InteractiveFly: GeneBrief

Ryanodine receptor 44F: Biological Overview | References

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. In this study, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity (Shakiryanova, 2007).

It has long been speculated that vesicle motion at synapses could be controlled to influence neurotransmission, but activity-dependent increases in vesicle mobility (i.e., mobilization) in nerve terminals were detected directly only recently. This was first accomplished with neuropeptide-containing dense-core vesicles (DCVs) in Drosophila neuromuscular junction (NMJ) by using two methods, fluorescence recovery after photobleaching (FRAP) and image correlation analysis of time-lapse movies (Shakiryanova, 2005). Subsequently, stimulus-induced vesicle motion was detected with mammalian neuroendocrine DCVs and frog NMJ small synaptic vesicles (SSVs) (Allersma, 2006; Gaffield, 2006). The increase in vesicle mobility in these diverse preparations shares common features. First, mobilized vesicles move randomly, in accordance with single particle-tracking studies that showed that releasable and reserve secretory vesicles move by diffusion, albeit at different rates Second, stimulation-induced mobility is unaffected by depolymerizing F-actin. Along with other experiments, this finding suggests that the long-standing question of whether F-actin is a barrier for vesicle immobilization or a track for motor-mediated translocation is not central to understanding stimulation-induced vesicle motion. Finally, the stimuli in each case are associated with Ca²⁺ influx, which was explicitly demonstrated to be required for enhancing vesicle mobility in two of the studies (Shakiryanova, 2005; Allersma, 2006). Although SSV mobilization is not seen in all neurons, these shared properties indicate that a conserved mechanism may underlie mobilization of both SSVs and DCVs at sites that are capable of this regulation (Shakiryanova, 2007).

Mobilization of neuropeptide-containing DCVs likely involves sustained signal transduction, because seconds of activity in Drosophila NMJ type Ib boutons induce many minutes of increased DCV mobility even after release is inhibited (Shakiryanova, 2005). However, the specific pathway activated by Ca²⁺ influx has not been identified. Thus, the role of this signaling pathway in synaptic function and plasticity is not known. Indeed, although pharmacological manipulations have been shown to produce DCV mobility increases that can account for increased release in vitro, inhibiting physiological vesicle mobilization has never been shown to block potentiation of release in vivo (Shakiryanova, 2007).

In this study, in vivo imaging of a green fluorescent protein (GFP)-labeled neuropeptide, which reports native neuropeptide secretion, shows that ryanodine receptor (RyR)-mediated Ca²⁺ release from presynaptic endoplasmic reticulum (ER) is necessary and sufficient for triggering DCV mobilization in the larval NMJ. Calmodulin kinase II (CaMKII) then acts as an effector of released ER Ca²⁺. Finally, RyR-mediated Ca²⁺ release and CaMKII are shown to be required for post-tetanic potentiation (PTP) of neuropeptide secretion (Shakiryanova, 2007).

It is thought that vesicle mobility is increased to facilitate secretion. In favor of this model, in vitro studies showed that DCV mobility limits neuropeptide release, and pharmacologically increasing DCV mobility enhances neuropeptide release. However, native signaling mechanisms that induce such DCV mobilization in a nerve terminal in vivo and the impact of this signaling on release have not been determined. Indeed, the importance of vesicle mobility has been obscured because the term mobilization has been co-opted to include almost any process that increases release. The recent direct detection of regulated vesicle mobility in motor neuron terminals has opened the door to probing the signaling involved in bona fide vesicle mobilization and its connection to synaptic plasticity. Specifically, it was reasoned that identifying the signaling that controls vesicle motion would serve two purposes: (1) to delineate regulatory mechanisms in the nerve terminal and (2) to produce the opportunity to test whether inhibiting mobilization affects release for the first time. In this study, synaptic DCV motion and neuropeptide release were imaged in vivo to show that both activity-dependent increases in DCV mobility and PTP require presynaptic ER Ca²⁺ release via RyRs leading to CaMKII activation. The use of a single signaling pathway for controlling DCV motion and release provides the first empirical indication of a mechanistic connection between mobilization and synaptic plasticity. Baseline DCV mobility and secretion evoked by low-frequency activity are not markedly affected by inhibiting RyRs or CaMKII. Therefore, this signaling pathway has a minor role until it is recruited by tetanic activity. The activity requirement for recruitment of RyR-CaMKII-activated mobilization and PTP is well matched to the native activity found at the larval NMJ [i.e., rhythmic bursting (Klose, 2005)] and the activity patterns known to be optimal for neuropeptide release. Given past in vitro studies of DCV mobility, the in vivo results presented in this study are consistent with the conclusion that physiological activity triggers RyR-activated CaMKII to increase DCV mobility, which in turn contributes to PTP of neuropeptide secretion (Shakiryanova, 2007).

The necessity for RyR-mediated Ca²⁺ release for mobilization and PTP is surprising because bulk Ca²⁺ is effectively elevated in Drosophila motor neuron terminals by Ca²⁺ influx through plasma membrane voltage-gated channels even when ER Ca²⁺ is depleted. Indeed, plasma membrane voltage-gated Ca²⁺ channels are sufficient for triggering some neuropeptide release at low levels of activity but apparently cannot induce sustained mobilization without the participation of RyRs. Thus, as far as mobilization is concerned, CaMKII is apparently more sensitive to Ca²⁺ released from intracellular stores than extracellular Ca²⁺ influx. Perhaps this specificity arises because of positioning of CaMKII closer to RyRs than voltage-gated channels in the presynaptic plasma membrane. Alternatively, a small amount of Ca²⁺ influx through a voltage-gated channel may trigger a much larger Ca²⁺ spark by opening multiple RyRs. This could be amplified by production of RyR-mediated Ca²⁺ waves. Thus, local Ca²⁺ elevation at the surface, which is sufficient for triggering exocytosis of docked vesicles, could give rise to a propagating Ca²⁺-induced Ca²⁺ release (CICR) from the ER to activate CaMKII in the cytoplasm where most DCVs reside. This positive feedback might also set the threshold activity required for activating CaMKII-induced mobilization. Hence, RyRs could control the activity dependence and spatial propagation of vesicle mobilization. Regardless of the underlying mechanism, the requirement for ER Ca²⁺ stores delineated in this study implies that the Ca²⁺ sources for triggering exocytosis (i.e., plasma membrane voltage-gated Ca²⁺ channels) and sustained mobilization (i.e., intracellular RyRs) are distinct (Shakiryanova, 2007).

These studies establish that RyR-CaMKII-dependent DCV mobilization is initiated in the presynaptic terminal. First, mobilization is blocked by disabling neuronal Sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) function, while leaving muscle SERCA intact. Second, the activity-induced increase in presynaptic DCV motion does not depend on standard synaptic transmission between nerve and muscle: mobilization persists after inhibiting exocytosis (Shakiryanova, 2005) or postsynaptic glutamate receptors with desensitizing doses of glutamate (Shakiryanova, 2005) or an antagonist. RyR and CaMKII are present in muscle as well as neurons (Hasan, 1992; Griffith, 1994; Haghighi, 2003; Lu, 2003), but it is not known whether muscle CaMKII, which participates in retrograde signaling (Haghighi, 2003), is activated by Ca²⁺ flux through RyRs. Further study may show that the intimate functional relationship between the RyR and CaMKII exists on both sides of the synapse (Shakiryanova, 2007).

It was also found that a short-lived elevation in Ca²⁺ induces long-lasting mobilization and PTP. Hence, residual Ca²⁺, which is important for synaptic plasticity, does not sustain these responses. The downstream involvement of CaMKII suggests that phosphorylation leading to a sustained increase in vesicle mobility and secretion persists after residual Ca²⁺ has dissipated. This could reflect either slow substrate dephosphorylation because of limited phosphatase activity or prolonged activation of CaMKII, possibly by autophosphorylation on T287 of the Drosophila CaMKII. The presence of some autophosphorylated CaMKII at the resting NMJ might also explain the small reduction in DCV mobility induced by CaMKII inhibitors applied at rest. Unfortunately, because it is very difficult to specifically measure presynaptic autophosphorylation of CaMKII with phosphospecific antibodies in the context of far greater amounts of CaMKII in the postsynaptic side of the Drosophila NMJ, the prevalence of presynaptic autophosphorylated CaMKII is not known. Likewise, the participation of other Ca²⁺-signaling proteins cannot be excluded. Nevertheless, it is clear that CaMKII-induced phosphorylation is critical for sustained DCV mobilization and PTP after Ca²⁺ has returned to baseline levels (Shakiryanova, 2007).

The results also hint that the RyR-CaMKII pathway may not be the sole signaling mechanism for regulating DCV mobility in synaptic boutons. First, inhibiting Ca²⁺ release revealed a sustained small decrease in DCV mobility, which was not detected in the absence of Ca²⁺ influx (Shakiryanova, 2005). Previously, it was showed that DCV mobilization in type Ib boutons begins to reverse after ~5 min (Shakiryanova, 2005). Given that CaMKII is required for sustained mobilization, it is hypothesized that dephosphorylation resets DCV dynamics. Future experiments could explore whether an activity-sensitive phosphatase such as calcineurin reduces DCV mobility. Such a mechanism could be responsible for reducing steady-state phosphorylation in the absence of Ca²⁺ release and reversing the effects of additional CaMKII-mediated phosphorylation when RyRs are active. It was also observed that there was a small transient increase in DCV motion in many of the experiments in which RyRs and CaMKII were inhibited. The simplest explanation for this result is that this is a consequence of incomplete inhibition of mobilization combined with the above described demobilization mechanism. However, it was not excluded that this represents the direct, but inefficient, activation of CaMKII by Ca²⁺ influx. It would be interesting to test whether DCV mobility is transient and limited to near the cell surface when RyRs are inhibited, but this is beyond the resolution of current methods that can be used with the intact living NMJ (Shakiryanova, 2007).

The signaling responsible for increasing DCV mobility and PTP of neuropeptide secretion in Drosophila motor neuron terminals may be widely relevant. For example, this pathway could be responsible for mobilization in neuroendocrine cells (Allersma, 2006). Likewise, CaMKII-mediated DCV mobilization may contribute to the increase in activity-dependent neuropeptide release by dendritic spines (Lochner, 2006) induced by release of ER Ca²⁺ (Ludwig, 2002). The signaling described in this study could also be important in synapses capable of SSV mobilization (e.g., the NMJ), which shares many features with DCV mobilization. In fact, presynaptic RyR and CaMKII contribute to synaptic potentiation in Aplysia synapses and the neurotrophin-induced increase in spontaneous neurotransmitter release in developing NMJs. SSV mobility studies should be able to test whether mobilization occurs and is necessary in these cases of synaptic plasticity (Shakiryanova, 2007).

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In C. elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. This study reports that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca²⁺ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. This study places Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca²⁺ sensor dSTIM (Stromal interaction molecule), and the Ca²⁺-release-activated Ca²⁺ channel dOrai (olf186-F) in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, these results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca²⁺ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway (Cuttell, 2008).

Phagocytosis is a crucial process during development and in innate immunity of all multicellular organisms. It allows for rapid engulfment of dying cells and pathogens by specialized phagocytes, such as macrophages and neutrophils in mammals. Phagocytosis is also an essential function of dendritic cells that present processed antigens to lymphocytes, thus linking innate and adaptive immunity. In C. elegans, the death genes ced-2, 5, 10, and 12 activate the small GTPase CED-10 that triggers actin cytoskeleton rearrangement during phagocytosis; the parallel CED1/6/7 pathway also converges on CED-10, but its precise role in phagocytosis remains elusive (Mangahas, 2005; Cuttell, 2008 and references therein).

During Drosophila embryogenesis, two macrophage receptors, Croquemort (CRQ), a CD36 homolog, and Draper (DRPR), a CED-1 homolog (Manaka, 2004), play a role in apoptotic cell clearance, much like their counterparts in mammals or C. elegans. The Drosophila homolog of CED-6, Dmel/Ced-6 (hereafter called drCed-6), and DRPR are also required in glial cells for axon pruning and the engulfment of degenerating neurons (Awasaki, 2006; MacDonald, 2006; Cuttell, 2008 and references therein).

In a deficiency screen, a mutant was characterized in which embryonic macrophages poorly engulfed apoptotic cells. In an RNAi screen using S2 cells, undertaker/retinophilin (uta) was identified as being responsible for this phenotype. uta encodes a membrane occupational and recognition nexus (MORN) repeat-containing protein with homology to mammalian Junctophilins (JPs). JPs form junctional complexes between the plasma membrane (PM) and the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca²⁺ storage compartment (Takeshima, 2000). These complexes allow for crosstalk between Ca²⁺ channels at the PM and the ER/SR Ca²⁺ channels, or Ryanodine receptors (RyRs), thus regulating Ca²⁺ homeostasis and functions of excitable cells (Takeshima, 2000). Although a role for Ca²⁺ in phagocytosis of various particles by mammalian phagocytes has been previously described, the molecular mechanisms underlying Ca²⁺ fluxes associated with these events are not known (Cuttell, 2008).

This study reports that, as for UTA, the Drosophila Ryanodine receptor, Rya-r44F (Xu, 2000), plays a role in phagocytosis of apoptotic cells in vivo. A requirement in phagocytosis was found for store-operated Ca²⁺ entry (SOCE) via dSTIM, a Ca²⁺ sensor of the ER/SR lumen (Roos, 2005), and CRACM1/dOrai, a Ca²⁺-release-activated Ca²⁺ channel (CRAC) (Feske, 2006; Vig, 2006). uta and rya-r44F genetically interact with drced-6 and drpr, and uta, drced6, and drpr are required for SOCE in S2 cells. Thus, these genes act in the same pathway that plays a general role in phagocytosis, as uta, dstim, dorai, drced-6, and drpr are also required for efficient phagocytosis of bacteria. These results provide a link between SOCE and phagocytosis, imply that UTA plays a similar role in macrophages to that of JPs in excitable cells, and shed light on a role for the CED1/6/7 pathway in Ca²⁺ homeostasis during phagocytosis (Cuttell, 2008).

Binding of various particles induces a rise in [Ca²⁺]i in mammalian phagocytes. In dendritic cells, [Ca²⁺]i increases upon apoptotic cell binding via integrin, and inhibition studies have suggested that both Ca²⁺ release from the ER/SR storage pool and extracellular Ca²⁺ entry into the cytosol are required for this process. Neutrophils also rely on such changes to promote particle engulfment. Yet, the molecular mechanisms underlying this rise in [Ca²⁺]i and what role it plays in phagocytes are poorly understood (Cuttell, 2008).

This study found that uta, a Drosophila gene encoding a JP-related protein, is required for phagocytosis of apoptotic cells. Genetic evidence is provided of a role for a Ryanodine receptor, Rya-r44F, and uta and rya-44F were genetically linked. SOCE via dstim and dorai promotes efficient apoptotic cell clearance. uta and rya-44F were genetically linked to drpr and drced-6, and a role was found for uta, drpr, and drced-6 in SOCE, thus demonstrating a functional link between the DRPR/drCed-6 pathway and SOCE during phagocytosis (Cuttell, 2008).

A model is proposed whereby apoptotic cell binding via DRPR (and possibly other receptors, such as CRQ) leads to ER Ca²⁺ release via Rya-r44F. DRPR, which bears an immunoreceptor tyrosine-based activation motif (ITAM) that is phosphorylated via Src and Syk family kinase-mediated signaling, appears to behave like an immunoreceptor (Ziegenfuss, 2008). In B and T lymphocytes, engagement of Fc immunoreceptors (the signaling of which also relies on phosphorylation on ITAMs) leads to a rise in [Ca²⁺]i. Thus, DRPR might play a similar role to that of Fc receptors in the signaling, leading to a rise in [Ca²⁺]i in macrophages (Cuttell, 2008).

UTA is localized in the ER and at the PM. Thus, it is proposed that, like JPs, UTA forms junctional complexes that link the PM events to the ER and trigger Ca²⁺ release from ER stores. The current studies, however, did not address whether the formation of UTA junctional complexes is required to trigger ER Ca²⁺ release via Rya-r44F, nor what triggers ER Ca²⁺ release. The resting membrane potential of mammalian phagocytes is depolarized upon contact with apoptotic cells. As in mammalian muscle cells, such changes in fly phagocytes might initiate ER Ca²⁺ release (Cuttell, 2008).

In S2 cells, Ca²⁺ imaging results with drpr and drced-6 RNAi and that of others with drced-6 RNAi (Vig, 2006) suggest that drpr and drced-6 are required for dOrai-mediated Ca²⁺ entry upon TG treatment (which bypasses the need for particle binding to the receptor). It is proposed that ER Ca²⁺ release feeds back onto DRPR and drCed-6 to activate their downstream signaling cascade. Although further studies will be required to test the validity of this proposal, several reports already support it: DRPR-mediated phagocytosis depends on Src and Syk family kinase signaling (Ziegenfuss, 2008), and the activity of such kinases can be Ca²⁺ dependent in mammalian cells (Cuttell, 2008).

It is then proposed that signaling downstream of DRPR and drCed-6 promotes and/or maintains the formation of UTA junctional complexes, thereby linking ER Ca²⁺ release to SOCE. dSTIM is indeed likely to act as an ER Ca²⁺ sensor that oligomerizes (Luik, 2008) and redistributes to ER-PM junctions upon ER Ca²⁺ depletion, as for its mammalian counterparts (Feske, 2007). It is proposed that UTA junctional complexes are needed to maintain a close proximity between the ER Ca²⁺ stores and the PM and to juxtapose dSTIM oligomers and dOrai, thereby promoting conformational changes and opening of dOrai. DRPR- and drCed-6-dependent signaling and/or UTA may also be required for dSTIM oligomerization. The resulting increase in [Ca²⁺]i then promotes engulfment of the particle (Cuttell, 2008).

Ca²⁺ may promote phagocytosis via several ways. It can enhance scavenger receptor (SR) activity: adhesion of mouse macrophages to a fibronectin-coated surface via integrin binding results in an increase in the number of SRs at their PM, which enhances their binding activity. This enrichment in SRs is dependent on extracellular Ca²⁺ influx, arguing in favor of a role for Ca²⁺ in SR trafficking and/or recycling. Several SRs or related receptors play a role in phagocytosis of apoptotic corpses, including the mammalian CD36 and its Drosophila homolog CRQ. Although no change was seen in CRQ expression in uta mutant macrophages, CRQ and UTA colocalize and genetically interact. One possible model is that CRQ is recruited to the phagocytic cup upon apoptotic cell binding after SOCE that depends on UTA, DRPR, and drCed-6, and that this might strengthen the binding and uptake of the corpse (Cuttell, 2008).

In C. elegans, CED-1 (DRPR homolog) is related to the endothelial scavenger receptor SREC. Its recruitment to the phagocytic cup depends on functional CED-7, and may occur by exocytosis. Components of the exocyst were implicated in phagocytosis. Moreover, Orai1 is required for degranulation of mast cells, which occurs by exocytosis. Thus, like Orai1, dOrai may be required for exocytosis and, whereas DRPR appears to always be present at the PM, CRQ may be recruited from its intracellular vesicular pool to the phagocytic cup by exocytosis, as previously proposed for CED-1, to promote apoptotic cell uptake (Cuttell, 2008).

A rise in Ca²⁺ was observed in mammalian neutrophils upon particle binding, and Ca²⁺ participates in phagocytosis by promoting F-actin breakdown and phagosome maturation. Mycobacterium tuberculosis is able to invade human macrophages without triggering an increase in [Ca²⁺]i: in the absence of Ca²⁺ signaling, phagosomes containing M. tuberculosis fail to mature, perhaps explaining the survival of this bacterium in the cell. A role for Ca²⁺ in particle binding and phagosome maturation in macrophages, however, was once discounted. uta, dstim, dorai, drCed-6, and drpr are required to trigger SOCE. Yet, although they are poorly phagocytic, macrophages in drced-6 hypomorphs and drpr null mutants engulf bacteria into fully matured phagosomes, arguing against Ca²⁺ being involved in phagosome maturation. This maturation, however, might still occur with lower efficiency when SOCE fails, as RNAi-treated S2 cells for all genes in this pathway poorly engulfed bacteria (Cuttell, 2008).

The findings that UTA links DRPR-mediated phagocytosis and Ca²⁺ homeostasis provide the opportunity to pursue the dissection of the DRPR pathway in Drosophila. DRPR is homologous to CED-1, which belongs to the CED1/6/7 pathway where CED-7 is an ABC transporter. Interestingly, an ABC transporter can modulate Ca²⁺ channel activity in plants, further supporting a link between the CED1/6/7-like pathways and Ca²⁺ homeostasis, which appears to have been conserved throughout evolution. Furthermore, a mutation in human Orai1 was found in some patients with severe combined immune deficiency (Feske, 2006). Thus, pursuing such studies might be relevant to mammalian systems and to human health (Cuttell, 2008).

Presynaptic ryanodine receptor-CamKII signaling is required for activity-dependent capture of transiting vesicles

Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca²⁺-induced Ca²⁺ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca²⁺/calmodulin-dependent kinase II (CamKII). This study shows that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase (SERCA) to deplete presynaptic ER Ca²⁺ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR-CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores (Wong, 2009).

The function of nerve terminals depends on vesicular delivery of proteins synthesized in the soma to synaptic boutons. Transport vesicles are known to contain channels, active zone constituents and neuropeptides. In contrast to synaptic membrane proteins and classical transmitters that are recycled following exocytosis, neuropeptide release is irreversible. Thus, peptidergic transmission depends on replacement of neuropeptide-containing dense core vesicles (DCVs). This is potentially a very slow process because delivery of vesicles synthesized in the soma to nerve terminals by fast axonal transport can take days. However, a cell biological strategy has been discovered that bypasses such delays. Activity-dependent capture of transiting vesicles utilizes a pool of DCVs that rapidly pass through the resting nerve terminal, but that are captured in response to a burst of activity (Shakiryanova, 2006). The onset of this capture, which is evident as decreased DCV efflux and increased neuropeptide content in synaptic boutons, occurs over a period of minutes instead of the hours that would be required for conventional steady state DCV replacement. Essentially, the nerve terminal can tap into the transiting DCV pool to rapidly replenish neuropeptide stores without any direct involvement of the soma. Hence, activity-dependent capture of transiting DCVs eliminates the delay in delivering nascent DCVs, apportions resources based on activity and places control of synaptic neuropeptide storage at sites of release instead of the site of synthesis (i.e., the soma) (Shakiryanova, 2006). A similar recruitment process also occurs with neurotrypsin-containing vesicles, which were concluded to rapidly undergo exocytosis following stimulated capture (Frischknecht, 2008). Likewise, vesicle capture appears to be involved in release of presynaptic Wnt/Wingless protein (Ataman, 2008). Therefore, activity-dependent capture of transiting vesicles supports synaptic neuropeptide, enzyme and developmental peptide release (Wong, 2009).

The signaling required for activity-dependent capture of transiting DCVs is unknown. The long duration of this response in Drosophila motor neurons (i.e., for ~0.5 hour) coupled with the requirement for electrical activity suggests a potential involvement of Ca²⁺-induced phosphorylation. In fact, recent experiments have shown that such signaling increases the mobility of resident DCVs in synaptic boutons. Mobilization, which is triggered by Ca²⁺ influx and persists for ~10 minutes (Shakiryanova, 2005), requires Ca²⁺-induced Ca²⁺ release from presynaptic endoplasmic reticulum (ER) via ryanodine receptors (RyRs) (Shakiryanova, 2007). Subsequently, Ca²⁺/calmodulin-dependent protein kinase II (CamKII) is activated as a necessary step for DCV mobilization (Shakiryanova, 2007). The overlapping onset of the capture and mobilization responses in the first minutes following a brief tetanus stimulated an investigation of whether the RyR-CamKII pathway participates in activity-dependent capture of transiting vesicles (Wong, 2009).

In this study a GFP (Green Fluorescent Protein)-tagged neuropeptide was imaged at the intact Drosophila neuromuscular junction. The rebound in synaptic neuropeptide stores following activity-evoked release, which is caused by capture of transiting vesicles (Shakiryanova, 2006), requires RyR-mediated Ca²⁺ efflux from presynaptic ER and activation of CamKII. Therefore, RyR-CamKII signaling initiates both mobilization of resident DCVs within synaptic boutons and capture of DCVs from the rapidly transiting pool (Wong, 2009).

In vivo imaging has shown that a brief bout of activity elicits prolonged DCV mobilization and capture. These processes are independent because capture requires axonal transport while mobilization does not (Shakiryanova, 2005; Shakiryanova, 2006). Nevertheless, the onsets of mobilization of resident DCVs and capture of transiting DCVs overlap (i.e., both develop over minutes following seconds of activity). This observation stimulated a test of the hypothesis that these two mechanisms are initiated by the same signaling. Previous studies had established that Ca²⁺ influx triggers DCV mobilization by activating RyR-mediated Ca²⁺ release from presynaptic ER that in turn stimulates CamKII (Shakiryanova, 2007). The pharmacological and genetic experiments presented in this study establish that RyR-CamKII signaling is also required for activity-dependent capture of transiting DCVs (Wong, 2009).

This finding raises the issue of how a single signaling pathway produces responses with different durations: after seconds of activity, DCV mobilization lasts ~10 minutes, while the capture response lasts ~40 minutes (Shakiryanova, 2005; Shakiryanova, 2006). One possible consideration is that these kinetic differences could originate in the processes responsible for reversal of mobilization and capture. Specifically, RyR-CamKII signaling could initiate the two processes in parallel, but dephosphorylation of distinct CamKII substrates might occur at different rates, possibly because of the involvement of different phosphatases. This potential explanation suggests that identifying the CamKII substrates that mediate mobilization and capture will be important for understanding the diversity in long-lasting responses initiated by activity-triggered presynaptic RyR-CamKII signaling. Recently, CamKII-dependent phosphorylation of kinesin superfamily protein 17 (KIF17) was found to be essential for unloading NMDA receptor-carrying cargoes from microtubules near the postsynaptic density (Guillaud, 2008). Therefore, CamKII might induce capture by triggering dissociation of transiting DCVs from their molecular motor dynactin complex, which would contain both a kinesin-3 family member UNC-104/Kif1 and a dynein retrograde motor, while mobilization might depend on another CamKII substrate. Alternatively, some process downstream of dephosphorylation might be rate determining for reversal of capture. For example, once captured vesicles are committed to return to the transiting pool, they might need to recruit an unoccupied motor complex to support rapid transiting. If such complexes are rare, then recovery from capture would be very slow. In contrast, recovery from mobilization, which does not require exiting from the bouton, would not be limited in the same way. Regardless of the specific basis for the diverse time courses of mobilization and capture, the use of the same signaling pathway to induce both of these effects is an elegant means to ensure that facilitation of release is coupled to replacement of depleted synaptic neuropeptide stores (Wong, 2009).

Prolonged presynaptic posttetanic cyclic GMP signaling in Drosophila motoneurons

Ca²⁺ can stimulate cyclic nucleotide synthesis, but it is not known whether this signaling occurs in nerve terminals in response to activity. In this study, in vivo imaging of Drosophila motoneuron terminals shows that activity rapidly induces a long-lasting signal from a transgenically expressed optical indicator based on the epac1 (exchange protein directly activated by cAMP 1) cAMP-binding domain. The epac1-cAMP sensor (camps) response in synaptic boutons depends on extracellular Ca²⁺ and ryanodine receptor-mediated Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum. However, mutations that inhibit rutabaga Ca²⁺-stimulated adenylyl cyclase and dunce cAMP-specific phosphodiesterase (PDE) have no effect. Instead, the activity-dependent presynaptic epac1-camps signal reflects elevation of cGMP in response to nitric oxide-activated guanylyl cyclase. Posttetanic presynaptic cGMP is long-lived because of limited PDE activity. Thus, nerve terminal biochemical signaling induced by brief bouts of activity temporally summates on a time scale orders of magnitude longer than fast transmission (Shakiryanova, 2008).

The mechanisms that control cyclic nucleotide levels in the nerve terminal are not known. Activity-induced presynaptic Ca²⁺ influx could be involved because some cyclases are directly activated by Ca²⁺. For example, the rutabaga gene, which affects synaptic plasticity and development in Drosophila, encodes a neuronal Ca²⁺-stimulated adenylyl cyclase. However, this isoform is also stimulated by the Gsα G protein. Thus, it is not clear whether the presynaptic effects of the rutabaga adenylyl cyclase reflect a permissive background effect or activation by Gsα or Ca²⁺. Determining the mechanisms responsible for activating presynaptic cyclases has been difficult because it has not been possible to directly measure cyclic nucleotides in living nerve terminals (Shakiryanova, 2008).

Recently, a ratiometric FRET-based cAMP sensor (camps) that uses the cAMP binding domain of epac (exchange protein directly activated by cAMP) was generated to report activation of adenylyl cylase by forskolin and receptors. Here, in vivo imaging shows that activity rapidly induces a Ca²⁺-dependent epac1-camps response in Drosophila motoneuron synaptic boutons. Surprisingly, this response is long-lasting and is unaffected by mutants that disrupt rutabaga, which lowers neuronal cAMP, or the dunce cAMP-specific phosphodiesterase (PDE), which elevates neuronal cAMP. Pharmacological and genetic experiments are presented that identify the presynaptic biochemical signaling detected by epac1-camps and explain how it temporally summates on a time scale of minutes (Shakiryanova, 2008).

The presynaptic epac1-camps response depends on Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release. First, removal of extracellular Ca²⁺ abolished the FRET change induced by a tetanus. Second, ryanodine (Ry) block of Ca²⁺-induced Ca²⁺ release by endoplasmic reticulum (ER) Ry receptors (RyRs), which eliminates activity-dependent vesicle mobilization and capture of transiting of transiting vesicles in Drosophila nerve terminals, reduced the epac1-camps response. To verify that RyRs were responsible for this partial inhibition, ER Ca²⁺ was depleted with thapsigargin (Tg). Under these conditions Ry had no effect, verifyng that RyR-mediated Ca²⁺-induced Ca²⁺ release was involved. Furthermore, Tg and Ry effects were statistically indistinguishable, implying that RyRs fully account for the participation of ER Ca²⁺ stores (i.e., there was no evidence of autoreceptor-induced inositol trisphosphate-mediated ER Ca²⁺ release). Together, these results show that Ca²⁺-induced Ca²⁺ release from the ER amplifies cyclic nucleotide signaling induced by Ca²⁺ influx (Shakiryanova, 2008).

In vivo imaging of epac1-camps in nerve terminals has yielded a number of striking results. First, activity-dependent cyclic nucleotide synthesis was demonstrated in synaptic boutons. This finding establishes a mechanism for coupling nerve terminal activity to cGMP signaling, which induces synaptic vesicle exocytosis at Drosophila motoneuron boutons and supports plasticity at other synapses. Second, previous studies of NOS-cGMP at synapses have focused on transynaptic effects (e.g., NO as a retrograde signal). However, because glutamatergic transmission was inhibited and postsynaptic muscle in this preparation does not produce cGMP in response to NO, NO generation and activation of guanylyl cyclase both occur presynaptically. This arrangement minimizes dilution of NO to produce robust activity-evoked cGMP responses in motoneuron terminals. Third, cGMP synthesized in response to brief bouts of activity is long-lived in synaptic boutons, because non-dunce PDE activity is limited in the nerve terminal. Hence, temporal summation of cGMP responses occurs on a time scale orders of magnitude longer than neurotransmission. Prolonged electrical activity-induced signaling may be important for a variety of NOS-dependent processes in insect nervous systems (Shakiryanova, 2008).

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam⁷) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. The biochemical properties of V91G D-CaM were investigated. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described the this paper establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM (Wang, 2004).

Drosophila Pkd2 is haploid-insufficient for mediating optimal smooth muscle contractility

Humans heterozygous for PKD1 or PKD2 develop autosomal dominant polycystic kidney disease, a common genetic disorder characterized by renal cyst formation and extrarenal complications such as hypertension and vascular aneurysms. Cyst formation requires the somatic inactivation of the wild type allele. However, it is unknown whether this recessive mechanism applies to life-threatening vascular aneurysms, which could involve weakening of the endothelial lining or surrounding vascular smooth muscle cells (SMCs). Drosophila Polycystic kidney disease gene-2 at 33E3 (Pkd2) encodes a PKD2 family of Ca(2+)-activated Ca(2+)-permeable cation channels. Loss-of-function Pkd2 mutations severely reduced the contractility of the visceral SMCs, which was restored by expressing wild type Pkd2 cDNA via a muscle-specific Gal4 driver. The effect of Pkd2 mutations alone on the skeletal muscle was minimal but was exacerbated by ryanodine-induced perturbation of intracellular Ca(2+) stores. Consistent with this, Pkd2 interacted strongly with a ryanodine receptor mutation, causing a synergistic reduction of larval body wall contraction rate that is normally regulated through Ca(2+) oscillation during excitation-contraction coupling in the skeletal muscle. These results suggest that PKD2 cooperates with the ryanodine receptor to promote optimal muscle contractility through intracellular Ca(2+) homeostasis. Further genetic analysis indicated that Pkd2 is strongly haploinsufficient for normal SMC contractility. Since Ca(2+) homeostasis is a conserved mechanism for optimal muscle performance, these results raise the possibility that inactivation of just one PKD2 copy is sufficient to compromise vascular SMC contractility and function in PKD2 heterozygous patients, thus explaining their increased susceptibility to hypertension and vascular aneurysms (Gao, 2004).

Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase

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The biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP3R) receptors and the endoplasmic reticulum Ca2+-ATPase (SERCA) were characterized by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. ³H-ryanodine binding in total membranes was enhanced by AMP-PCP, caffeine and xanthine, whereas Mg2+, Ruthenium Red and dantrolene were inhibitors. ³-ryanodine binding showed a bell-shaped curve with increasing free [Ca2+], without complete inhibition at millimolar levels of [Ca2+]. ³-IP3 binding was inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca2+-ATPase activity was inhibited by thapsigargin. Confocal microscopy demonstrated abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca2+-ATPase in Drosophila embryos and adults. Ryanodine receptor was expressed mainly in the digestive tract and parts of the nervous system. Maximum parsimony and Neighbour Joining were used to generate a phylogenetic classification of Drosophila ryanodine and insitol triphosphate receptors and Ca2+-ATPase based on 48 invertebrate and vertebrate complete sequences. The consensus trees indicated that Drosophila proteins grouped with proteins from other invertebrates, separately from vertebrate counterparts. Despite evolutionary distances, functional results demonstrate that Drosophila ryanodine and inositol triphosphate receptors and Ca2+-ATPase are reasonably similar to vertebrate counterparts. Protein expression data are consistent with the known functions of these proteins in the Drosophila digestive tract and nervous system. Overall, results show Drosophila as a valuable tool for intracellular Ca2+ dynamics studies in eukaryotes (Vázquez-Martínez, 2003).

The ryanodine receptor is essential for larval development in Drosophila

The study investigated role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. A P element insertion was identified in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F (Ryr), and it was used to generate the hypomorphic allele Ryr¹⁶. An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr ¹⁶ larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca²⁺ stores had previously been implicated in phototransduction; to address this, Ryr¹⁶ mutant clones were generated in the adult eye and whole-cell, patch-clamp recordings were performed on dissociated ommatidia. The results do not support a role for Ryr in normal light responses (Sullivan, 2000).

The Drosophila genome contains a single RyR gene at cytological position 44F. Genomic sequence was obtained flanking each P element in the 44F region and it was determined that P{lacW}l(2)k04913 was inserted in an intron of Ryr, 255 bp upstream of the first coding exon. l(2)k04913 was excised, and it was found that 80% of the independent excision lines were viable, indicating that the lethal lesion of l(2)k04913 corresponded to the P element insertion site and that there were no other lethal mutations on the chromosome. All but one of the lethal excisions failed to complement l(2)k04913 and Df(2R)Np3, a deficiency that uncovers Ryr^k04913, confirming that these mutations were allelic to the P element. Using PCR, four lethal excision lines were identified that had deletions specifically in Ryr. The largest, Ryr ¹⁶, removed the first coding exon and extended into the first and second introns (Sullivan, 2000).

Ryr encodes a protein >5,000 amino acids in length that contains many methionine residues. The transmembrane, channel-forming domain is found at the extreme C terminus, and it has been shown for RyR1 that the C-terminal third of the protein is sufficient to make a functional channel (Sullivan, 2000).

Ryr is widely expressed. RNA in situs were performed on embryos using both genomic- and cDNA-derived probes. Ryr expression was first detected in the mesoderm around stage 9 and then increased starting at stage 13. The highest levels were seen in hypodermal muscles and in the visceral muscles surrounding the gut. The transcript was also detected at lower levels in other tissues, notably the central nervous system (Sullivan, 2000).

Based on the in situ results, the role of Ryr was examined in hypodermal or body wall muscles, which are analogous in structure and function to vertebrate skeletal muscle. Forward movement is initiated by contraction of the posterior body wall muscles, moving the tail up, forward, and down. The contraction propagates as a constriction anteriorly, narrowing and lengthening the larval body, and finally terminates on extension of the mouth hooks up and forward. Initiation of a new body wall contraction (BWC) typically does not occur until termination of the previous one; however, initiation and propagation appear to be independent processes. For example, in calmodulin null mutants, initiation of BWCs was greatly decreased, whereas propagation occurred at normal speeds once initiated (Sullivan, 2000).

BWC was quantified by counting the number of end-to-end contractions a single larva underwent per minute. In Ryr ¹⁶ larvae, the timing of BWC initiation appeared normal, but the contractions propagated more slowly than in wild-type and Ryr ¹⁶/CyO-GFP ^arm controls, such that the rate of BWC was reduced by 50%. Furthermore, contraction in Ryr ¹⁶ animals was attenuated and often had associated tremors; this muscle weakness is probably responsible for the altered larval appearance. As expected, Ryr ¹⁶/Df(2R)Np3 larvae had a more severe defect in BWC propagation, which was reduced by over 90%. The weakest allele, Ryr ^k04913, had a small but significant decrease in the rate of BWC propagation compared with wild-type and Ryr ^k04913/CyO-GFP ^arm controls. By the second instar, Ryr^k04913 larvae had visible contraction defects in hypodermal muscle, which included tilting and dragging of the mouth hooks (Sullivan, 2000).

The drug ryanodine is a highly specific modulator of the RyR channel. In vitro studies have demonstrated that low doses of ryanodine (<10 microM) activate the channel, medium doses (>10 microM) lock the channel into a subconductance state, and high concentrations (~100 microM) completely inactivate it. A range of ryanodine concentrations was fed to newly hatched larvae in yeast paste, and the effect on BWC rates was determined. Low concentrations of ryanodine (<5 microM) had no significant effect on BWC rates when compared to larvae fed yeast paste doped with solvent alone. Higher ryanodine levels (5-100 microM) decreased the rate of BWC propagation, but not initiation, and at the highest doses (~100 microM), BWC was completely inhibited. Ryanodine similarly inhibited BWC in second and third instar larvae. Ryanodine concentrations ~10 microM also caused larvae to round up, altering their appearance in a manner similar to that of Ryr ¹⁶. These results demonstrate that Ryr ¹⁶ is phenocopied by inhibitory concentrations of ryanodine, which is expected if the drug and the mutation both target Ryr. The complete inhibition of BWC by ryanodine provides further evidence that Ryr¹⁶ is not a null allele and suggests that the ryanodine receptor is essential for ECC in hypodermal muscles. In contrast, C. elegans (Maryon, 1998) can undergo muscle contraction even in the absence of RyR activity (Sullivan, 2000).

Visceral muscle function was analyzed in wild-type and Ryr ¹⁶ larvae by assaying the ingestion and excretion of food. The dye bromophenol blue is nontoxic, readily ingested, and completely excreted by larvae, as well as easy to detect through the cuticle. In a typical ingestion assay, 96%-100% of wild-type, Itp-r ¹, and Ryr ¹⁶/CyO-GFP ^arm larvae scored positive for ingestion within 30 min. A very mild, but reproducible, ingestion defect was seen in Ryr^k04913 larvae. In sharp contrast, Ryr ¹⁶ larvae ingested food much more slowly, and the percentage that scored positive failed to reach 100 even after 2 days. Additionally, Ryr ¹⁶ larvae accumulated food in the pharynx, which was never seen in controls, and those that failed to ingest food were indistinguishable in growth and movement from those that did. Ryr¹⁶ mutants may have additional defects in nutrient absorption or metabolism; however, it may simply be that none of the Ryr ¹⁶ larvae ingest sufficient food for growth. These results demonstrate that Ryr ¹⁶ mutants have a severe defect in the ingestion and passage of food into the gut, suggesting that the head and visceral muscles are impaired (Sullivan, 2000).

Pulse-chase and defecation assays were used to measure visceral muscle function specifically. Newly hatched larvae were fed blue yeast for 4 h, then transferred to undyed yeast and scored for complete loss of the dye. In a typical excretion time course, 100% of Ryr ¹⁶/CyO-GFP ^arm, Itp-r ¹, and wild-type larvae had completely excreted the dye within 3 h. In contrast, Ryr ¹⁶ larvae excreted the dye extremely slowly, such that roughly half still retained dye in the gut when assayed for up to 12 h. As a second test for visceral muscle function, the rate of defecation was measured. Wild-type and heterozygous larvae on average defecated once every 4 min, Ryr ^k04913 once every 7 min, and Ryr¹⁶ less than once per 60 min. Both assays demonstrate that Ryr ¹⁶ larvae have a severe defect in excretion, consistent with impaired visceral muscle function (Sullivan, 2000).

Drosophila visceral muscles, although considered striated muscles, are distinct from those of the hypodermus: they are much smaller and mononucleate; the contractile elements are reduced in density and more disorganized; and the sarcoplasmic reticulum (SR) is less developed. It has been proposed that the extent of SR in a given muscle type correlates with the requirement for intracellular Ca²⁺, and thus the RyR, in contraction. However, this hypothesis is inconsistent with the severe visceral defect seen in Ryr¹⁶ larvae. One possibility is that visceral muscle ECC requires Ca²⁺ release from internal stores, but not the rapid, widespread release enabled by extensively developed stores. Alternatively, intracellular Ca²⁺ stores may be essential for some other process equally vital for the passage of food through the gut, such as Ca²⁺ homeostasis or muscle relaxation (Sullivan, 2000).

The DV is the major pulsatile organ of the Drosophila circulatory system. The contractile region or heart is a tube-shaped chamber of cardial cells in the posterior segments that circulates the hemolymph by lateral constriction. The aorta extends anteriorly and connects to the ring gland and lymph nodes. Little is known about the structure or physiology of the circulatory muscle, although L-type channels have been implicated in DV contraction, as is the case in vertebrate cardiac cells. DV function and development have been compared with that of the vertebrate heart, although it has recently been argued that visceral muscle development is the more analogous (Sullivan, 2000).

By expressing GFP in muscles, the larval heartbeat was examined in vivo for all three larval stages; it remained relatively consistent. The heartbeat frequency ranged from slow to fast to fibrillating, with the heart occasionally pausing for up to several seconds; more rarely, localized contractions or twitches occurred. There was no obvious correlation between larval behavior or movement and heart rate. Ryr ¹⁶/CyO and wild-type heart rates and behavior were indistinguishable. In Ryr ¹⁶ larvae, the heart rate was reduced by ~75% relative to the heterozygotes. The decrease in heart rate was caused by loss of the fast and fibrillating contractions but not an increase in pausing (Sullivan, 2000).

The effect of ryanodine on the dorsal vessel was examined in wild-type first and second instar larvae. Feeding larvae ~25 microM ryanodine decreased heart rates, and ~100 microM ryanodine reduced it by ~85% relative to wild type. It was not possible to completely inhibit circulatory muscle contraction with ryanodine as it was in the case of hypodermal muscles. This may reflect experimental limitations, because the larvae must be washed to observe the GFP signal. As detected by increasing BWC rates, larvae rapidly recovered when washed out of ryanodine. Alternatively, the ryanodine receptor may not be essential for ECC in circulatory muscles (Sullivan, 2000).

In previous studies (Amon, 1997), it was observed that depletion of Ca²⁺ stores in dissected photoreceptor cells by ryanodine inhibited subsequent light responses. This effect could be rescued by the addition of Ca²⁺-calmodulin, which inhibits Ca²⁺ release by the ryanodine receptor. Based on these results, it was proposed that Ca²⁺ release through the RyR is required for phototransduction. However, these experiments could not distinguish a pleiotropic effect of the pharmacological depletion of internal stores from an actual role for the RyR in light transduction. To test this, Ryr¹⁶ mutant eye clones were generated in heterozygous adults by using the Flp/FRT system. Clones representing >90% of the eye were generated using eyFlp and a marker chromosome carrying the minute (M53) mutation, which slows cellular growth. The eyeless enhancer drives Flp expression specifically in the eye disc, and the resulting Ryr¹⁶ mutant cells outgrow both M53/Ryr ¹⁶ and M53/M53 cells in mosaic tissue. Ryr¹⁶ clones appeared morphologically normal; furthermore, whole-cell, voltage-clamp recordings on Ryr ¹⁶ ommatidia showed responses indistinguishable from wild type. Taken together, these results are inconsistent with the postulate that Ryr is required for phototransduction (Sullivan, 2000).

To examine the role of Ryr in embryonic development, females were generated with germ-line clones homozygous for Ryr ¹⁶ using the dominant female sterile technique. However, the progeny of these females, when crossed to Ryr ¹⁶ males, were phenotypically identical to either wild type or Ryr¹⁶. Consistent with this, RNA interference using double-stranded RNA transcribed from Ryr cDNA had no apparent effect on embryogenesis, although a majority of the injected embryos that hatched did have muscle contraction defects. The overall behavior of Ryr ¹⁶ larvae was indistinguishable from wild type: they detected and migrated to food sources; had an enhanced rate of food ingestion after starvation; had normal salt avoidance and response to mechanical stimulation; and showed no spontaneous avoidance behavior. BrdUrd labeling of Ryr¹⁶ larval brains showed that cell cycle progression is normal in these mutants. Thus, at present these results provide no evidence that Ryr has any nonmuscle roles in Drosophila development (Sullivan, 2000).

Search PubMed for articles about Drosophila Ryanodine receptor

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Feske, S., et al. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441: 179-185. PubMed Citation: 16582901

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Gao, Z., Joseph, E., Ruden, D. M. and Lu, X. (2004). Drosophila Pkd2 is haploid-insufficient for mediating optimal smooth muscle contractility. J. Biol. Chem. 279(14): 14225-14231. PubMed Citation: 14732716

Griffith, L. C., Wang, J., Zhong, Y., Wu, C. F. and Greenspan, R. J. (1994). Calcium/calmodulin-dependent protein kinase II and potassium channel subunit eag similarly affect plasticity in Drosophila. Proc. Natl. Acad. Sci. 91: 10044-10048. PubMed Citation: 7937834

Guillaud, L., Wong, R. and Hirokawa, N. (2008). Disruption of KIF17-Mint1 interaction by CaMKII-dependent phosphorylation: a molecular model of kinesin-cargo release. Nature cell biology 10: 19-29. PubMed Citation: 18066053

Haghighi, A. P., et al. (2003). Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction. Neuron 39: 255-267. PubMed Citation: 12873383

Hasan, G. and Rosbash, M. (1992) Drosophila homologs of two mammalian intracellular Ca2+-release channels: identification and expression patterns of the inositol 1,4,5-triphosphate and the ryanodine receptor genes. Development 116: 967-975. PubMed Citation: 1338312

Klose, M. K., Chu, D., Xiao, C., Seroude, L. and Robertson, R. M. (2005). Heat shock-mediated thermoprotection of larval locomotion compromised by ubiquitous overexpression of Hsp70 in Drosophila melanogaster. J. Neurophysiol. 94: 3563-3572. PubMed Citation: 16093328

Lochner, J. E., et al. (2006). Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging. J. Neurobiol. 66: 564-577. PubMed Citation: 16555239

Lu, C. S., Hodge, J. J., Mehren, J., Sun, X. X. and Griffith, L. C. (2003). Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation. Neuron 40: 1185-1197. PubMed Citation: 14687552

Ludwig, M., et al. (2002). Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites. Nature 418: 85-89. PubMed Citation: 12097911

Luik, R. M. et al. (2008). Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature 454: 538-542. PubMed Citation: 18596693

MacDonald, J. M., et al. (2006). The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 50: 869-881. PubMed Citation: 16772169

Manaka, J., et al. (2004). Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages. J. Biol. Chem. 279: 48466-48476. PubMed Citation: 15342648

Mangahas, P.M. and Zhou, Z. (2005). Clearance of apoptotic cells in Caenorhabditis elegans. Semin. Cell Dev. Biol. 16: 295-306. PubMed Citation: 15797839

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Shakiryanova, D., et al. (2005). Activity-dependent liberation of synaptic neuropeptide vesicles. Nat. Neurosci. 8: 173-178. PubMed Citation: 15643430

Shakiryanova, D., Tully, A. and Levitan, E. S. (2006). Activity-dependent synaptic capture of transiting peptidergic vesicles. Nat. Neurosci. 9: 896-900. PubMed Citation: 16767091

Shakiryanova, D., et al. (2007). Presynaptic ryanodine receptor-activated calmodulin kinase II increases vesicle mobility and potentiates neuropeptide release. J. Neurosci. 27(29): 7799-806. PubMed Citation: 17634373

Shakiryanova, D. and Levitan, E. S. (2008). Prolonged presynaptic posttetanic cyclic GMP signaling in Drosophila motoneurons. Proc. Natl. Acad. Sci. 105(36): 13610-3. PubMed Citation: 18765813

Sullivan, K. M., Scott, K., Zuker, C. S. and Rubin, G. M. (2000). The ryanodine receptor is essential for larval development in Drosophila melanogaster. Proc. Natl. Acad. Sci. 97(11): 5942-7. PubMed Citation: 10811919

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Vázquez-Martínez, O., et al. (2003). Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase. J. Cell Sci. 116(Pt 12): 2483-94. PubMed Citation: 12766186

Vig, M., et al. (2006). CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312: 1220-1223. PubMed Citation: 16645049

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Wang, B., et al. (2004). Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila. Protein Sci. 13(12): 3285-97. PubMed Citation: 15557269

Wong, M. Y., Shakiryanova, D. and Levitan E. S. (2009). Presynaptic ryanodine receptor-CamKII signaling is required for activity-dependent capture of transiting vesicles. J. Mol. Neurosci. 37(2): 146-50. PubMed Citation: 18592416

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date revised: 6 June 2010

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