New aspects of nuclear calcium signalling

Nuclear calcium signalling has been a controversial battlefield for many years and the question of how permeable the nuclear pore complexes (NPCs) are to Ca2+ has been the subject of a particularly hot dispute. Recent data from isolated nuclei suggest that the NPCs are open even after depletion of the Ca2+ store in the nuclear envelope. Other research has suggested that a new Ca2+-releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), can liberate Ca2+ only from acidic organelles, probably lysosomes, rather than from the traditional Ca2+ store in the endoplasmic reticulum (ER). Recent work indicates that NAADP can release Ca2+ from the nuclear envelope (NE), which has a thapsigargin-sensitive, ER-type Ca2+ store. NAADP acts in a manner similar to inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] or cyclic ADP-ribose (cADPR): all three messengers are equally able to reduce the Ca2+ concentration inside the NE and this is associated with a transient rise in the nucleoplasmic Ca2+ concentration. The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3.

. Strong stimulation can induce large Ca 2+ waves that can overcome this mitochondrial barrier (Fig. 1C) and therefore quickly penetrate the nucleus. In this case, the difference between the Ca 2+ concentration in the cytosol and the nucleus is marginal (Al-Mohanna et al., 1994;Lipp and Niggli, 1994).
Accurate measurements of the nuclear Ca 2+ concentration are extremely difficult and require separate calibration of the nuclear dye owing to many possible artefacts, such as increased brightness of most of the fluorescent indicators in the nucleoplasm (Al-Mohanna et al., 1994;Bootman et al., 2000;Perez-Terzic et al., 1997). In addition to exhibiting differences in dissociation constant and dynamic range, the indicators can also be bound inside the nucleoplasm and sequestered in Ca 2+ stores (Al-Mohanna et al., 1994;Burnier et al., 1994;Ikeda et al., 1996;Ronde and Nichols, 1997). These problems effectively invalidate the majority of the observations of nuclear-cytoplasmic Ca 2+ gradients. There is thus probably no persistent nuclear-cytoplasmic gradient (Badminton et al., 1996;Badminton et al., 1998;Brown et al., 1997;Bootman et al., 2000;Bootman et al., 2002;Tovey et al., 1998). Indeed, this is convincingly shown by luminescence measurements of targeted aequorin (Brini et al., 1993).

The Ca 2+ permeability of NPCs
If sustained nuclear-cytoplasmic Ca 2+ gradients do not exist, then NPCs must be easily permeable to small ions, including Journal of Cell Science 117 (15) Fig. 1. Transient nuclear-cytoplasmic Ca 2+ gradients in pancreatic acinar cells [modified figure reproduced with permission from Springer-Verlag (Gerasimenko et al., 1996b)]. (A) A typical localized Ca 2+ spike induced by the short application of acetylcholine (ACh) in the secretory granule area does not enter the nucleus or basal area of a doublet of pancreatic acinar cells: the first image is the transmitted light picture, the second image is the same cluster, stained with the nuclear dye Hoechst 33342. Yellow boxes, secretory granule areas; blue boxes, nuclei; purple boxes, basal areas. Colour images show confocal recording of propagation of Ca 2+ spike in time (two images per second) from left to right. Cells were stained with Fura Red in AM form. Bar, 5 µm. (B) Traces of Ca 2+ concentration changes: upper traces are for secretory granule area (yellow boxes in A), middle traces are from the nuclei (blue boxes in A), lower traces are from the basal areas (purple boxes in A). (C) A long application of ACh induces global responses in the same cluster. The Ca 2+ concentration in the nucleus only temporarily differs from that in the cytoplasm. Bar, 5 µm. (D) The temporary Ca 2+ gradient along the line between nucleus and cytoplasm during a local Ca 2+ spike can be quite high: ~400 nM/µm. (I) Transmitted light picture of the cell. Dark areas correspond to secretory granules. [Ca 2+ ] was measured along the diagonal line. Bar, 5 µm. (II) Position of the nucleus was verified by staining with Hoechst 33342. (III) The graph corresponds to changes of [Ca 2+ ] along the line shown in I; 1 at rest, 2 at the peak of the local Ca 2+ response. N indicates the position of the nucleus. Ca 2+ . Unfortunately, there are contradictory data regarding the Ca 2+ permeability of NPCs. Electrophysiological recordings in isolated nuclei (Assandri et al., 1997) (reviewed by Santella, 1996) have shown that the ionic conductance of NPCs can change dramatically and that only a small proportion of NPCs are open under patch-clamp conditions. Danker and collaborators (Danker et al., 1999;Danker et al., 2001) have shown that most NPCs are in an open state and are freely permeable to ions. Atomic force microscopy experiments suggest that NPCs change their conformation when the central plug blocks the pore (Stoffler et al., 1999), but there is no proof that the plugs actually prevent Ca 2+ diffusion through NPCs. Even if they do, the available evidence indicates that there are substantial numbers of NPCs without plugs at any time. We have shown (Gerasimenko et al., 1995) that Ca 2+ concentration changes outside the isolated nucleus are quickly reproduced by similar Ca 2+ changes in the nucleoplasm, both in isolated liver nuclei (Fig. 2) and in isolated pancreatic nuclei (Fig. 3), (Gerasimenko et al., 2003). Generally speaking, NPCs must thus be open to passive diffusion of small ions in these systems. There might of course be special conditions under which NPCs are closed, and we cannot conclude that all NPCs are open all the time for passive diffusion. But, even if half of the NPCs were closed, the NE would still be very permeable to small ions and molecules. This could resolve the apparent discrepancy between single NPC observations (reviewed by Santella, 1996) and nuclear electric conductance measurements of large surfaces of nuclei using the nuclear hourglass technique, which indicated that the NE conductance is always high (Danker et al., 1999).
The NPC is a supramolecular assembly that has a molecular weight of ~125,000 kDa (Greber and Gerace, 1995) and a large aqueous channel with a diameter of ~9 nm. Normally, molecules <40 kDa pass through such a channel without the need for a nuclear localization signal. Some studies suggest that free diffusion of such molecules could be inhibited by the depletion of the nuclear Ca 2+ store (Greber and Gerace, 1995;Lee et al., 1998;Lyman and Gerace, 2001;Perez-Terzic et al., 1999). The mechanism might involve binding of intra-ER Ca 2+ to the NPC protein gp210 (Greber and Gerace, 1995). However, we and others have not observed this (Strubing and Clapham, 1999;Stehno-Bittel et al., 1995;Perez-Terzic et al., 1996;Wei et al., 2003;Gerasimenko et al., 2003). Very convincing data in favour of free diffusion through NPCs have been obtained recently using fluorescence recovery after photobleaching (FRAP) of 27 kDa enhanced green fluorescence protein (EGFP) in intact cells (Wei et al., 2003). These recent studies employed a transfection technique that does not affect nuclear transport; by contrast, microinjection, which was used previously, can disrupt the cytoskeleton (Swaminathan et al., 1997).
Our own results indicate that, after depletion of the NE Ca 2+ stores with Ins(1,4,5)P3 or NAADP, subsequent external application of a high Ca 2+ concentration followed by the Ca 2+ chelator EGTA induces a large rise and thereafter a fast decrease in the nucleoplasmic Ca 2+ concentration (Gerasimenko et al., 2003). These measurements demonstrate rapid movement of Ca 2+ across the NE, most likely through the NPCs. On the basis of all these data, we favour the argument that the NPCs are permeable to Ca 2+ even after depletion of the NE Ca 2+ stores.
Interesting data in favour of such a system (Clubb et al., 1998;Fricker et al., 1997) show that the NE has invaginations into the nucleoplasm in many cell types and could explain Ca 2+ release sites inside the nucleoplasm. Recent data by Echevarria et al. confirm these suggestions and show that local Ca 2+ release from the nucleoplasmic reticulum occurs through Ins(1,4,5)P3Rs and induces translocation of protein kinase C (PKC) to the NE membrane (Echevarria et al., 2003). These new data argue for an independent intranuclear Ca 2+ signalling system.
Unfortunately, because accurate measurement of the Ca 2+ concentration in the nucleoplasm of intact cells is difficult (as discussed above), there are no reliable Ca 2+ measurements that unequivocally show independent activation of an intranuclear Ca 2+ signalling system with a physiological agonist.

RyRs in nuclear membranes
The intracellular Ca 2+ -releasing messenger cADPR (Lee et al., 1989) has been shown to be an endogenous activator of RyR Ca 2+ channels (Meszaros et al., 1993) and plays a role similar to Ins(1,4,5)P3 in Ca 2+ signalling (Ehrlich et al., 1994;Fitzsimons et al., 2000;Solovyova et al., 2002;Wakui et al., 1990). The demonstration that the NE releases Ca 2+ in response to cADPR and ryanodine indicates that RyRs are present on the inner nuclear membrane (Gerasimenko et al., 1995). Indeed, the presence of RyRs has been confirmed both by RyRspecific antibodies (Adebanjo et al., 1999;Adebanjo et al., 2000;Santella and Kyozuka, 1997) and by specific staining with fluorescent BODIPY FL ryanodine (Fig. 3D) (Gerasimenko et al., 2003). Release of Ca 2+ through cADPR induces a reduction of the free Ca 2+ concentration in the NE Ca 2+ store, similar to that evoked by Ins(1,4,5)P3. These data indicate that the cADPR-induced Ca 2+ release in the nucleus can also be directed into the nucleoplasm (similar to what has been shown for Ins(1,4,5)P3-induced Ca 2+ release). However, we cannot exclude the possibility that Ca 2+ released outside the outer membrane of the NE can diffuse into the nucleoplasm through open NPCs (Gerasimenko et al., 1995;Lipp et al., 1997;Gerasimenko et al., 2003).

Conclusions
Given the evidence for intranuclear Ins(1,4,5)P3 (Cocco et al., 1994;Divecha et al., 1991) and cADPR production systems (Adebanjo et al., 1999), the existence of a completely independent intranuclear Ca 2+ signalling system is proposed, which would not require any cytoplasmic release although subsequent cytoplasmic Ca 2+ uptake would be needed (Bootman et al., 2000). In spite of the evidence for the existence of such a complete Ca 2+ release system in the nucleus, there is little evidence (Lui et al., 1998;Santella and Kyozuka, 1997;Santella et al., 1998) of its independent involvement in nuclear Ca 2+ signal generation. In fact, there are more reports indicating that Ca 2+ released primarily into the cytoplasm (Ca 2+ puffs) can diffuse into the nucleoplasm (Bootman et al., 1997;Bootman et al., 2000;Hennager et al., 1995;Lipp et al., 1997;Shirakawa and Miyazaki, 1996). The existence of a complete, independent, intranuclear Ca 2+ signalling system is potentially very interesting, but its physiological significance is currently questionable and requires convincing evidence of activation by physiological agonists. Future work in this area will undoubtedly bring more clarity and understanding of mechanisms involved. In the intact pancreatic acinar cells, the Ca 2+ store in the NE is part of one lumenally continuous ER store ; however, it is highly specialized in different parts of the cell (Gerasimenko et al., 2002). The data on isolated nuclei (Gerasimenko et al., 2003), as compared with the studies on local Ca 2+ spiking in the secretory pole in intact cells (Cancela et al., 2000;Cancela et al., 2002;Park et al., 2001;Petersen et al., 1991;, reveal that local control of Ca 2+ release can operate differently in different parts of the cell. All three messengers can induce Ca 2+ release at both sites. The local Ca 2+ spiking in the secretory region of pancreatic acinar cells [as well as the global Ca 2+ -induced Ca 2+ waves (Ashby and Tepikin, 2002;Ashby et al., 2003;Thorn et al., 1993;Thorn et al., 1994)] are highly dependent on cooperative interaction of Ins(1,4,5)P3Rs and RyRs (Ashby et al., 2002;Boittin et al., 1998;Cancela et al., 2000;Kidd et al., 1999;Koizumi et al., 1999;Lipp et al., 2000). By contrast, there is little cooperativity of Ins(1,4,5)P3Rs and RyRs in the nucleus; in fact, the receptors can function independently, inducing similar release of Ca 2+ into the nucleoplasm (Gerasimenko et al., 2003).
The difference between the NAADP effects in the nucleus and the secretory granule area are even more striking: whereas there is a mutually potentiating interaction of Ins(1,4,5)P3, cADPR and NAADP in the secretory granule area (Cancela et al., 2002), there is no such potentiation in the nucleus  (Gerasimenko et al., 2003)]. Ca 2+ released from the NE enters the nucleoplasm either directly through Ins(1,4,5)P3Rs or RyRs in the inner nuclear membrane (INM) or through the NPCs when released through Ins(1,4,5)P3Rs or RyRs in the outer nuclear membrane (ONM). Three messengers can be produced locally inside the NE: Ins(1,4,5)P3 generated by phospholipase C (PLC) or cADPR and NAADP generated by the CD38/ADP ribosyl cyclase (ARC). cADPR and NAADP bind different binding sites or receptors (marked by ?) and activate RyR Ca 2+ channels. All three Ca 2+ messengers can also be produced in the cytosol and then enter the nucleoplasm through the NPCs. Ca 2+ is pumped into the NE and ER by the sarco-endoplasmic reticulum Ca 2+ -activated ATPase (SERCA) on the ONM. IP3R, inositol (1,4,5)trisphosphate receptor. (Gerasimenko et al., 2003). These differences could be explained in two different ways. Three separate channels (receptors) could interact. Such a model would depend on the presence of so-far-uncharacterized NAADPR Ca 2+ channels. A much simpler hypothesis (Fig. 4) is that there are only two types of Ca 2+ release channels -Ins(1,4,5)P3Rs and RyRsin a single store. Ca 2+ can enter the nucleoplasm through Ins(1,4,5)P3Rs or RyRs in the inner nuclear membrane, although we cannot exclude Ca 2+ release from the Ins(1,4,5)P3Rs or RyRs in the outer nuclear membrane and subsequent entry through NPCs. All three Ca 2+ -releasing messengers, Ins(1,4,5)P3, cADPR and NAADP, can be produced inside the NE, although they can also enter the nucleoplasm through the NPCs. Ca 2+ is pumped into the NE by SERCA on the outer nuclear membrane. Potentiating or non-potentiating interactions of Ins(1,4,5)P 3Rs and RyRs (see discussion above) in this simpler model would depend on the degree of closeness of the two types of Ca 2+ release channel and of the cADPR and NAADP receptors. This exciting area of research will definitely attract more attention in the future.