| Gene name - neither inactivation nor afterpotential C
Cytological map position - 27F3-27F3
Function - motor domain protein
| Symbol - ninaC
FlyBase ID: FBgn0002938
Genetic map position - chr2L:7377718-7384344
Classification - Myosin motor domain, ATPase, Serine/Threonine protein kinase
Cellular location - cytoplasmic
Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, this study shows that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ~100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabelling revealed that Arr2 in the cell body colocalizes with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. It is proposed that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction (Hardie, 2012).
Photoreceptors are highly polarized cells with membrane-rich photosensory compartments separated from the rest of the cell body. It has recently become widely recognized that several phototransduction proteins translocate between these compartments in response to light, representing a form of long-term light and dark adaptation. One of the best-studied examples is arrestin, which terminates the light response by binding to photo-isomerized rhodopsin (metarhodopsin). In dark-adapted photoreceptors most arrestin localizes to the cell body in both vertebrate and insect photoreceptors, but on illumination translocates to the photosensory compartmen. In fly photoreceptors the photosensory compartment is represented by the rhabdomere, a light-guiding, rod-like stack of ~30,000 densely packed apical microvilli loaded with rhodopsin and proteins of a phototransduction cascade mediated by heterotrimeric Gq protein, phospholipase C (PLC), and Ca2+-permeable 'transient receptor potential' (TRP) channels (Hardie, 2012).
Although the possible role of active transport by molecular motors remains debated, recent evidence in both vertebrate rods and Drosophila microvillar photoreceptors supports an essentially passive diffusional model of arrestin translocation, down gradients established by light-regulated 'sinks'. Recent studoes provided evidence that metarhodopsin (M) is the major light-activated sink in fly rhabdomeres by showing that the dominant arrestin isoform (Arr2) translocated in a 1:1 stoichiometric relationship to the number of rhodopsin photo-isomerizations (Satoh, 2010). This study also showed that Arr2 translocation was very rapid (τ ~10 s), but profoundly slowed in mutants of various phototransduction proteins including Gq, phospholipase C (PLC) (norpA), and the major Ca2+-permeable TRP channel (Satoh, 2010). The evidence suggested that Ca2+ influx via the light-sensitive channels was required to accelerate Arr2 translocation, possibly by releasing Arr2 from a Ca2+-dependent cytosolic sink (Satoh, 2010). However, direct evidence for the role of Ca2+ was lacking, while the identity of the putative cytosolic sink and the mechanism(s) mediating the acceleration remained unresolved (Hardie, 2012).
This study shows directly that Ca2+ is both necessary and sufficient to accelerate Arr2 translocation and provides evidence that the Ca2+-regulated cytosolic sink is the cytosolic isoform of NINAC, a calmodulin (CaM) binding myosin III. The evidence also suggests the existence of another potential Ca2+ dependent cytosolic sink, which sequesters and releases arrestin on a much slower timescale, and that NINAC protects Arr2 from sequestration and immobilization by this site. The data support a mechanism for the Ca2+-dependent translocation of Arr2 that is remarkably similar to a previously proposed disinhibitory mechanism of Ca2+-dependent inactivation of M (Liu, 2008) required for rapid termination of the light response (Hardie, 2012).
Previously studies have proposed that Ca2+ influx via the light-sensitive TRP channels is required for rapid Arr2 translocation, because the slow translocation in trp mutants could be rescued by genetic elimination of Na+/Ca2+ exchanger activity (Satoh, 2010). The present study confirmed the role of Ca2+ directly by imaging Arr2-GFP translocation in dissociated ommatidia, and showing that extracellular Ca2+ is required for rapid Arr2 translocation. Ca2+ was not only required, but also sufficient to enable rapid translocation without any products of PLC activity, since the Ca2+ ionophore, ionomycin, fully rescued translocation in blind norpA mutants lacking PLC. Significantly, it was found that the requirement of Ca2+ for rapid translocation was obviated in null mutants of NINAC (CaM binding MyoIII), with the cytosolic p132 isoform of NINAC alone being sufficient to slow down translocation in Ca2+-free conditions. This suggests that cytosolic NINAC p132 acts as a Ca2+/CaM-dependent 'brake' on translocation by binding Arr2, releasing it in response to Ca2+ influx associated with the photoresponse. This conclusion was further supported by finding that the ninaC mutation rescued rapid translocation in trp mutants (in ninaC;trp double mutants). However, the failure to rescue translocation in norpA;ninaC mutants, except under special conditions, and the demonstration of significant slowing of translocation following prolonged dark adaptation in ninaC mutants also indicated the existence of a second Ca2+-dependent cytosolic sink (Hardie, 2012).
Despite an earlier study reporting that Arr2 translocation was impaired in ninaC mutants, as shown here and previously (Satoh, 2005; Satoh, 2010), Arr2 translocation, whether of endogenous Arr2 or GFP-tagged Arr2, appears essentially intact in ninaC-null mutants. In fact, far from being impaired, the results indicate that translocation can be rescued by ninaC mutations under conditions where translocation is slowed down by reduced Ca2+ influx. Although translocation was significantly slower in ninaC mutants following prolonged dark adaptation, it was never prevented and full translocation was always achieved within ~2-3 min of appropriate illumination (Hardie, 2012).
A novel phenotype of ninaCP235-null mutants, and also ninaCΔ174 mutants lacking only the rhabdomeric p174 isoform, was the complete absence of an early rapid increase in fluorescence routinely observed during the first ~500 ms of measurements of Arr2-GFP fluorescence from the DPP of wild-type photoreceptors or dissociated ommatidia. With a time constant of ~260 ms, this rapid phase was ~40× faster than the overall translocation (τ ~10 s) itself the fastest protein translocation reported in a photoreceptor to date, and probably diffusionally limited (Satoh, 2010). It therefore seems unlikely that the fast phase represents a 40x faster, ninaC-dependent movement of Arr2 from cell body into the rhabdomere. Instead we suggest that it represents a change in the fluorescence efficiency of Arr2-GFP as it is released (via Ca2+ influx) from the rhabdomeric p174 NINAC isoform. A lower fluorescence when bound to NINAC might reflect crowding of the GFP-fluorophore, or could be due to some other feature of the nano-environment of the fluorophore when Arr2 is bound to NINAC in the microvilli. This interpretation was supported by the ability to eliminate the rapid phase by pre-illumination with long wavelength light, which induces Ca2+ influx without net change in M. The rapid phase then re-emerged with a time constant of ∼3 s in the dark, presumably representing rapid rebinding of Arr2-GFP to NINAC (Hardie, 2012).
After more than a few minutes in the dark, Arr2 translocation into the rhabdomere became progressively slower, with clear functional consequences in a parallel slowing of the decay of the ERG. This gradual slowing was considerably more pronounced in ninaC-null mutants, where it si proposed that the slowing represents binding or sequestration of Arr2 via one or more NINAC-independent target(s) or compartment(s). Release from such sites also requires activation of the phototransduction cascade, and translocation could be accelerated back to levels typical of short dark-adaptation times by pre-illumination with bright orange light, which itself does not generate a net increase in M. It seems likely that the rise in Ca2+ is also responsible for release from this site; however, the involvement of other products of the phototransduction cascade cannot be excluded. The identity of this second site or compartment remains a subject for future investigation. Given previous reports that Arr2 can bind to phosphoinositides, negatively charged phosphoinositide species on endomembranes, which could be screened by Ca2+, might represent promising candidates. Drosophila Arr2 is an unusually basic (positively charged) protein and may thus have a strong tendency to bind to such sites. The finding that the slowing of translocation with dark adaptation was more pronounced in ninaC mutants suggests that one of the functions of cytosolic NINAC may be to prevent immobilization of Arr2 by this alternative potential sink. Because the Ca2+-dependent release of Arr2 from NINAC occurs on a subsecond timescale, this then allows more rapid translocation (and hence recovery of the electrical response) after a period in the dark (Hardie, 2012).
These results demonstrate that Arr2 translocation is accelerated by Ca2+ influx, and suggest that this is mediated by a disinhibitory mechanism, whereby NINAC p132 binds to Arr2 under low Ca2+ conditions in the dark, rapidly releasing it in response to Ca2+ influx associated with the photoresponse. Although inferred from essentially independent experiments, this mechanism is strikingly similar to one previously proposed for the rapid, Ca2+-dependent inactivation of M during the light response itself (Liu, 2008). In that study the time constant of M inactivation by Arr2 was found to be accelerated from ~200 ms under Ca2+-free conditions to ~20 ms following Ca2+ influx. This Ca2+ dependence was eliminated in both ninaC-null mutants, and in ninaCΔ174 mutants lacking the rhabdomeric p174 (but not in ninaCΔ132 mutants lacking cytosolic p132). The results also indicated a disinhibitory mechanism, leading to the proposal that Arr2 in the microvilli was bound to rhabdomeric NINAC p174 under low Ca2+ conditions in the dark, thus hindering its diffusional access to activated M. Ca2+ influx via the first activated TRP channels, then rapidly releases Arr2, allowing it to diffuse, bind to, and inactivate M (Hardie, 2012).
NINAC p132 and p174 share a common CaM binding site (CBS) and although p174 has a second CBS not found in p132 (Porter, 1993; Porter, 1995), the pronounced slowing of translocation with dark adaptation in null ninaCP235 mutants was recapitulated in mutants lacking the common CBS. It is therefore suggested that essentially the same mechanism underlies the Ca2+-dependent rapid translocation of Arr2, but now acting via NINAC p132 rather than p174 and working over much larger distances (several micrometers as opposed to the nanometer dimensions of single microvilli) and hence slower timescales (Hardie, 2012).
The proposed interaction between NINAC and Arr2 finds some support from biochemical data reporting coimmunoprecipitation of Arr2 and NINAC in extracts from whole heads (Lee, 2004). However, that study also reported that both Arr2 and NINAC had significant in vitro affinity for phosphoinositides. It was proposed that the NINAC/Arr2 association was indirect and mediated by both Arr2 and NINAC binding to phosphoinositide-rich membrane. Although the current results clearly indicate that Ca2+-dependent modulation of Arr2 binding to M (Liu, 2008) and Ca2+-dependent translocation of Arr2 are both dependent upon NINAC, the possibility cannot be excluded that the interaction is mediated indirectly via a NINAC-dependent target. Ultimate verification will require direct demonstration of NINAC/Arr2 binding and its dependence upon Ca2+/CaM (Hardie, 2012).
The regulated multisink model proposed in this study differs fundamentally from an earlier model in which NINAC was proposed as a molecular motor transporting Arr2 in phosphoinositide-rich vesicles (Lee, 2004). By contrast it shows strong parallels with current models for arrestin translocation in vertebrate rods (Calvert, 2006; Slepak, 2008). Here, phosphorylated rhodopsin represents the light-activated sink in the outer segments, while microtubules have been proposed as the cytosolic sink in the inner segments. There is also evidence indicating light-regulated acceleration of translocation in vertebrate rods. The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Hardie, 2012 and references therein).
Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy. While it can be conveniently studied in photoreceptors with their distinctive polarized morphologies and high concentrations of transduction machinery, translocation according to the same general principles may represent a general and elegant solution to the problem of directed movements of signaling proteins (Hardie, 2012).
Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches in Drosophila led to the identification of several proteins required for termination including protein kinase C (PKC), NINAC p174, which consists of fused protein kinase and myosin domains, and a PDZ scaffold protein, INAD. This study describes a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss of retin resulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse, typically lead to a reduction in levels of the major rhodopsin, Rh1, to attenuate signaling. Consistent with the slower termination in retin1, the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin1 was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Since PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age-dependent, and paralleled the onset of the termination phenotype in retin1 mutant flies. It is concluded that the slower termination of the photoresponse in retin1 resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC (Venkatachalam, 2010).
This study describes the identification of Retin, a protein required for termination of the photoresponse. Unlike other proteins that function in termination, the retin phenotype is age-dependent. Slow termination leads to increased endocytosis and degradation of the major rhodopsin, Rh1, which serves as a negative feedback mechanism to attenuate the visual response. Consistent with a defect in termination, the age-dependent impairment in the photoresponse in retin1 is associated with greater endocytosis of Rh1 and an age-dependent reduction in the concentration of Rh1 (Venkatachalam, 2010).
A central question concerns the basis for the age-dependent decrease in the termination rate in retin deficient flies. Retin has been reported to function in macrophages through a pathway that involves the ryanodine receptor, a store-operated channel, Orai, and the interacting protein, STIM1 (Cuttell, 2008), which is present in the endoplasmic reticulum (ER) and senses changes in ER Ca2+. However, Ca2+ release from the ER, the ryanodine receptor and the IP3-receptor do not appear to function in Drosophila visual transduction. Furthermore, knockdown of stim1 RNA using a photoreceptor cell GAL4 in combination with UAS-stim1-RNAi transgene had no effect on phototransduction, the concentration of Retin, or other proteins reduced in retin1 mutant eyes. The decrease in termination in retin1 mutant flies was not due directly to loss of Retin, since the Retin protein was absent in young flies that exhibited normal termination. The retin phenotype also was not a consequence of a reduction in NINAC p174, since both 3 and 7 day-old retin1 flies displayed similarly low levels of p174; however, only the 7 day-old flies exhibited the slow termination phenotype (Venkatachalam, 2010).
It is concluded that the age-dependent termination phenotype in retin1 results from a reduction in PKC levels. Consistent with this proposal, the decline in PKC concentration paralleled the appearance of the termination phenotype. In young retin1 flies, which displayed normal termination, PKC was not reduced significantly from wild-type. However, in older retin1 flies, the PKC concentration declined two-fold. In further support of the conclusion that the 50% decrease in PKC is responsible for the termination defect in retin1, a similar impairment in termination occurs in heterozygous flies, which are missing copy of the gene encoding the eye-enriched PKC (Venkatachalam, 2010).
The following mechanism is proposed through which Retin affects the concentration of PKC. First, Retin forms a complex with NINAC p174, and this interaction is required for the stability of p174. Both proteins co-immunoprecipitated from head extracts, and loss of Retin resulted in a lower concentration of p174. The requirement for Retin and NINAC was mutual since Retin was undetectable in flies missing p174. Second, NINAC is required for stabilizing the PDZ-containing scaffold protein INAD. NINAC and INAD interact, and it was found that a single amino acid mutation that disrupts the INAD binding site in p174 (ninaCI1501E) causes a reduction in INAD. Third, PKC binds stoichiometrically to INAD and requires this interaction for stability. As a result, INAD and PKC displayed indistinguishable two-fold decreases in protein levels. It was found that PKC also declined to a similar extent in flies expressing NINACI1501E. Because INAD was reduced in ninaCI1501E flies, but not Retin or NINAC p174, the instability of PKC was not due to non-specific effects resulting from changes in the concentrations either Retin or p174. Thus, loss of Retin causes a reduction in the level of p174, which in turn affects the concentration of INAD, leading to instability of PKC, which underlies the slower termination (Venkatachalam, 2010).
Despite the defect in termination, retin1 flies exhibited only minor effects on retinal morphology. There are multiple examples of mutations that are associated with termination defects that display relatively minor alterations in rhabdomere morphology. These include rac2, ninaC, and stops. Of particular relevance, flies heterozygous for a mutation disrupting the eye-enriched PKC (inaCP209/+ flies), which exhibit a termination phenotype similar to retin1, do not undergo retinal degeneration (Venkatachalam, 2010).
Finally, both Retin and myosins with fused N-terminal protein kinase domains are found in other organisms including humans. Protein kinase/myosins (myosin IIIs) and Retin are both expressed in the mammalian retina. This raises the possibility that Retin and myosins related to NINAC may form a complex in mammalian photoreceptor cells, and are required for signaling (Venkatachalam, 2010).
Absolute visual thresholds are limited by 'dark noise,' which in Drosophila photoreceptors is dominated by brief (~10 ms), small (~2 pA) inward current events, occurring at ~2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the Gq α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca2+ in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. These results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca2+-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency (Chu, 2013).
Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. This study investigated the mechanism and role of rhodopsin inactivation. The lifetime of activated rhodopsin (metarhodopsin = M*) was determined in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M* was inactivated rapidly (τ ~20 ms) under control conditions, but ~10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M*. These results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling (Liu, 2008).
This study exploited the bistable nature of invertebrate rhodopsins to measure the lifetime of activated metarhodopsin in Drosophila. The approach measures the time window during which photoreisomerization of M* can suppress the response to light. The relative lack of overlap of the R and M spectra in UV opsins has been exploited by recording from the UV-sensitive photoreceptors in Limulus median ocelli. This strategy was adapted for Drosophila by using flies engineered to express the UV opsin Rh3; the effective M* lifetime was found to be very short (τdec ≈20 ms) under physiological conditions. Strikingly, M* lifetime was prolonged ~10-fold in the absence of Ca2+ influx, indicating that M-Arr2 binding is Ca2+ dependent and that M* lifetime is the rate-limiting step in response deactivation in Ca2+-free solutions. Further experiments led to proposal of a mechanism for Ca2+-dependent M* inactivation by Arr2, mediated by calmodulin (CaM) and myosin III NINAC (Liu, 2008).
Photoisomerization of rhodopsin (R) by short-wavelength light (480 nm for Rh1 or 330 nm for Rh3) generates active metarhodopsin (M*). M* continues to activate Gq until it binds arrestin (Arr2) or is reconverted to R by long-wavelength illumination (570/460 nm). M is serially phosphorylated by rhodopsin kinase (RK), but this is not required for M* inactivation or Arr2 binding. CaMKII-dependent phosphorylation of Arr2 at Ser366 and photoreconversion of Mpp to Rpp is required for the release of Arrp. Phosphorylation of Arr2 also prevents endocytotic internalization of M-Arr2. In Arr2S366A or mutants defective in CamKII, photoreconversion fails to release Arr2. Finally, Rpp is dephosphorylated by the Ca-CaM-dependent rhodopsin phosphatase (rdgC) to recreate the ground state, R. The results suggest that under low-Ca2+ conditions Arr2 is prevented from rapid binding to M* because it is sequestered by NINAC or a NINAC-regulated target; however, Ca2+ influx acting via CaM rapidly releases Arr2. Each microvillus contains ~70 Arr2 molecules, ensuring rapid quenching of M* once they are free to diffuse. The role of M* phosphorylation remains uncertain but may be involved in Rh1 internalization by the minor arrestin, Arr1 (Liu, 2008).
Ca2+ dependence of M* lifetime had not previously been demonstrated in an invertebrate photoreceptor, and the consensus from data in Drosophila suggested no obvious mechanism by which M* lifetime could be regulated by Ca2+. The finding that M* inactivation is strongly Ca2+ dependent prompted a re-examination of possible roles of Rh1 and Arr2 phosphorylation as well as CaM. Although M* lifetime remained strongly Ca2+ dependent in mutants defective in rhodopsin and arrestin phosphorylation, the Ca2+ dependence of M* inactivation was effectively eliminated in hypomorphic cam mutants. This requirement for CaM appeared to be mediated by the myosin III NINAC protein, since the Ca2+ dependence of M* inactivation was effectively abolished in both the null ninaCP235 mutant and an allele (ninaCKD) in which CaM levels in the microvilli were unaffected. NINAC, which is the major CaM-binding protein in the photoreceptors, has long been known to be required for normal rapid response deactivation, but the mechanistic basis remained unresolved. These results now strongly suggest that it is specifically required for the Ca2+- and CaM-dependent inactivation of M* by Arr2 (Liu, 2008).
How might NINAC regulate the Ca2+-dependent inactivation of M*? A clue comes from the finding that Arr2 levels were substantially reduced in ninaC mutants. After taking this into account, the lack of Ca2+ dependence of M* inactivation in ninaC mutants was in fact associated with a very pronounced acceleration of response inactivation under Ca2+-free conditions. This was most clearly revealed in ninaCKD, which appears to be specifically defective only in Ca2+-dependent M* inactivation and does not show the additional response defects of the null ninaC phenotype (e.g., Hofstee, 1996). This suggests a disinhibitory mechanism whereby Ca2+-dependent inactivation of M* may be achieved, at least in part, by the NINAC-dependent prevention of Arr2-M* binding under low-Ca2+ conditions. Specifically, it is suggested that in Ca2+-free solutions, or in the low-Ca2+ conditions prevailing during the latent period of the quantum bump under physiological conditions, Arr2 in the microvilli is predominantly bound to NINAC or a NINAC-regulated target, thus restricting its access to M*. However, following Ca2+ influx, CaCaM would bind to NINAC, causing NINAC to release Arr2, which, as a soluble protein, could then rapidly diffuse to encounter and inactivate M* (Liu, 2008).
Interestingly, a recent study reported that NINAC can interact with Arr2 in a phosphoinositide-dependent manner (Lee, 2004). This interaction was described in the context of a role of NINAC in light-induced translocation of Arr2, which was reported to be disrupted in ninaC mutants. However, involvement in translocation was challenged by a subsequent study reporting that Arr2 translocation was unaffected in ninaC mutants (Satoh, 2005). It will be interesting to see whether the Arr2-NINAC interactions described by Lee (2004) reflect a role in the CaCaM- and NINAC-dependent inactivation of M* reported in this study (Liu, 2008).
It has long been known that responses under Ca2+-free conditions decay ~10-fold more slowly than in the presence of Ca2+. The current results establish that the inactivation of M* by Arr2 is the rate-limiting inactivation step in such Ca2+-free responses, with a time constant of ~200 ms in wild-type photoreceptors. Following inactivation of M* by photoreisomerization under Ca2+-free conditions, the response decayed with a time constant of ~80 ms. This also provides a unique and direct measure of the time constant(s) of the downstream mechanisms of inactivation, which presumably include GTP-ase activity of the Gq-PLC complex and removal of DAG by DAG kinase. It will be interesting so see whether Ca2+ also accelerates these inactivation mechanisms (Liu, 2008).
By contrast, the failure to accelerate response decay by overexpressing Arr2 in the presence of Ca2+ indicates that inactivation of M* is not rate limiting under physiological conditions. This can be understood by recognizing that the macroscopic kinetics are determined by the convolution of the bump latency distribution and bump waveform, the latter probably terminated by Ca2+-dependent inactivation of the light-sensitive channels. Until the Ca2+ influx associated with the quantum bump, the phototransduction machinery in each microvillus is effectively operating under Ca2+-free conditions. The results suggest that it is the Ca2+ influx associated with each quantum bump that promotes M* inactivation, and hence the timing of M* inactivation will be determined by the bump latency distribution and not vice versa. This leads to the, perhaps counterintuitive, concept that response termination is rate limited, not by any specific inactivation mechanism, but rather by the time course with which the cumulative probability of bump generation approaches 100% (Liu, 2008).
Clearly, rapid quenching of M* is essential to maintain the fidelity and high temporal resolution of phototransduction. In wild-type cells, an effectively absorbed photon generates only one quantum bump, but never (or extremely rarely) two or more; yet the multiple bump trains observed in arr2, cam, and ninaC mutants show that additional bumps are readily generated within 50-100 ms if M* fails to be inactivated. To prevent such multiple bumps without Ca2+-dependent feedback would require such a high rate of Arr2 binding that many M* molecules would be inactivated before they had a chance to activate sufficient G proteins to generate a quantum bump. This would result in an effective reduction in sensitivity, as is directly illustrated by the phenotype of p[Arr2] flies overexpressing Arr2. These show not only a 5-fold reduction in quantum efficiency. but also a reduction in bump amplitude and even an increase in bump latency, which is attributed to a decreased rate of second messenger generation. The mechanism proposed in this study provides an elegant solution to this dilemma. The analysis suggests that in the low-Ca2+ environment prior to Ca2+ influx, much of the Arr2 in the microvillus is bound to NINAC (or NINAC-regulated target), thus allowing M* to remain active long enough to activate sufficient G proteins to guarantee production of a full-sized quantum bump with high probability. Only after the bump has been initiated does Ca2+ influx accelerate the inactivation of M* by releasing Arr2, thus ensuring that only one bump is generated. This strategy is complemented and enabled by the ultracompartmentalization afforded by the microvillar design, which ensures that the Ca2+ rise is both extremely rapid and largely confined to the affected microvillus (Liu, 2008).
Drosophila visual transduction has served as a paradigm to characterize G protein-coupled neuronal signaling. As in mammals, light-activated rhodopsin is phosphorylated and interacts with a rhodopsin regulatory protein, arrestin, which facilitates deactivation of the receptor. However, unlike mammalian phototransduction, light activation in Drosophila is coupled to stimulation of phospholipase C rather than a cGMP-phosphodiesterase. The visual arrestin, undergoes light-dependent trafficking in mammalian and Drosophila photoreceptor cells, though the mechanisms underlying these movements are poorly understood. In Drosophila, the movement of the visual arrestin, Arr2, functions in long-term adaptation and is dependent on interaction with phosphoinositides (PIs). However, the basis for the requirement for PIs for light-dependent shuttling has been unclear. This study demonstrates that the dynamic trafficking of Arr2 into the phototransducing compartment, the rhabdomere, requires the eye-enriched myosin III, NINAC. Defects in ninaC result in a long-term adaptation phenotype similar to that which occurs in arr2 mutants. The interaction between Arr2 and NINAC is PI dependent and NINAC binds directly to PIs. These data demonstrate that the light-dependent translocation of Arr2 into the rhabdomeres requires PI-mediated interactions between Arr2 and the NINAC myosin III (Lee, 2004).
The activity of signaling cascades can be profoundly affected by modulating the levels of regulatory proteins in specialized cellular compartments. The concentrations of such proteins can be altered through changes in protein synthesis or degradation. However, a faster mechanism for regulating the levels of a signaling protein involves protein trafficking in response to agonist stimulation. Dynamic movements of signaling proteins are of particular importance to neurons, such as photoreceptors, which are highly polarized and respond to their external stimulus, light, with great rapidity (Lee, 2004).
During the last two decades, it has become clear that several key signaling proteins in mammalian and Drosophila photoreceptor cells undergo light-dependent translocations in and out of the phototransducing compartments, the outer segments, and rhabdomeres, respectively. These include the Drosophila TRPL cation channel, which are concentrated in the phototransducing compartments only in dark-adapted animals. Upon exposure to light stimulation, these proteins shuttle into the cell bodies or inner segments over the course of several minutes. Visual arrestin, which participates in termination of the photoresponse, also undergoes dramatic light-dependent translocation, though the direction of movement is opposite to that of the TRPL and the Gα and Gβ subunits. In response to light stimulation, arrestin migrates into the outer segments/rhabdomeres of vertebrate and Drosophila photoreceptor cells (Lee, 2004 and references therein).
The light-dependent translocations of signaling proteins in photoreceptor cells appear to function in long-term light adaptation. Photoreceptor cells adapt to increasing intensities of background illumination by increasing the rate of termination of the photoresponse. The rate of termination of the light response is relatively slow in animals that are initially dark-adapted. Prior exposure to background illumination accelerates the rate of response termination. However, mutations that decrease the rate of translocation of the Drosophila visual arrestin from the cell body to the rhabdomere cause corresponding defects in this mode of adaptation (Lee, 2004 and references therein).
A critical question concerns the mechanisms underlying the light-dependent shuttling of signaling proteins, such as visual arrestin. Since mammalian rhodopsin undergoes light-dependent phosphorylation, which promotes binding to visual arrestin, it has been proposed that visual arrestin becomes concentrated in the outer segments due to binding to phosphorylated rhodopsin. However, it has been shown recently that translocation of visual arrestin into the outer segments occurs normally in rhodopsin kinase-deficient mice. It has been shown that the major Drosophila visual arrestin, Arr2, binds to phosphoinositides (PIs) and that this interaction is necessary for light-induced trafficking. However, the basis for this PI requirement and the identity of proteins that promote the light-dependent translocation of Arr2 or any other signaling protein have not been previously identified (Lee, 2004).
It has now been shown that the NINAC myosin III, which consists of linked protein kinase and myosin head domains), is required for movement of Arr2 into the rhabdomeres. NINAC is expressed as two isoforms, p132 and p174, which are detected exclusively in the cell bodies and rhabdomeres, respectively. While p174 is required for response termination, no role for p132 in the photoresponse has been described. This study shows that p132 is the primary isoform necessary for light-dependent trafficking of Arr2 into the rhabdomeres. Moreover, flies that do not express p132 display a defect in long-term adaptation, consistent with its role in Arr2 shuttling. This study shows that the interaction between Arr2 and PIs is required for Arr2 to interact with NINAC, NINAC is also a PI binding protein, and that these interactions promoted the light-dependent movement of Arr2 into the rhabdomeres. These data indicate that the light-dependent shuttling of Arr2 into the rhabdomeres requires an association with the NINAC myosin III that is mediated through PIs (Lee, 2004).
In contrast to the Lee (2004a) study, a study by Satoh (2005) found no requirement for NINAC in Arr1 or Arr2 translocation. The Satoh study concludes that the two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. The majority arrestin, Arr2, quenches rhodopsin signaling, while Arr1 promotes light-induced rhodopsin endocytosis. It is proposed that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin, thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration (Satoh, 2005). Light-dependent subcellular translocation of Gqalpha in Drosophila photoreceptors has been shown to be facilitated by NINAC (Cronin, 2004).
Therefore, NINAC represents the first protein required for light-dependent movement of any signaling protein in vertebrates or invertebrates. In the ninaC null mutant (ninaCP235), there was no detectable increase in rhabdomeral Arr2, even after a 1 hr exposure to light. Consistent with the requirement for NINAC for Arr2 translocation, long-term light adaptation is severely disrupted in the ninaC null mutant, ninaCP235. This defect is more pronounced than that observed in mutant flies expressing a derivative of Arr2 (Arr23K/Q), which displays a large reduction in PI binding (Lee, 2003). The stronger light adaptation phenotype in ninaCP235 than in arr23K/Q is consistent with the findings that the PI/Arr2 interaction and Arr2 translocation is reduced but not eliminated in arr23K/Q photoreceptor cells (Lee, 2004).
The current results also address a long-standing question concerning the role for the cell body-enriched isoform of NINAC p132. NINAC p174 is enriched in the rhabdomeres and is required for termination of the phototransduction. However, a role for p132 in the photoresponse had not been described, which was not surprising given that p132 is detected exclusively in the cell bodies and phototransduction takes place in the rhabdomeres. Nevertheless, while both NINAC proteins participate in the light-dependent translocation of Arr2 into the rhabdomeres, it was found that p132 is the primary isoform required. This conclusion is further supported by the long-term adaptation defect in ninaCΔ132 flies (Lee, 2004).
Both NINAC isoforms consist of linked protein kinase and myosin domains, either of which might promote Arr2 shuttling. It is suggested that the myosin domain functions in the trafficking of Arr2, since a lysine to arginine mutation in the protein kinase domain, which eliminates enzymatic activity in other protein kinases, has no impact on either Arr2 translocation or long-term adaptation. The relatively rapid movement of Arr2 into the rhabdomeres, which occurs over the course of a few minutes, suggests that the myosin motor activity may promote the movement. Human photoreceptor cells express a NINAC homolog, Myo3A, which moves toward the plus end of actin filaments. As is typical of other microvillar structures, the plus end of the filaments is oriented near the distal tips of the microvilli. By analogy to Myo3A, Drosophila NINAC also is likely to be a plus-ended myosin, which could potentially shuttle cargo from the cell bodies into the rhabdomeres. However, it was not possible to demonstrate that the NINAC motor activity per se was required for movement of Arr2; point mutations in conserved residues required for motor activity of other myosins, such as those in the actin and ATP binding sites, cause instability of NINAC in vivo (Lee, 2004).
Translocation of Arr2 has been shown to require direct interaction with PIs; however, the basis of this requirement has been unclear. In the current work, the association of Arr2 and NINAC was shown to be dependent on PIs and NINAC was shown to bind PIs. In support of this conclusion, it was found that the Arr2/NINAC interaction is disrupted by detergent and further augmented by the addition of exogenous PIs. Furthermore, NINAC binds to PIs in vitro. The interaction between NINAC and Arr2 is dependent on PIs and not on calmodulin. Calmodulin could not serve as a link between NINAC and Arr2; it was shown that Arr2 does not bind calmodulin. Although the NINAC proteins used for the biochemistry were characterized as partially purified, p132 and p174 were the only proteins clearly seen on a Coomassie-stained gel. Therefore, all other proteins were present at substoichiometric concentrations and would therefore not be effective linkers between NINAC and Arr2. In addition, p132 has only one calmodulin binding site. Consequently, the association of Arr2 to p132 immobilized on calmodulin-agarose could not have occurred through a second calmodulin bound to p132 (Lee, 2004).
It is proposed that p132 binds to PI-containing vesicles, which bind simultaneously to Arr2, facilitating the trafficking of Arr2 into the rhabdomeres. Interestingly, there is an age-dependent accumulation of vesicles in the cell bodies of ninaCΔ132, which may result from the absence of p132-dependent vesicular movement into the rhabdomeres of these mutant flies. Although the null allele, ninaCP235, displays slow termination of the light response and retinal degeneration, which could indirectly affect Arr2 movement, ninaCΔ132 flies do not undergo retinal degeneration or exhibit defects in activation or termination of the photoresponse. While it cannot be excluded that changes in calmodulin distribution associated with elimination of p132 contribute to the defect in Arr2 translocation, several observations support the conclusion that p132 functions directly in light-dependent movement of Arr2. These include the findings that p132 associates with Arr2, the demonstration that this association is dependent on PIs, and the previous report that the Arr2-PI interaction is required for translocation (Lee, 2004).
A remaining question concerns the mechanism underlying the reciprocal translocation of Arr2 from the rhabdomeres to the cell bodies in the dark. While p132 is required for the rapid light-dependent shuttling of Arr2 into the rhabdomeres, it was dispensable for the dark-associated movement of Arr2 out of the rhabdomeres. One possibility is that the transfer of Arr2 from the rhabdomeres to the cell bodies may occur via myosin VI, which is a minus end-directed motor. However, photoreceptor cell-specific expression of a myosin VI antisense transgene, which has been shown to suppress myosin VI activity in vivo, has no impact on Arr2 shuttling back to the cell bodies. Alternatively, the retrograde movement of Arr2 out of the rhabdomeres may occur by diffusion, since this movement occurs on the order of several hours, while the NINAC-dependent trafficking into the rhabdomeres is essentially complete in 10 min (Lee, 2004).
The current study raises the question as to whether light-stimulated translocation of vertebrate visual arrestin into the outer segment of photoreceptor cells occurs through interaction with a myosin or kinesin and, if so, whether the association is PI dependent. Mammalian visual arrestin binds to inositol phosphates and associates with vesicle-like structures in the inner segments. Kinesin-II is one candidate that could potentially participate in light-stimulated trafficking, because both visual arrestin and opsin accumulate in the inner segments in KIF3A knockout mice. However, it is not known if the kinesin-II participates in light-induced translocation of visual arrestin or whether the observed effect on arrestin localization is due to retinal degeneration. Unlike kinesin-II-deficient photoreceptor cells, absence of NINAC p132 in Drosophila does not lead to retinal degeneration. It is intriguing to speculate that the vertebrate Myo3A or Myo3B might function in a manner analogous to NINAC. Consistent with this possibility are the observations that the cilium connecting the inner and outer segments contains actin, in addition to tubulin. Moreover, Myo3B is expressed in the retina, and Myo3A is enriched in photoreceptor cells in addition to the cochlea. Recently, one form of nonsyndromic deafness has been attributed to mutations in human MYO3A. Whether these individuals also have a defect in long-term light adaptation is an open question, which remains to be addressed (Lee, 2004 and references).
This study examined the light-dependent subcellular translocation of the visual Gqalpha protein between the signaling compartment, the rhabdomere and the cell body in Drosophila photoreceptors. The translocation of Gqalpha was characterized, and the first evidence is provided implicating the involvement of the photoreceptor-specific myosin III NINAC in Gqalpha transport. Translocation of Gqalpha from the rhabdomere to the cell body is rapid, taking less than 5 minutes. Higher light intensities increased the quantity of Gqalpha translocated out of the rhabdomeres from 20% to 75%, consistent with a mechanism for light adaptation. Translocation of Gqalpha requires rhodopsin, but none of the known downstream phototransduction components, suggesting that the signaling pathway triggering translocation occurs upstream of Gqalpha. Finally, it was show that ninaCqalpha transport from the cell body to the rhabdomere, suggesting that NINAC might function as a light-dependent plus-end motor involved in the transport of G(q)alpha (Cronin, 2004).
This study reports the light-dependent translocation of Gqα between the rhabdomeric compartment, where phototransduction occurs, and the cell body of Drosophila photoreceptors. In dark-raised flies, Gqα is localized to the rhabdomeres; illumination triggers massive translocation of Gqα to the cell body within 5 minutes. The quantity of Gqα transported is dependent on light intensity: increasing the light intensity over five orders of magnitude leads to the translocation of ~20%-75% Gqα. Similarly, ~90% of vertebrate transducin is reported to be shuttled from the outer segment to the inner segment of rod photoreceptors under saturating light intensities. The results are consistent with Gqα translocation as a mechanism for light adaptation. Previous reports have shown that a reduction in the quantity of Gqα or the light-activated TRP channel in mutant photoreceptors results in a decrease in amplification. Light-activated translocation of the TRPL channel has also been suggested to contribute to long-term light adaptation. trpl mutants, however, do not display a complete loss of adaptation. It is suggested that the translocation of Gqα and TRPL between the rhabdomere and cell body both contribute to light adaptation in Drosophila photoreceptors (Cronin, 2004).
Genetic analyses show that Gqα translocation from the rhabdomere to the cell body requires the activation of rhodopsin to meta-rhodopsin, but not any of the known signaling components downstream of the G-protein, including PLC, TRP, TRPL, eye-PKC and Arr2. In agreement with these studies, others have shown that a constitutively activated rhodopsin leads to the persistence of non-membrane-bound Gqα and that norpA mutants display a light-dependent shift of Gqα from the membrane-associated fraction to the soluble fraction of head homogenates. In contrast to these data, however, a previous study reported a requirement for the TRP channel in Gqα translocation. The reasons for these different results are unclear. Different experimental conditions, including light intensity, illumination time or fixation procedures following illumination, might have contributed to these conflicting results. Thiss study further demonstrated that the translocation of Gqα from the cell body to the rhabdomere requires the photoconversion of meta-rhodopsin to rhodopsin (Cronin, 2004).
The signaling pathways following the light activation of rhodopsin and leading to Gqα translocation between the rhabdomere and cell body remain to be determined. Activation of Gqα might or might not be necessary for translocation. Pharmacological agents that block Gqα activation might be useful in determining whether activation of Gqα is required for translocation. One possibility is that, even without activating Gqα, meta-rhodopsin might signal some other unidentified component leading to the mobilization of Gqα. Another possibility is that activation of Gqα by rhodopsin is the trigger for translocation itself. Activated Gqα remains in the rhabdomeres to activate PLC and might then become a target for transport. Alternatively, activated Gqα might signal the translocation of inactive G-protein heterotrimers. In vertebrates and crayfish, Gβγ has been reported to translocate out of the outer segment and rhabdomeres, respectively, with illumination. The half-time to complete translocation of the transducin-α subunit from the outer to the inner segment was reported to be three times faster than that for the transducin-β subunit, suggesting that the α and βγ subunits translocate separately. In knockout mice lacking the transducin-α subunit, however, transducin-βγ was unable to redistribute to the inner segment with light, suggesting that transducin translocates as a heterotrimer. It has not yet been demonstrated whether the Gβγ subunit in Drosophila photoreceptors also moves out of the rhabdomere with illumination. If so, association with Gqα before, during and after transport will need to be assayed (Cronin, 2004).
How is Gqα transported between the rhabdomere and cell body? Three potential mechanisms are envisioned: (1) endocytosis of membrane-associated Gqα; (2) transport by a myosin; and/or (3) diffusion of free Gqα from one compartment to the other. Because Gqα returns to the rhabdomeres significantly more slowly than it leaves, translocation in each direction might involve different components and/or mechanisms (Cronin, 2004).
The results show that translocation of Gqα is unaltered in the shits1 mutant at the restrictive temperature, suggesting that shibere-mediated endocytosis is an unlikely mode of translocation. Endocytosis might instead eliminate unwanted components from the rhabdomere, because accumulated rhodopsin-arrestin complexes in norpA and rdgC mutants are removed by endocytosis (Cronin, 2004).
Another possibility for transport of Gqα is a mechanism involving myosin(s). This study shows that the photoreceptor-specific myosin NINAC is required for a normal rate of Gqα transport from the cell body to the rhabdomere. It cannot, however, be ruled that NINAC, which contains a protein-kinase domain and has been implicated as a signaling protein in phototransduction (Hofstee, 1996; Porter, 1995; Porter, 1993; Wes, 1999), is involved in signaling Gqα translocation. ninaC null mutants have also been shown to exhibit a loss of the axial cytoskeleton from rhabdomeres and undergo retinal degeneration (Hicks, 1992; Matsumoto, 1987), making it possible that slowed Gqα transport is due in part to rhabdomeric cytoskeletal degeneration. However, if slowed Gqα transport is indeed a secondary effect of retinal degeneration, we would expect the effect to be rather non-specific. Because the results show that only plus-end-directed translocation is affected in ninaC mutants, whereas minus-end-directed translocation is unaltered, it is suggested that the slowed rate of Gqα transport is a direct effect of the loss of NINAC protein. Future analyses of additional ninaC mutant alleles will determine whether NINAC does indeed function as a motor protein in Gqα transport (Cronin, 2004).
Interestingly, ninaC mutants do not display a complete blockage of Gqα translocation from the cell body to the rhabdomere. One possibility is that multiple myosins contribute to the transport of Gqα. There are 12 other myosins in Drosophila. It will be important to determine whether any of these myosins are expressed in photoreceptors. Alternatively, in the absence of NINAC, Gqα might ultimately be translocated to the rhabdomeres by another molecular mechanism, such as diffusion. In wild type, NINAC might function in concert with diffusion to increase the rate of Gqα transport. Diffusion might contribute to translocation of Gqα in either direction. For example, when meta-rhodopsin activates the Gq protein in phototransduction, the concentration of free Gqα is suddenly increased in the rhabdomeres, perhaps driving Gqα down its concentration gradient into the cell body. If diffusion drives Gqα back into the rhabdomeres with dark incubation then we expect that proteins binding to Gqα might play a role in regulating the concentration of free Gqα. It will be important to determine whether, in Drosophila photoreceptors, Gqαβγ translocates as an inactive heterotrimer, Gqα and Gβγ translocate independently or Gβγ does not translocate. Other binding proteins of Gqα and Gβγ might also play essential roles in regulating the concentration of free G-protein subunits and their direction of translocation (Cronin, 2004).
Subcellular localization of transduction proteins has proved to be crucial for signaling because mislocalization of components often results in the severe impairment of function. Dynamic regulation of protein localization might be an important strategy for controlling the quantity of transduction components available for signaling. In this way, cells can adjust their sensitivity and prevent overstimulation. The subcellular translocation of Gqα in Drosophila photoreceptors provides an attractive model for further investigation into the signaling pathway leading to translocation and the molecular mechanisms of transport (Cronin, 2004).
Many of the proteins that are critical for Drosophila phototransduction assemble into a signaling complex, signalplex, through association with the PDZ-domain protein InaD. Some of these proteins depend on InaD for proper subcellular localization to the phototransducing organelle, the rhabdomere, making it difficult to assess any physiological function of this signaling complex independent of localization. InaD binds directly to the NINAC myosin III, yet the subcellular localization of NINAC is normal in inaD mutants. Nevertheless, the InaD binding site is sufficient to target a heterologous protein to the rhabdomeres. Disruption of the NINAC/InaD interaction delays termination of the photoreceptor response. Thus one role of this signaling complex is in rapid deactivation of the photoresponse (Wes, 1999).
Several lines of evidence demonstrated that NINAC p174 interacts with INAD. NINAC p174 bound directly to INAD in an affinity column-binding assay, NINAC and INAD interacted in the yeast two-hybrid system, and NINAC and INAD co-immunoprecipitated in vivo. Furthermore, the INAD-binding site of NINAC specifically recruited an exogenous protein (β-galactosidase) into the rhabdomeres. A point mutation (I->E) disrupted the NINAC/INAD interaction in the yeast two-hybrid system and in co-immunoprecipitation experiments in vivo, and abolished the ability of the INAD-binding site to target or retain β-galactosidase in the rhabdomeres. Moreover, the β-galactosidase-p174 tail fusion was not detected in the rhabdomeres in inaD1 mutants flies (Wes, 1999).
Currently, there is no evidence that any signaling protein binds INAD for a purpose other than targeting to or retention in the rhabdomeres. TRP, PLC and PKC all seem to require INAD for localization to the rhabdomeres. Therefore, the apparent defects in activation and deactivation of phototransduction in inaD mutants could be due to reductions in the concentration of these signaling proteins within the rhabdomeres (Wes, 1999).
This study found that NINAC p174 did not require interaction with INAD for rhabdomere localization, providing a unique opportunity to assess the consequences of specifically disrupting an INAD−target protein interaction without concerns that any observed effects were due to profound mislocalization. The observation that the association of INAD and NINAC was critical for deactivation suggests that one function of the signalplex is to promote rapid deactivation. Moreover, because there were no defects in other aspects of the photoresponse, the INAD/NINAC interaction seems to function specifically in deactivation. In contrast to ninaC null mutants, which show reduced adaptation, delayed deactivation, light- and age-dependent retinal degeneration and severe alterations in the subcellular distribution of calmodulin, the only phenotype associated with P[ninaCI1501E] flies was defective deactivation. The specific features of the delayed termination resulting from perturbation of the NINAC−INAD association suggest that the defect occurs at a step in phototransduction subsequent to rhodopsin. Mutations in proteins that mediate inactivation of rhodopsin, such as arrestin, cause a prolonged depolarization under moderate light conditions, whereas no such defect occurs in P[ninaCI1501E] flies (Wes, 1999).
Interestingly, although NINAC did not require INAD for rhabdomere localization, the INAD-binding site of NINAC did possess the capacity for rhabdomere targeting and/or retention. Fusion of the INAD-binding site in p174 to β-galactosidase facilitated rhabdomere localization of the chimeric protein. It seems then, that NINAC contains more than one rhabdomere localization signal. The second localization signal may serve primarily to target NINAC p174 to the rhabdomeres, whereas the INAD-binding site may function to recruit NINAC into the signalplex, possibly in a regulated fashion. Redundant localization signals may also be present in the NMDA receptor subunit, NR2, which interacts with the PDZ protein, PSD-95. Even though PSD-95 colocalizes with NMDA receptors in excitatory synapses and is capable of clustering NMDA receptors in heterologous cells, suggesting a role of PSD-95 for receptor localization, NMDA receptors are still localized normally in vivo when the PDZ-binding domains are mutated (Wes, 1999).
The finding that NINAC p174 associated with INAD suggests that the complexity and size of the signalplex may be considerably larger than previously envisioned. Most of the abundant rhabdomeral membrane proteins bind directly to INAD. The major protein component in the rhabdomeral cytoplasm is F-actin. Because NINAC binds both INAD and actin, the actin-based cytoskeleton may be linked indirectly to the signalplex (Wes, 1999).
It is proposed that the NINAC−cytoskeletal interaction functions in termination and the INAD−NINAC association serves as a critical link between the NINAC−actin-based cytoskeleton and other signaling proteins. Consistent with a role for the actin−NINAC interaction in deactivation, it was found that deletion of the myosin domain, but not the protein kinase domain, caused a delay in deactivation similar to that in P[ninaCI1501E] photoreceptor cells. Moreover, transgenic flies expressing a derivative of NINAC containing several point mutations in the myosin domain also showed a defect in termination. Functions for actin/myosin force in negative feedback regulation of the auditory response have been proposed. Interestingly, a myosin Iβ located primarily at the tips of stereocilia has been suggested to indirectly associate with the transduction complex through an unknown rafting protein. In an analogous manner, INAD may be such a link that couples the NINAC/actin-based cytoskeleton with the signalplex (Wes, 1999).
A potential role for PDZ proteins in linking signaling molecules with the actin cytoskeleton is not unique to INAD. The mammalian PDZ-containing scaffold protein, syntrophin, binds directly to Na+ channels and to the actin-associated protein dystrophin. However, this latter interaction is not mediated by a PDZ domain. Other mammalian PDZ domain proteins, such as GRIP, bind to signaling molecules and have been proposed to anchor to the cytoskeleton. However, the functional significance of the association between PDZ-domain complexes and the actin cytoskeleton remains unclear (Wes, 1999).
In conclusion, the current data indicate that association of NINAC with INAD is critical in enhancing the speed of deactivation. It is likely that other signaling proteins, such as PLC, bind to INAD to facilitate activation, although it remains to be shown that an effect on activation occurs independent of any profound changes in spatial distribution. It is intriguing to speculate that dual functions in activation and deactivation may be common features among many macromolecular signaling complexes (Wes, 1999).
The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin-binding proteins in the retina and the NinaC-calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo-transduction. The current report presents evidence for two calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, transgenic flies were generated expressing ninaC genes deleted for either C1 or C2. It was found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction (Porter, 1995; full text of article).
Search PubMed for articles about Drosophila NinaC
Calvert, P. D., et al. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends Cell Biol. 16: 560-568. PubMed ID: 16996267
Chu, B., Liu, C. H., Sengupta, S., Gupta, A., Raghu, P. and Hardie, R. C. (2013). Common mechanisms regulating dark noise and quantum bump amplification in Drosophila photoreceptors. J. Neurophysiol. 109(8): 2044-55. PubMed ID: 23365183
Cronin, M. A., Diao, F. and Tsunoda, S. (2004). Light-dependent subcellular translocation of Gqalpha in Drosophila photoreceptors is facilitated by the photoreceptor-specific myosin III NINAC. J. Cell Sci. 117(Pt 20): 4797-806. PubMed ID: 15340015
Hardie, R. C., Satoh, A. K. and Liu, C. H. (2012). Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors. J. Neurosci. 32(27): 9205-16. PubMed ID: 22764229
Hicks, J. L. and Williams, D. S. (1992). Distribution of the myosin I-like NinaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes. J. Cell Sci. 101: 247-254. PubMed ID: 1569127
Hofstee, C. A., Henderson, S., Hardie, R. C. and Stavenga, D. G. (1996). Differential effects of NinaC proteins (p132 and p174) on light-activated currents in Drosophila photoreceptors. Vis. Neurosci. 13: 897-906. PubMed ID: 8903032
Lee, S. J., et al. (2003). Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin. Neuron 39(1): 121-32. PubMed ID: 12848937
Lee, S. J. and Montell, C. (2004). Light-dependent translocation of visual arrestin regulated by the NINAC myosin III. Neuron 43(1): 95-103. PubMed ID: 15233920
Liu, C.-H., et al. (2008). Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC Myosin III. Neuron 59: 778-789. PubMed ID: 18786361
Matsumoto, H., Isono, K., Pye, Q. and Pak, W. L. (1987). Gene encoding cytoskeletal proteins in Drosophila rhabdomeres. Proc. Natl. Acad. Sci. 84: 985-989. PubMed ID: 3103129
Porter, J. A., Yu, M., Doberstein, S. K., Pollard, T. D. and Montell, C. (1993). Dependence of calmodulin localization in the retina on the NINAC unconventional myosin. Science 262: 1038-1042. PubMed ID: 8235618
Porter, J. A., Minke, B. and Montell, C. (1995) Calmodulin binding to Drosophila NinaC required for termination of phototransduction. EMBO J. 14: 4450-4459. PubMed ID: 7556088
Satoh, A. K. and Ready, D. F. (2005). Arrestin1 mediates light-dependent rhodopsin endocytosis and cell survival. Curr. Biol. 15(19): 1722-33. PubMed ID: 16213818
Satoh, A. K., Xia, H., Yan, L., Liu, C. H., Hardie, R. C. and Ready, D. F. (2010). Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors. Neuron 67: 997-1008. PubMed ID: 20869596
Slepak, V. Z. and Hurley, J. B. (2008). Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: interaction-restricted diffusion. IUBMB Life 60: 2-9. PubMed ID: 18379987
Venkatachalam, K., et al. (2010). Dependence on a retinophilin/myosin complex for stability of PKC and INAD and termination of phototransduction. J. Neurosci. 30(34): 11337-11345. PubMed ID: 20739554
Wes, P. D., Xu, X. Z., Li, H. S., Chien, F., Doberstein, S. K. and Montell, C. (1999). Termination of phototransduction requires binding of the NINAC myosin III and the PDZ protein INAD. Nat. Neurosci. 2: 447-453. PubMed ID: 10321249
date revised: 6 August 2013
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