indicated above, cytosolic and nuclear activities can be coordinated by the spread of Ca2+ between these compartments. In addition, the Ca2+-dependent movement of enzymes (such as calcineurin) or other signalling mediators can convey signals into and out of the nucleus. One such moiety is the ubiquitous Ca2+-binding protein calmodulin, which has been proposed to translocate into the nucleus following cellular stimulation. Although translocation of calmodulin from the cytosol into the nucleus has been observed in numerous cell types following elevation of cytosolic Ca2+ (Mermelstein et al., 2001; Thorogate and Torok, 2004; Wu and Bers, 2007), the mechanism by which calmodulin enters the nucleus is not clear. Calmodulin does not contain an obvious motif for nuclear localisation, and might therefore `piggy-back' on other proteins that are translocated into the nucleus (Thorogate and Torok, 2007).

Although calmodulin is constitutively present in the nucleus, translocation of additional calmodulin, together with an increase in Ca2+ levels, could be crucial for nuclear signalling pathways. For example, calmodulin translocation in depolarised neurons occurred over the same time-course as CREB phosphorylation (Deisseroth et al., 1998). Buffering nuclear calmodulin, or inhibiting the nuclear influx of calmodulin, prevented the depolarisation-induced phosphorylation of CREB on Ser133. Furthermore, transgenic mice expressing a calmodulin-binding protein that was specifically expressed in the nuclei of forebrain neurons had a decreased level of CREB phosphorylation and impaired long-term memory consolidation (Limback-Stokin et al., 2004). Calmodulin is one example of many proteins that probably move into the nucleus to allow Ca2+ signals to function. Ca2+ causes its many functions by binding to proteins and altering their configurations to make them active or inactive. Therefore, the movement of signalling proteins concomitant with nucleoplasmic Ca2+ changes is important for Ca2+ to trigger downstream events.

Can NPCs inhibit Ca2+ diffusion?

Nuclear pore complexes (NPCs) are the major gateway for ions and macromolecules to pass between the cytosol and nucleoplasm (Box 3). Measurements of the size of the NPC central channel indicate that particles smaller than 9 nm should pass through without restriction. In particular, Ca2+ ions, which have a hydrated radius of ∼4 Å, should pass through easily. Although the central NPC channel can become partially occluded during the transport of various cargos, which could plausibly affect Ca2+ movement, ions and small molecules can pass unhindered through other channels that are thought to exist at the periphery of NPCs (Kramer et al., 2007). From these simple considerations, it would be expected that NPCs provide a constitutive conduit for Ca2+ moving between the nucleoplasm and the cytosol.

However, it has been suggested that NPCs can attain a conformation in which they do not permit ion transport. Using patch-clamp techniques, it is possible to form giga-ohm seals on the outer NE for periods of many minutes without any evident ion flux (Bustamante et al., 2000). This is a striking finding, as even a conservative estimate of NPC density suggests that a typical patch-clamp experiment should encompass 10-100 NPCs. As the pores have a conductance of ∼1 nanosiemen each, there should be a considerable current whenever a patch electrode is attached (Danker et al., 1997). However, electrophysiological recordings often detect only a few (about six per patch) operative channels (Bustamante et al., 2000). These observations have led to the suggestion that NPCs mainly exist in a closed conformation in which they are not ion conductive.

Box 3. Nuclear pore complexes

NPCs are large (∼125 MDa) protein complexes with an eightfold-symmetric architecture. They are approximately 120 nm in diameter, 80 nm in height and are studded around the circumference of the NE. A roughly cylindrical channel runs from top to bottom in the middle of each NPC (Stoffler et al., 2006). It is generally accepted that the central channel forms the pathway for the movement of large molecules, and it was assumed that the central channel would also allow the passage of small molecules and ions. However, many studies have shown that the binding of proteins that occlude the central channel and prevent active transport do not affect the passive diffusion of small molecules (Kramer et al., 2007). It has been demonstrated that there is an alternative pathway for the passage of ions and small molecular weight species through ∼eight symmetrical pores that are located around the periphery of NPCs (Shahin et al., 2001).

NPCs allow metabolites, nucleic acids and many proteins to pass efficiently across the NE via passive diffusion (Lee et al., 1998). Proteins in excess of ∼50 kDa require energy-dependent movement across the NPC. Such large proteins are often designated for nuclear entry or exit by a specific NLS or NES. NLS and NES domains are constitutively displayed by some proteins to ensure continuous nuclear or cytosolic distribution. In other proteins, these targeting sequences can be revealed by covalent modification of the protein, thereby providing a mechanism for dynamic control of protein subcellular localisation. A classic example is the transcription factor NFAT (see main text for details) (Shaw, 1988; Hallhuber et al., 2006).

A commonly observed but controversial feature of NPCs is the presence of a `central plug' that appears to occupy, and perhaps occlude, the channel (see figure). The nature of the putative NPC plug has been widely debated. Although it has been described as a dynamically regulated component of the NPC that somehow moves into place to occlude the pore, it has also been suggested that the plug is in fact cargo caught in transit. Support for the latter idea comes from the observation that there is an increased number of NPCs containing plugs when nuclei are cooled or if ATP is withdrawn, which prevents active transport (Stoffler et al., 1999). If the central plug were made up of randomly bound cargo, it could not be part of a deliberate Ca2+-sensitive mechanism for regulating nuclear-cytosolic exchange of macromolecules. However, data that oppose the idea that the central plug is made up of randomly bound cargo include AFM studies showing that there are two reproducible conformations of the NPC. Given that different types of cargo probably have various shapes, AFM images of NPCs that had a greater variety of central plug conformations would be expected. However, it must be considered that most cargos are probably much smaller than the NPC, and therefore it is possible that they would not be evident in AFM studies.

An alternative viewpoint has arisen from studies carried out using a different electrophysiological approach to monitor NPC activity. Instead of using membrane-attached patches, Oberleithner and colleagues employed an `hourglass' technique in which an isolated nucleus was sandwiched in a thin capillary tube. Using this method, it was observed that NPCs are constitutively open under physiological conditions (Danker et al., 2001). It was possible to modulate the NPC current recorded using the hourglass technique. For example, the addition of ATP or the depletion of the NE Ca2+ store increased the conductance of the NPC, but the NPCs always permitted cation flux (Shahin et al., 2001).

These latter findings arguably convey the physiological properties of NPCs most accurately. It is likely that patch-clamping of the NE distorts the membrane and somehow causes the NPCs to become non-conductive, whereas, under native conditions that are maintained using the hourglass technique, NPCs allow the constitutive passage of Ca2+ and other ions. Therefore, NPCs cannot be considered a complete diffusion break for Ca2+. Rather, ions in the nucleoplasm or cytosol have permanent access to the other compartment (Eder and Bading, 2007). At most, NPCs might act as a diffusion filter and introduce a kinetic delay in the equilibration of nuclear-cytosolic Ca2+ concentrations. The extent of the kinetic delay might be subject to modulation: although NPCs do not close, their conductance can change in response to factors such as Ca2+ and ATP. Furthermore, the extent of NPC expression on the NE can vary from 1-50 NPCs per μm2, depending on cell type (Wang and Clapham, 1999). A greater expression of NPCs would allow a more rapid equilibration of cytosolic and nuclear Ca2+ concentrations.

Regulation of NPCs – a role for Ca2+?

Numerous studies have proposed that NPCs are regulated by changes in Ca2+ concentration either within the lumen of the NE, or at the cytosolic-nucleoplasmic face. However, there is little consistency in the nature of the observed effect. A widely cited example is the demonstration that altering the Ca2+ concentration in the lumen of the NE regulates both passive diffusion and NLS-mediated protein transport through NPCs (Greber and Gerace, 1995): depletion of the NE Ca2+ store was found to attenuate the nuclear influx of proteins bearing a NLS and of a non-specific fluorescent dextran molecule. It was suggested that the Ca2+ sensor in the NE was the integral membrane protein gp210, a nucleoporin of 210 kDa that functions to anchor NPCs. gp210 interacts with NPCs via its cytosolic C-terminus, whereas the bulk of the protein projects into the lumen of the NE, where it can sense Ca2+ levels and thereby mediate changes in NPC structure (Greber et al., 1990). In support of this idea, an antibody specific for the lumenal domain of gp210 was found to inhibit both passive diffusion and signal-mediated transport into the nucleus (Greber and Gerace, 1992).

A similar finding was reported when the nuclear import of histone H1 (a ∼21 kDa constitutively expressed nuclear protein) and two smaller fluorescently tagged dextran molecules (of 3 and 10 kDa) was compared in intact cardiomyocytes (Perez-Terzic et al., 1999). Reducing the Ca2+ concentration in the NE lumen by treating cells with a Ca2+ chelator, a Ca2+-pump inhibitor or ionomycin prevented the energy-dependent import of the histone. The same treatments prevented the passive influx of 10 kDa dextran, but not the 3 kDa dextran species. These data indicate that NPC function is sensitive to the concentration of Ca2+ within the NE, and that altering lumenal Ca2+ concentration can regulate both active and passive flux of large, but not small, molecules. However, a similar study examining the diffusion of GFP between the nucleoplasm and cytosol of intact Xenopus oocytes reached the opposite conclusion; the NE reduced diffusion of GFP by ∼100-fold, but depletion of Ca2+ from the lumen of the NE made no difference (Wei et al., 2003). Yet a different finding was reported in a study of the movement of photo-activated GFP across the NE in hepatocytes (O'Brien et al., 2007). In this case, a rise in cytosolic Ca2+ levels, and not changes in NE Ca2+ content, increased the influx (but not the efflux) of photo-activated GFP across the NE. In this latter study, it was also demonstrated that Ca2+ signals evoked by InsP3Rs regulated the influx of GFP into the nucleus, suggesting a putative link between cell activation by InsP3-producing stimuli and the control of nuclear trafficking.

These discrepant studies highlight some of the controversy surrounding how NPC opening is regulated. As yet, there is little consensus about what effects cytosolic Ca2+, or Ca2+ within the NE, have on NPC structure. However, although the results of the various studies are not entirely consistent, they suggest that Ca2+ is somehow important in regulating nuclear traffic. The mechanism by which Ca2+ might regulate NPC permeability is unclear. One of the most commonly proposed theories is that a central `plug' moves and blocks the channel in response to changes in the Ca2+ concentration in either the cysotol or the lumen of the NE (see Box 3). Consistent with this idea, several studies reported Ca2+-dependent changes in the shape of NPCs (Stoffler et al., 1999) and, in particular, alterations in the number of NPCs bearing a central plug. A key technique in these investigations was atomic force microscopy (AFM), which can be used to scan the three-dimensional topography of the cytosolic face of NPCs. AFM can resolve changes in the surface of large macromolecules, and has been used to show that the depletion of Ca2+ from the lumen of the NE significantly and reversibly increased the proportion of NPCs with a central plug (Wang and Clapham, 1999). The observed change in NPC structure that is caused by lumenal Ca2+ depletion is in line with the Ca2+-dependent alterations in NPC transport described above (Perez-Terzic et al., 1999), and supports the concept of allosteric regulation of NPC gating by Ca2+. However, the reported changes in NPC structure are not the same in different studies (Erickson et al., 2006), and alteration in NPC structure following Ca2+ release from the NE is not universally observed (Stoffler et al., 2006).

Even if changes in NPC permeability do indeed occur under physiological conditions, it is not clear what difference this makes to cellular growth or activity. It has been argued that simply altering the permeability of the nucleus to species of different molecular masses is a rather crude mechanism for regulating nuclear-to-cytosolic flux (Torok, 2007). Much greater specificity for NPC transit can be achieved by cellular signalling mechanisms that either promote or diminish the exposure of molecular tags (such as the NLS and NES) that promote NPC transport.

Nuclear Ca2+ signalling in intact cells

It has been established that Ca2+ signals occur in the nucleus of intact cells. For example, confocal imaging studies have shown that as Ca2+ waves `sweep' through the cytosol of hormone-stimulated cells, the nucleus becomes engulfed by the signal (Lipp et al., 1997). However, a major controversial issue concerning nuclear Ca2+ signalling is whether Ca2+ transients are generated autonomously in the nucleoplasm, and whether they can be distinct from cytosolic Ca2+ changes. Numerous studies have examined Ca2+ signalling in isolated nuclei (Adebanjo et al., 2000; Bezin et al., 2008b; Gerasimenko et al., 1995). The advantage of studying nuclei in isolation is that changes in nucleoplasmic Ca2+ only occur if Ca2+ is released from channels within the inner NE. Although the NPCs are present in this type of preparation, it is unlikely that Ca2+ released from channels on the outer NE (that face away from the nucleus), or from potential remnants of the ER, can diffuse into the nucleoplasm. Rather, any Ca2+ that is released outside of the NE is rapidly dissipated and diluted. Isolated nuclei therefore represent a system in which nucleoplasm-directed Ca2+-release channels can be examined. However, although these studies are useful for examining the potential of nuclei to generate their own Ca2+ signals, they do not determine whether these signals are at all significant compared with the larger fluxes of Ca2+ that occur in the cytosol of intact cells. The key issue, therefore, is to establish how nuclear Ca2+ signals arise within the physiological context of intact cells, and whether Ca2+ channels facing the nucleoplasm have a significant role.

Measuring nuclear Ca2+ signals: key considerations

The idea that nuclear Ca2+ levels are regulated independently from those in the cytosol emerged as soon as imaging technology advanced to allow monitoring of Ca2+ changes simultaneously in different cellular compartments (Bkaily et al., 1997). Using both wide-field and confocal fluorescence imaging of living cells, several groups began to report that nuclear Ca2+ concentration differed from that in the cytosol. The extent to which the Ca2+ signals in these two compartments differed was highly variable between studies. In some cases, the nuclear and cytosolic Ca2+ levels were found to be entirely independent, whereas others reported modest differences in the kinetics or amplitude of the nucleoplasmic and cytosolic responses (Leite et al., 2003; Santella et al., 2003). Although the examples of differential Ca2+ concentration in the nucleus and cytosol are too numerous to mention in detail, some are discussed below, and Table 1 lists some of the reported findings.

Table 1.

Selected examples of observed differences in cytosolic and nucleoplasmic Ca2+ levels

Nature of difference in nuclear and cytosolic Ca2+ signals Cell type Experimental method References
Temporally independent Ca2+ signals occurred in each compartment   Smooth muscle   Fluorescence imaging   (Fedoryak et al., 2004)  
Lower threshold for InsP3-evoked Ca2+ release in the nucleus   PC12, NIH3T3 cells   Fluorescence imaging   (Huh et al., 2007)  
Nucleoplasmic Ca2+ signal lagged ∼4 seconds behind elevation in cytosolic Ca2+  Starfish oocytes   Fluorescence imaging   (Santella et al., 2003)  
Nucleoplasmic Ca2+ signal lagged ∼100 milliseconds behind elevation in cytosolic Ca2+  Cardiac myocytes   Fluorescence imaging   (Bootman et al., 2007; Kockskamper et al., 2008a)  
Lower threshold for InsP3-evoked Ca2+ release in the nucleus   Liver-cell line   Fluorescence imaging   (Leite et al., 2003; Echevarria et al., 2003)  
Larger increase in Ca2+ in the nucleoplasm than in the cytosol, but with the same kinetics   HeLa cells   Bioluminescence (aequorin)   (Brini et al., 1993)  
Smaller increase in Ca2+ in nucleoplasm than in the cytosol, but with the same kinetics   HeLa cells   Bioluminescence (aequorin)   (Badminton et al., 1996; Badminton et al., 1995)  
Nucleoplasmic Ca2+ signals occurred in the absence of Ca2+ changes in the surrounding cytosol   Cardiac myocytes   Fluorescence imaging   (Higazi et al., 2009)  
Nature of difference in nuclear and cytosolic Ca2+ signals Cell type Experimental method References
Temporally independent Ca2+ signals occurred in each compartment   Smooth muscle   Fluorescence imaging   (Fedoryak et al., 2004)  
Lower threshold for InsP3-evoked Ca2+ release in the nucleus   PC12, NIH3T3 cells   Fluorescence imaging   (Huh et al., 2007)  
Nucleoplasmic Ca2+ signal lagged ∼4 seconds behind elevation in cytosolic Ca2+  Starfish oocytes   Fluorescence imaging   (Santella et al., 2003)  
Nucleoplasmic Ca2+ signal lagged ∼100 milliseconds behind elevation in cytosolic Ca2+  Cardiac myocytes   Fluorescence imaging   (Bootman et al., 2007; Kockskamper et al., 2008a)  
Lower threshold for InsP3-evoked Ca2+ release in the nucleus   Liver-cell line   Fluorescence imaging   (Leite et al., 2003; Echevarria et al., 2003)  
Larger increase in Ca2+ in the nucleoplasm than in the cytosol, but with the same kinetics   HeLa cells   Bioluminescence (aequorin)   (Brini et al., 1993)  
Smaller increase in Ca2+ in nucleoplasm than in the cytosol, but with the same kinetics   HeLa cells   Bioluminescence (aequorin)   (Badminton et al., 1996; Badminton et al., 1995)  
Nucleoplasmic Ca2+ signals occurred in the absence of Ca2+ changes in the surrounding cytosol   Cardiac myocytes   Fluorescence imaging   (Higazi et al., 2009)  

Owing to their brightness, Ca2+-sensitive fluorescent indicators have been used in most studies that compared the properties of nuclear and cytosolic Ca2+ signals. However, it is certain that many of these studies are invalid as they failed to recognise that organic fluorochromes have different characteristics in the nuclear and cytosolic compartments (Birch et al., 1992; Bkaily et al., 2006). Indeed, one of the most prominent early studies of nuclear Ca2+ regulation – which suggested that Ca2+ concentration in the nucleus tracked that in the cytosol up to ∼300 nM, after which they were insulated against further rises (al-Mohanna et al., 1994) – is compromised by the lack of in situ calibration (O'Malley et al., 1999). We (Thomas et al., 2000b) and others (O'Malley et al., 1999; Perez-Terzic et al., 1997) have demonstrated that commonly used fluorescent Ca2+ indicators are much brighter in the nucleus than in the cytosol; they differ with respect to both their affinity for Ca2+ and their dynamic range. Therefore, to obtain absolute values for Ca2+ concentration in different cellular compartments, it is crucial to perform independent calibrations with the indicator in exactly the same environment in which it will be used (Thomas et al., 2000b). Typically, when quiescent cells are loaded with a Ca2+ indicator such as fura2 or fluo3, it is evident that the nucleus has a greater fluorescence than the cytosol. The difference in the intensity of the indicator fluorescence between the nucleus and cytosol is maintained or even exaggerated as the Ca2+ concentration globally increases. This exaggerated nuclear fluorescence has led some studies to erroneously conclude that nuclei amplify cellular Ca2+ signals, and even that there are intranuclear Ca2+ gradients (Birch et al., 1992). It is not known why fluorescent Ca2+ indicators behave differently in the nucleus, although it has been established that factors such as ionic strength, viscosity and pH can alter Ca2+ binding to the indicator. In addition, it appears that Ca2+ indicators interact with nuclear contents; for example, in Xenopus oocyte nuclei that had been loaded with fluo3, it was found that the indicator did not diffuse freely because it became somehow `trapped' in chromatin and/or DNA (Perez-Terzic et al., 1997). These technical artefacts are one of the main reasons why nuclear Ca2+ signalling has such a controversial history.

Perinuclear Ca2+ release can affect nucleoplasmic Ca2+ levels

The observation of a nucleoplasmic Ca2+ signal does not necessarily indicate that the nuclei themselves are the actual source of Ca2+. It is evident that Ca2+ diffuses more slowly in the cytosol than in the nucleoplasm because of the much greater buffering and sequestration capacity in the cytosolic compartment (Fox et al., 1997). This means that Ca2+ signals that arise immediately outside of the nucleus have a greater potential to diffuse through NPCs and into the nucleus than to permeate across the cytosol. Consistent with this idea, it was observed that mobilisation of Ca2+ from intracellular stores surrounding the nucleus of various cell types caused simultaneous Ca2+ elevations within the cytosol and nucleoplasm (Chamero et al., 2002), whereas distal Ca2+ signals that arose from voltage-operated Ca2+ channels at the plasma membrane induced cytosolic Ca2+ transients, but only modest and delayed increases of nucleoplasmic Ca2+.

An emerging concept in Ca2+ signalling is that localised perinuclear Ca2+ release – that is, microscopic Ca2+ release events that occur next to the nucleus – can affect nuclear activities (Fig. 3A). It has been shown that brief openings of InsP3Rs or RyRs induce microscopic Ca2+ events that do not diffuse farther than a few micrometres. If such events occur in the cytosol at sites that are distant from the nucleus, they probably have only a modest impact on cell behaviour, but if they occur next to the nucleus, then they can have a long-lasting and integrating effect on nucleoplasmic Ca2+. Such observations have been made using both excitable and non-excitable cell types (Lipp et al., 1997). Indeed, many of the studies that proposed the existence of independent nuclear Ca2+ signals could be explained by such a perinuclear cytosolic Ca2+ release, followed by anisotropic diffusion of Ca2+ through NPCs into the nucleoplasm. In particular, this probably applies to studies in which the nuclear Ca2+ signal lags behind the cytosolic Ca2+ response (Huh et al., 2007).

Fig. 3.

Perinuclear Ca2+ channels can affect nucleoplasmic Ca2+ concentration. (A) The differential effect of perinuclear versus distal Ca2+ release on nucleoplasmic Ca2+ levels. When Ca2+ puffs are activated distal to the nucleus, they are constrained to a distance of ∼2-4 μm owing to cytosolic Ca2+ buffering and sequestration. These Ca2+ puffs do not diffuse through the cytosol, and therefore do not reach the nucleus. By contrast, when a Ca2+ puff of similar amplitude occurs next to the nucleus, it diffuses anisotropically into the nucleoplasm, engulfs the entire nucleus and can even spill out of the nucleus on the opposite side to where the Ca2+ signal originated. Further details can be found in (Lipp et al., 1997). (B) Perinuclear expression of type 2 InsP3Rs in a neonatal myocyte.

Fig. 3.

Perinuclear Ca2+ channels can affect nucleoplasmic Ca2+ concentration. (A) The differential effect of perinuclear versus distal Ca2+ release on nucleoplasmic Ca2+ levels. When Ca2+ puffs are activated distal to the nucleus, they are constrained to a distance of ∼2-4 μm owing to cytosolic Ca2+ buffering and sequestration. These Ca2+ puffs do not diffuse through the cytosol, and therefore do not reach the nucleus. By contrast, when a Ca2+ puff of similar amplitude occurs next to the nucleus, it diffuses anisotropically into the nucleoplasm, engulfs the entire nucleus and can even spill out of the nucleus on the opposite side to where the Ca2+ signal originated. Further details can be found in (Lipp et al., 1997). (B) Perinuclear expression of type 2 InsP3Rs in a neonatal myocyte.

Perinuclear Ca2+ release, and the consequent elevation in nuclear Ca2+ that it induces, has recently become a particular focus in studies of cardiac myocytes (Higazi et al., 2009; Kockskamper et al., 2008b; Wu et al., 2006). Ca2+ signals occur rhythmically in cardiac myocytes and promote the interaction of protein filaments that make the cells contract and allow the heart to pump blood. This process of excitation-contraction (EC) coupling in the heart relies on the interplay between voltage-gated Ca2+ channels on the plasma membrane and RyRs on the sarcoplasmic reticulum (the major internal Ca2+ store). The voltage-gated Ca2+ channels respond to the depolarizing action potential that sweeps over the heart with each beat by opening and thereby permitting the influx of a small amount of Ca2+. This Ca2+ signal is greatly amplified by RyRs that are close to the voltage-gated Ca2+ channels. Ca2+ diffuses from the RyRs to the actin/myosin filaments and initiates cell contraction. RyRs are therefore considered to be the main Ca2+-release channel in the heart. However, cardiac myocytes also express InsP3Rs (Lipp et al., 2000; Perez et al., 1997), and recent studies of neonatal (Garcia et al., 2004; Guatimosim et al., 2008; Luo et al., 2008b; Luo et al., 2006), atrial (Bootman et al., 2007; Kockskamper et al., 2008a; Zima et al., 2007) and ventricular myocytes (Higazi et al., 2009; Wu et al., 2006) demonstrated that InsP3-generating agonists, or InsP3 itself, have specific effects on nucleoplasmic Ca2+ signals. Indeed, it appears that a substantial proportion of InsP3Rs in neonatal and adult cardiomyocytes is expressed on membranes close to the nucleus or on the NE (Bare et al., 2005; Higazi et al., 2009; Liu et al., 2001) (Fig. 3B). This strategic positioning of InsP3Rs might allow the generation of nucleus-specific Ca2+ signals (even though they originate in the cytosolic compartment), which could have a significant role in regulating cardiac gene transcription, and thereby be involved in controlling processes such as cardiac hypertrophy. In support of this, it has been demonstrated that the release of perinuclear InsP3-sensitive Ca2+ stores promotes the activation of calcineurin and nuclear transport of NFAT into the nucleus to trigger a hypertrophic gene programme (Higazi et al., 2009). In addition, perinuclear InsP3-mediated Ca2+ release promotes the phosphorylation of histone deacetylase 5 via Ca2+-calmodulin-dependent kinase II, causing it to be exported out of the nucleus (Wu et al., 2006) and thereby de-repressing genes that underlie the hypertrophic growth of myocytes.

It is well known that the stimulation of myocytes with hormones that activate InsP3 production can promote hypertrophy (Sugden and Clerk, 2005), but it is not clear how hormones induce subtle changes in Ca2+-dependent gene transcription within cells that already experience periodic surges of Ca2+ during EC-coupling (Roderick et al., 2007). Perinuclear InsP3Rs might explain how this occurs, as they can provide a source of Ca2+ that is spatially and temporally distinct from the Ca2+ signals that are involved in EC-coupling (Molkentin, 2006). Consistent with this model, stimulation of rat neonatal myocytes with phenylephrine evoked InsP3R-mediated perinuclear Ca2+-release events that entered the nucleoplasm (Luo et al., 2008). Together with the finding that inhibiting InsP3R opening prevented hypertrophic growth in response to phenylephrine stimulation (Luo et al., 2006), it is logical to conclude that phenylephrine promotes hypertrophy by activating perinuclear InsP3Rs and, consequently, causing nucleoplasmic Ca2+ signals. Comparable observations have been made in cardiac cells stimulated with another InsP3-generating agonist, endothelin-1; InsP3Rs in close proximity to the NE triggered nucleoplasmic Ca2+ signals and hypertrophic gene transcription, whereas EC-coupling did not (Garcia et al., 2004; Higazi et al., 2009). Although InsP3Rs conduct significantly less Ca2+ and are expressed at levels that are ∼100-fold lower than RyRs, the activation of InsP3Rs can clearly have a dramatic effect on gene transcription in cardiac myocytes owing to their specific impact on nuclear Ca2+. It remains to be determined whether this model of perinuclear Ca2+ release is involved in regulating gene transcription and other nuclear activities in other cell types. However, there are numerous studies that have examined InsP3R distribution in various cell types and shown that these Ca2+ channels are often most densely concentrated around the nucleus (Bootman et al., 2001; Shirakawa and Miyazaki, 1996; Thomas et al., 2000a; Vermassen et al., 2003). It therefore seems probable that modulation of nucleoplasmic Ca2+ concentration by perinuclear Ca2+ release is a generic signalling paradigm.

Independent nuclear Ca2+ signalling

The studies described above substantiate the idea that perinuclear Ca2+ release significantly affects nucleoplasmic Ca2+ concentration. However, the observations also raise questions about the function of Ca2+ channels located on the inner NE and in the nucleoplasmic reticulum. In particular, why don't the Ca2+ channels on the inner NE, or other putative nuclear Ca2+ stores, generate a Ca2+ signal that is independent from the cytosol? Or do Ca2+ signals that arise in the cytosol always dominate what happens in the nucleus? As mentioned earlier, it has been shown that isolated nuclei have the machinery to release Ca2+, but the majority of intact cell studies indicate that nucleoplasmic Ca2+ signals originate outside the nucleus (Lipp et al., 1997; Luo et al., 2008). Indeed, in one notable study, InsP3 was introduced specifically to the nucleus or cytosol to examine the relative ability of this messenger to trigger Ca2+ signals in each compartment. As expected, when released in the cytosol, InsP3 evoked a cytosolic Ca2+ signal that invaded the nucleus via diffusion. However, when InsP3 was released in the nucleus, the resulting Ca2+ signal also initiated in the cytosol, and then invaded the nucleus (Shirakawa and Miyazaki, 1996). This indicates that even when InsP3 is given immediate access to the InsP3Rs on the NE, the messenger diffuses out of the nucleus and preferentially triggers cytosolic Ca2+ release. A similar conclusion was reached when an InsP3R antagonist was introduced in the cytosol of cells during stimulation with an InsP3-generating agonist. The antagonist, which was too large to enter the nucleus, blocked both cytosolic and nucleoplasmic Ca2+ transients (Allbritton et al., 1994). The obvious conclusion from such studies is that, although nuclei have the capacity to generate Ca2+ transients, their contribution is `swamped' by the larger cytosolic Ca2+ release [but see Lui et al. (Lui et al., 1998) for a contradictory observation].

Perhaps it is only under specific stimulation conditions that substantial Ca2+ responses are evoked from nuclear InsP3Rs. For example, a comparison between the Ca2+ signals that are evoked by vasopressin and hepatocyte growth factor (HGF) in hepatic carcinoma cells revealed that vasopressin triggered Ca2+ release in the cytosol, whereas HGF caused nuclear Ca2+ mobilisation (Gomes et al., 2008). In the latter case, it was suggested that the HGF receptor was internalised and translocated to the nucleus, where it triggered local InsP3 production. Vasopressin, on the other hand, stimulated InsP3 production in the cytosol. It is therefore plausible that different stimuli activate InsP3Rs in alternative cellular compartments, depending on where in the cell the InsP3 is generated.

An example of nuclear-specific Ca2+ signalling is presented in Fig. 4A, which shows the spatial profile of spontaneous Ca2+ transients that occur in a neonatal cardiac myocyte. Such Ca2+ signals are largely generated by the stochastic activation of InsP3Rs and RyRs (Luo et al., 2008). The black trace shows substantial nuclear Ca2+ elevations, but a corresponding increase in cytosolic Ca2+ on either side of the nucleus is not observed. It was not determined whether the nuclear Ca2+ signals were due to activation of Ca2+ channels on the inner NE, or to perinuclear Ca2+ release. However, the figure presents an example in which substantial Ca2+ changes can be clearly observed solely in the nucleus.

Autonomous generation of Ca2+-mobilising messengers in the nucleus

If nuclei are truly independent of cytosolic signalling, they must have the ability to generate Ca2+-mobilising messengers that then act locally on nucleoplasmic InsP3Rs, RyRs and NAADPRs. Numerous studies have demonstrated that the biochemical machinery that is required for the generation of Ca2+-mobilising messengers is present in the nucleus. For example, the ADP-ribosyl cyclase enzyme that is responsible for the production of cyclic ADP ribose (cADPR) and/or NAADP is localised in the nucleus (Adebanjo et al., 1999; Bezin et al., 2008a; Trubiani et al., 2008). In addition, it has been well established that nuclei possess phosphoinositide signalling mechanisms that are similar to, but distinct from, those that occur at the plasma membrane, and which can lead to InsP3 production (Visnjic and Banfic, 2007; Ye and Ahn, 2008). Indeed, nuclei have substantial amounts of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphoinositide-specific phospholipase C (PtdIns-PLC) (Downes et al., 2005). Interestingly, it has been reported that PtdIns(4,5)P2 and PtdIns-PLC localise in particles known as nuclear speckles (see Fig. 1). These small nuclear substructures are believed to be to be the sites of storage and/or assembly of pre-mRNA splicing factors. The exact function of PtdIns(4,5)P2 and PtdIns-PLC in nuclear speckles is currently under investigation, but they are involved in linking the production of diacylglycerol (DAG) and InsP3 (and their metabolites) to the control of RNA processing (Alcazar-Roman and Wente, 2008).

Fig. 4.

Cytosolic and nuclear Ca2+ signals can be independent. (A) An example of Ca2+ signals that occur in the nucleus of a neonatal rat cardiac myocyte independently of Ca2+ signals in the cytosol. In the inset, two adjacent cells are outlined in white. The cells showed spontaneous Ca2+-release events, which is typical of neonatal cardiomyocytes in culture. The Ca2+ signals were recorded by loading the cells with fluo4 and monitoring changes in fluorescence emission using confocal microscopy, and the responses were recorded from the positions indicated in the left-hand cell. Calcium increase is shown as the fluo4 fluorescence normalised to the baseline (F/F0). (B) Difference in the kinetics of cytosolic and nuclear Ca2+ in an adult atrial cardiac myocyte during regular electrical pacing. Cells were paced to steady state before the recording was started. The traces were obtained by confocal line-scanning of fluo4 fluorescence across a region perpendicular to the long axis of the cell. Line scans were obtained at 2-ms intervals using an Olympus FluoView 1000. The regions of the line scan from which the cytosolic and nuclear Ca2+ traces in C were obtained are indicated on the left of the line scan image (marked by `C'). (C) Spatial properties of the cytosolic and nuclear Ca2+ signals. The intensity of the white pixels is proportional to Ca2+ concentration, as shown in the right-hand panel.

Fig. 4.

Cytosolic and nuclear Ca2+ signals can be independent. (A) An example of Ca2+ signals that occur in the nucleus of a neonatal rat cardiac myocyte independently of Ca2+ signals in the cytosol. In the inset, two adjacent cells are outlined in white. The cells showed spontaneous Ca2+-release events, which is typical of neonatal cardiomyocytes in culture. The Ca2+ signals were recorded by loading the cells with fluo4 and monitoring changes in fluorescence emission using confocal microscopy, and the responses were recorded from the positions indicated in the left-hand cell. Calcium increase is shown as the fluo4 fluorescence normalised to the baseline (F/F0). (B) Difference in the kinetics of cytosolic and nuclear Ca2+ in an adult atrial cardiac myocyte during regular electrical pacing. Cells were paced to steady state before the recording was started. The traces were obtained by confocal line-scanning of fluo4 fluorescence across a region perpendicular to the long axis of the cell. Line scans were obtained at 2-ms intervals using an Olympus FluoView 1000. The regions of the line scan from which the cytosolic and nuclear Ca2+ traces in C were obtained are indicated on the left of the line scan image (marked by `C'). (C) Spatial properties of the cytosolic and nuclear Ca2+ signals. The intensity of the white pixels is proportional to Ca2+ concentration, as shown in the right-hand panel.

Mammalian cells express at least 13 different PtdIns-PLC isozymes that can be grouped into six subfamilies (β, γ, δ, ϵ, η and ζ) (Cockcroft, 2006; Suh et al., 2008), of which PtdIns-PLCβ1 is considered to be the predominant nuclear form (Faenza et al., 2008; Ye and Ahn, 2008). In some cell types, PtdIns-PLCβ1 is constitutively present in nucleus, whereas in others, growth-factor stimulation causes the enzyme to translocate from the cytosol to the nucleus. This is facilitated by C-terminal sequences in PtdIns-PLCβ1 that act either as a NLS or cause the retention of the protein in the nucleus (Kim et al., 1996). It is known that PtdIns-PLCβ isoforms are activated at the plasma membrane by G-protein-coupled receptors and are responsible for rapid InsP3 production and acute Ca2+ signals (see Box 2). In the nucleus, however, PtdIns-PLCβ1 plays an important role in signalling events that are evoked by growth-factor-receptor stimulation (Visnjic and Banfic, 2007). For example, PtdIns-PLCβ1 was found to be essential during the differentiation of skeletal muscle myoblasts (Ramazzotti et al., 2008). The expression of the enzyme increases during myogenesis, and is required for the induction of cyclin D3 expression and the formation of the retinoblastoma-cyclin D3 complex that underlies differentiation (De Santa et al., 2007). The nuclear localisation of PtdIns-PLCβ1 is essential in this context, as a cytosolically targeted form does not support myoblast differentiation (Faenza et al., 2007).

These studies highlight the importance of nuclear PtdIns-PLCs in growth factor responses. However, they mainly describe the roles of nuclear DAG, protein kinase C translocation or inositol polyphosphate formation that results from PtdIns-PLC activation (Divecha et al., 1991). Considerably less attention has been focussed on whether changes in the levels of nuclear Ca2+ participate in growth-factor-mediated responses. However, several recent reports have suggested a crucial role for Ca2+ downstream of nuclear PtdIns-PLC activation. For example, insulin receptors are found in the nucleus following insulin stimulation of cells, where they promote PtdIns(4,5)P2 turnover and nuclear Ca2+ signalling independently of cytosolic Ca2+ signalling (Rodrigues et al., 2008). The specific action of insulin on nuclear InsP3 production was confirmed by expressing the InsP3-binding portion of the InsP3R (known as an InsP3 `sponge') to buffer changes in InsP3 concentration. To affect its subcellular location, the InsP3 sponge was fused to either a NLS or a NES. The expression of the nucleus-targeted InsP3 sponge significantly attenuated responses to insulin, whereas the cytosol-targeted InsP3 sponge did not (Rodrigues et al., 2008). The opposite effect was observed if the cells were stimulated with vasopressin, which acts on the plasma membrane pool of PtdIns(4,5)P2 via cytosolic PtdIns-PLC isozymes (Gomes et al., 2008).

In addition to growth factor receptors being present in the nucleus, a number of studies have demonstrated that functional hormone receptors are also internalised and move across the NE. For example, a C-terminal NLS directs the nuclear import of the bradykinin B2 receptor (Savard et al., 2008). Specific bradykinin-binding sites are present in hepatocyte nuclear extracts, and the addition of bradykinin to isolated nuclei was found to evoke a rapid, transient nucleoplasmic Ca2+ signal. Furthermore, it has been demonstrated that endogenous and ectopically expressed mGluR5 metabotropic glutamate receptors are present on the nuclear membranes of primary neurons and of cultured cells, respectively (Kumar et al., 2008). Studies of isolated nuclei indicate that these receptors activate PtdIns-PLCs in the nucleus, leading to nucleoplasmic InsP3 generation and Ca2+ signals.

Exactly how G-protein-coupled receptors in the nucleus become activated is unclear. The lumen of the NE is topologically the same as the extracellular space, so the ligand-binding domain of nuclear hormone receptors would be exposed within the NE lumen. It has been proposed that specific transporters allow the uptake of neurotransmitters such as glutamate into the NE, where they can bind to their cognate receptors and activate signalling (Jong et al., 2005). Similarly, receptors for endothelin, angiotensin and prostaglandin, the activation of which can also stimulate Ca2+-mobilising pathways, are reportedly expressed on nuclear membranes (Bkaily et al., 2006; Coffey et al., 1997).

Although many studies have focussed on investigating the nuclear functions of PtdIns-PLCβ1, other PtdIns-PLC isoforms are also found in the nucleus, in particular PLCδ1 (Okada et al., 2005) and PtdIns-PLCγ1 (Gomes et al., 2008). In quiescent cells, PtdIns-PLCδ1 is localised at the plasma membrane and in the cytosol, but following stimulation, it translocates to the nucleus in a Ca2+-dependent manner (Okada et al., 2005), where it plays a role in regulating PtdIns(4,5)P2 levels, DNA synthesis and cell proliferation (Stallings et al., 2008). Growth-factor-induced Ca2+ signals, as well as non-specific Ca2+ transients caused by ionophores, promote nuclear PtdIns-PLCδ1 translocation, thereby acting as positive feedback for further nuclear Ca2+ signalling (Yagisawa, 2006). Although data regarding the functions of different PtdIns-PLC isozymes in the nucleus are still emerging, current evidence suggests that they all play roles in cell survival, division and differentiation.

PtdIns-PLCζ is also known to accumulate in the nucleus owing to an NLS, but in this case, the translocation of the enzyme prevents Ca2+ signalling (Ito et al., 2008). Repetitive Ca2+ oscillations occur immediately following fertilisation in mammalian oocytes, and are believed to be driven by InsP3 production that is caused by the introduction of PtdIns-PLCζ into the oocyte from the fused sperm. The Ca2+ oscillations cease around the time when the pronucleus is formed, owing to the sequestration of PtdIns-PLCζ in the nucleus (Larman et al., 2004). Unlike the other PtdIns-PLC isozymes described above, when PtdIns-PLCζ is in the nucleus, it does not evoke InsP3 production and Ca2+ signalling.

The Ca2+-mobilising pathways described above have well-characterised effects on nuclear Ca2+ signalling. However, there are numerous additional messengers and signalling pathways by which cellular Ca2+ signals can be evoked (Bootman et al., 2002), and some of these have been shown to function in the nucleus. Another example of a putative nuclear messenger is arachidonic acid: this lipid is known to increase cytosolic Ca2+ concentration. Although little is known about the effect of arachidonic acid on nucleoplasmic Ca2+, it is plausibly an additional regulator of nuclear Ca2+ concentration. Indeed, phospholipase A2, an enzyme involved in the production of aachidonic acid, translocates into the nucleus in a Ca2+-dependent manner (Schievella et al., 1995). Furthermore, arachidonic acid can be metabolised into further Ca2+ signalling moieties by the action of cyclooxygenase-2, which translocates into the nucleus in response to growth-factor-stimulation (Coffey et al., 1997). Although the bulk of studies have focussed on InsP3-mediated Ca2+ release in the nucleus, it is probable that nuclear Ca2+ signalling utilises the same diverse range of messengers and channels as the cytosol.

Recovery of nucleoplasmic Ca2+ signals

It has been noted in several studies that nuclear Ca2+ signals persist considerably longer than equivalent cytosolic Ca2+ transients (Bootman et al., 2007; Kockskamper et al., 2008a; Lipp et al., 1997). The reason for this is the lack of Ca2+ ATPases on the inner NE (Humbert et al., 1996). Because of the slow dissipation of nuclear Ca2+, it is plausible that rapid cellular stimulation could cause a progressive accumulation of Ca2+ in the nucleoplasm. Fig. 4B,C shows an example of the kinetic delay in both the rise and dissipation of nuclear Ca2+. The recordings were obtained by imaging fluo4 fluorescence from a single atrial cardiac myocyte that was activated by regular electrical depolarisation. In addition to the kinetic delay in recovery of the nuclear Ca2+ transient, it is evident that the nuclear fluo4 fluorescence was consistently higher than that in the cytosol, even when both signals had recovered. This is because fluo4 is brighter in the nucleus than in the cytosol (as described above), not because there was a persistent gradient of Ca2+ between the cytosol and nucleus. The calibrated diastolic Ca2+ levels are actually very similar in the cytosol and nucleus. The differences in the kinetics of the nuclear and cytosolic Ca2+ signals are illustrated in the line-scan image shown in Fig. 4C; it is evident that the nucleus takes longer to fill with Ca2+, and considerably longer for the Ca2+ signal to dissipate.

A major mechanism for the dissipation of nuclear Ca2+ signals is via simple diffusion through NPCs. To leave the nucleus, a Ca2+ ion has to pass through the nucleoplasm and then encounter and traverse a NPC. Once outside the nucleus, Ca2+ can be quickly sequestered by Ca2+ ATPases on the outer NE, ER and plasma membrane, or by other buffers such as mitochondria. The NE can be regarded as a diffusion barrier for both Ca2+ entry and exit. Although Ca2+ ATPases are not expressed on the inner NE, there is growing evidence that a splice variant of type 1 sodium-calcium exchanger (NCX) is expressed on the inner NE (Ledeen and Wu, 2007). It is suggested that the nuclear NCX uniquely associates with a ganglioside known as GM1, which potentiates Ca2+ transport and prevents Ca2+ overloading within the nucleus (Ledeen and Wu, 2008). NCX is a mechanism of Ca2+ transport that is usually associated with electrically excitable cells in which large and rapid fluxes of Ca2+ occur. However, many non-excitable cells also express NCX and, in some cells, expression of the transporters appears to be restricted to the nucleus (Xie et al., 2004). The configuration of the NCX on the inner NE is the same as on the plasma membrane, but the interaction with GM1 is atypical. In the presence of a Na+ gradient, nuclei sequester Ca2+, verifying the functionality of NCX (Xie et al., 2002). The Na+ gradient might be driven by a Na+/K+ ATPase or Na+H+ exchanger, all of which are also present on the NE (Bkaily et al., 2006; Garner, 2002). Therefore, in addition to passive diffusion out of the nucleus, nucleoplasmic Ca2+ signals can be reversed by active uptake processes.

Replenishment of Ca2+ in the NE lumen is probably mediated by ER-localised Ca2+ ATPases. Although these enzymes sequester cytosolic Ca2+ into the ER, the fact that the lumen of the NE and ER are connected means that Ca2+ can diffuse from one membrane system to the other (Wu and Bers, 2006). In addition, it has been reported that Ca2+ accumulates in the NE via a curious mechanism that is driven solely by InsP4 (Hsu et al., 1998; Malviya and Klein, 2006). It has been reported that specific InsP4 receptors are present on the outer NE (Malviya, 1994), but exactly how these binding sites activate Ca2+ uptake is unknown. A major problem with this proposed uptake pathway is that the ER and NE possess a substantially higher Ca2+ concentration than the cytosol, and, unless the Ca2+ content of these stores was completely emptied, the flux of Ca2+ would always be towards the cytosol. Complete depletion of ER Ca2+ is a pathological state that culminates in cell death.

In the process of signal transduction, the reversal of a signalling event is as important as its induction. Exactly how nucleoplasmic Ca2+ signals decline back to pre-stimulation levels is not entirely clear, as the majority of studies have focussed solely on the generation of nucleoplasmic Ca2+ transients. Currently, passive diffusion through NPCs and uptake into the NE by NCX appear to be the main mechanisms by which nuclear Ca2+ transients are limited and reversed.

Conclusions

Over the past decade there has been a tremendous increase in the knowledge of Ca2+ signalling systems in the nucleus, and progress has been made in unravelling the impact of changes in nuclear Ca2+ on cellular activity. Recent studies have clearly highlighted the idea that gene transcription and cell-cycle progression can be specifically influenced by nuclear Ca2+ signals. It is probable that many more cellular processes will be shown to be dependent on nucleoplasmic Ca2+ changes. Despite this understanding of the functions of nuclear Ca2+ signals, there is still some uncertainty concerning how nuclear Ca2+ signals are generated and whether Ca2+ signalling in the nucleus is really autonomous. This is a surprisingly contentious issue that has been debated for many years. It has been shown that NPCs do not prevent Ca2+ fluxes from crossing the NE, which would imply that Ca2+ signalling in the nucleus must follow cytosolic Ca2+ changes (providing that cytosolic Ca2+ changes persist for sufficient time). Yet, several studies have shown that Ca2+ signals initiate within, and are restricted to, the nucleus. Exactly how Ca2+ signals within the nucleus or cytosol are prevented from equilibrating with the other compartment remains unclear.

In future studies, it will be important to work towards defining unified views on several topics, including nuclear Ca2+-release mechanisms and the regulation of NPC structure and function by Ca2+. As some of the controversy in nuclear Ca2+ signalling might be owing to the use of different cell types in different laboratories, it will also be necessary to consider cell-type-specific versus generic Ca2+ signalling paradigms. Finally, another goal of future research will be to link putative nucleoplasmic Ca2+ signals with cellular processes.

The authors would like to thank Simon Walker and Hattie O'Nions for their help in the preparation of Fig. 2 and Daniel Higazi for preparation of Fig. 3B. This work was supported by the BBSRC and the British Heart Foundation (grants PG/06/034/20637 and PG/07/040). H.L.R. is a Royal Society University Research Fellow. I.S. is a BBSRC-funded PhD student.

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