Using antibodies specific for the Stoned proteins, the distribution of StnA and StnB in the nervous system was examined. Both proteins are strikingly expressed at synaptic connections both in the CNS and at the neuromuscular junction in the mature embryo (20-22 hr AEL) and throughout larval development. In the third instar NMJ, both Stoned proteins are highly expressed in all synaptic bouton types, including type I, II, and III boutons. Both StnA and StnB proteins show precise colocalization with presynaptic markers, such as the synaptic vesicle-associated cysteine string protein (CSP), suggesting a presynaptic localization. Double-labeling experiments using antibodies against known postsynaptic proteins, such as the membrane-associated Discs large, show the presynaptic StnA and StnB proteins surrounded by a halo of the postsynaptic marker (Discs large), consistent with restriction of the Stoned proteins to the presynaptic region. Similar results were obtained using a different postsynaptic marker, the GluRII glutamate receptor, which also shows a halo of GluR protein expression surrounding the Stoned labeling. These results suggest that both Stoned A and B are present exclusively at the presynaptic compartment where the proteins colocalize with SV pools (Fergestad, 1999).
StnA and StnB act cooperatively to regulate synaptic vesicle recycling events in Drosophila. Both proteins localize to the presynaptic compartment and occupy common subsynaptic domains. Recent work from a variety of laboratories has precisely defined spatial and functional domains within synaptic boutons at the Drosophila NMJ. These domains include the active zone, periactive zone, membrane-associated and internal vesicular pools, and a well defined 'network' or 'lattice' domain that exclusively localizes endocytotic proteins. The endocytotic domain is of particular interest because of the hypothesis that the Stoned proteins mediate vesicular recycling. Previous studies have shown that alpha-Adaptin, a subunit of the endocytotic AP2 Clathrin-associated adapter complex, and Dynamin, the GTPase 'pinchase' mediating endocytosis, both localize to the highly characteristic lattice occupying the area surrounding the active zone domains (Gonzalez-Gaitan, 1997; Fergestad, 2001).
The localization of StnA and StnB relative to these well defined presynaptic domains has been determined. Both StnA and StnB proteins colocalize tightly with the endocytotic proteins alpha-Adaptin and Dynamin. All four proteins lie within the endocytotic lattice that surrounds but excludes the exocytotic active zones. Like Dynamin, both StnA and StnB are tightly associated with the plasma membrane and do not occupy cytosolic domains in the bouton interior. Markers of the active zone and vesicular pools, such as the SV-associated Cysteine String Protein (Csp), do not colocalize with the Stoned proteins but rather occupy the domains within the endocytotic lattice. This confocal analysis supports the localization of both StnA and StnB proteins with the endocytotic network and not with SV pools and areas of exocytosis (Fergestad, 2001).
The shibireTS1 mutation disrupts Dynamin function and provides a temperature-dependent block in the vesicle-budding step of endocytosis. Stimulation of the Drosophila NMJ in shibireTS1 mutants at the restrictive temperature (30°C) depletes the SV population because SVs are driven into the plasma membrane in the absence of endocytosis. Immunological staining of these vesicle-depleted shibireTS1 terminals shows that SV markers, such as Csp, become associated exclusively with the plasma membrane. shibireTS1 SV-depleted terminals were labeled with antibodies against StnA, StnB, and alpha-Adaptin. No alteration in the endocytotic network, including the distribution of the Stoned proteins, was observed. These studies confirm that both StnA and StnB are associated with the plasma membrane and do not associate with internal vesicles. Returning SV-depleted shibire TS1 terminals to the nonrestrictive temperature (22°C) allows endocytosis to resume, resulting in mass membrane retrieval from the plasma membrane. After SV depletion (30°C for 10 min) and brief recovery to permit massed endocytosis (22°C for 10 min), no detectable alteration in the expression pattern of the endocytotic proteins, including both StnA and StnB is observed. These data support the conclusion that the Stoned proteins occupy only the endocytotic domain within synaptic boutons and are tightly associated with the plasma membrane (Fergestad, 2001).
There is a prominent mislocalization of Synaptotagmin I protein in stoned mutants; stoned function is required to maintain Synaptotagmin I in tight synaptic bouton domains and to prevent its loss throughout the arbor and proximal regions of the axons and its eventual degradation. It was of interest to determine whether this relationship is reciprocal by testing whether the Stoned proteins are mislocalized and/or degraded in the absence of Synaptotagmin. Immunohistochemical studies in sytAD4, a null allele of synaptotagmin, have revealed no detectable alteration in either StnA or StnB expression at the embryonic NMJ. Both Stoned proteins are maintained in tight bouton puncta in the complete absence of Synaptotagmin. These data suggest that the Stoned proteins are specifically required for the recycling of Synaptotagmin but do not require Synaptotagmin for their localization within the endocytotic domains. Furthermore, these data suggest that the phenotypes observed in synaptotagmin mutants do not result from aberrant localization of the Stoned proteins (Fergestad, 2001).
stoned mutants exhibit impaired synaptic transmission and a reduced number of morphologically abnormal SVs, suggesting a defect in vesicular recycling at the synapse. To assay for SV recycling defects directly, the fluorescent lipophilic dye FM1-43 was used in membrane retrieval and SV-recycling assays at the NMJ. Extracellularly applied FM1-43 dye is incorporated into SVs after endocytosis, reliably maintained in SVs and vesicular intermediates, and released during stimulated exocytosis. Incubating Drosophila larval NMJ preparations with FM1-43 in a depolarizing solution of high K+ (90 mM) results in specific dye incorporation in SVs of the presynaptic boutons that can be released after subsequent depolarization and fusion. This analysis was used, in various experimental paradigms, to assay endocytosis and SV recycling in stoned mutant NMJs (Fergestad, 2001).
Wild-type and stoned mutant third instar animals were dissected in the same chamber and stimulated identically with 5 min of high-K+ saline in the presence of FM1-43. Control boutons revealed robust endocytosis and strongly incorporated dye, whereas stoned mutant boutons loaded dye very poorly under the same conditions. The mean density of FM1-43 incorporation in larval NMJ boutons (>5 µm) was quantified and normalized to that of the control. The viable stnC mutant animals display a significant impairment in dye uptake. Examination of two lethal stoned alleles, stn13-120 and stnPH1, reveals an even more profound defect in endocytosis. Because severe stoned mutations are all embryonic lethal, normalized comparisons of FM1-43 dye uptake to control were done at the embryonic NMJ. Wild-type embryonic NMJs can be loaded with FM1-43 by high-K+ depolarization, and all synaptic boutons appear to label, generating a signal comparable with antibody staining against SV proteins at the same stage (21-23 hr AF). In sharp contrast, both lethal stoned alleles show a >90% reduction in dye incorporation, making measurable endocytosis essentially undetectable. These studies show that three different alleles of stoned all show severe defects in exo-endo SV recycling at the NMJ synapse and that the severity of the recycling defect correlates with the severity of the mutant allele examined (Fergestad, 2001).
Although such a dye uptake impairment implies a direct defect in membrane retrieval, the possibility that the reduced exocytosis observed in stoned mutants causes a coupled reduction in endocytosis cannot be excluded. To address this possibility, spaced durations of depolarization stimulation and FM1-43 dye loading were studied. Brief dye loading with high K+ for <1 min (30 sec and 1 min intervals assayed) resulted in a similar defect in stoned dye loading. Dye loading with 5 min of stimulation provided a slight increase in bouton fluorescence intensity in both control and mutant NMJ terminals, but the stoned-specific defect in endocytosis remained unaltered. Furthermore, 10 min of high-K+ application and dye labeling resulted in no further increase in synaptic dye incorporation of either control or stoned mutant animals, suggesting that the cycling SV pool is maximally saturated after <5 min of high-K+ stimulation. Similarly, to determine whether the striking defects in endocytosis in the embryonic lethal mutants were caused by delayed endocytosis, dye was applied for 5 min in calcium-free saline after 5 min of high-K+ stimulation with dye. Longer periods of dye application did not improve the FM1-43 bouton labeling in either wild-type controls or stoned mutants. These studies suggest that the recycling SV pool is saturated at these loading times and that stoned mutants have a specific and severe reduction in SV endocytosis. These findings show that the defect in dye uptake is independent of the stimulation duration and probably results from a smaller recycling pool of SVs in stoned mutants (Fergestad, 2001).
Large NMJ boutons (>3 µm, typically 3-5 µm) in normal Drosophila third instar larvae show characteristic patterns of SV pools. Wild-type boutons loaded with high K+ always incorporate FM1-43 dye in a circular pattern, with the fluorescence restricted to cortical regions underlying the plasma membrane and an absence of signal in the central regions of the bouton. This dye incorporation shows that the recycling SV pool filled via high-K+ stimulation is spatially restricted in a characteristic peripheral ring. In contrast, the bouton interior contains a reserve pool of SVs that are accessed only under conditions of intense transmission demand. The reserve pool can only be loaded with FM1-43 after high-frequency (>30 Hz) stimulation or after complete elimination and mass renewal of the SV population with the Dynamin mutant shibireTS1. It was of interest to determine whether the ready/reserve SV pool boundary is maintained in stoned mutants and whether Stoned proteins may play a role in the spatial dynamics of SV recycling (Fergestad, 2001).
In clear contrast to the normal condition, standard high-K+ FM1-43 labeling in stnC boutons results in the dye filling the entire bouton, including the center of the bouton where reserve pool vesicles are normally located. Recycled vesicles in stoned mutants lack the normal spatial restriction defining the readily releasable and reserve SV pools. This spatial distribution pattern is reminiscent of the FM1-43 signal after dye uptake in the Dynamin mutant shibireTS1. After temperature-dependent depletion of all SVs, shibireTS1 animals return to the permissive temperature undergo mass membrane retrieval coinciding with the formation of early endosomes/cisternae and repopulation of the entire bouton with SVs. shibireTS1 SV dynamics were assayed by first loading the NMJ terminal at the permissive temperature (22°C) and then reloading the same terminal after temperature-dependent (30°C) depletion of all SVs. Before unloading, the shibireTS1 boutons display a labeled circular pool of SVs corresponding to the readily releasable SV pool identical to that of wild-type controls. However, after total SV depletion, shibireTS1 terminals load both ready and internal reserve pools comparable with the pattern observed in stoned mutants. Thus, stoned mutants display aberrant trafficking of newly endocytosed membrane, which may be inappropriately targeted into sorting endosomes in the bouton interior. This conclusion is consistent with the significantly increased incidence of enlarged vesicles and multivesicular bodies observed in stoned mutants at the EM level. This conclusion also supports the hypothesis that loss of stoned function results in increased segregation of membrane and/or protein to the sorting and degradation pathways, at the expense of the recycling SV pool (Fergestad, 2001).
Application of high-K+ saline to FM1-43-labeled synaptic boutons results in a second round of exocytosis that releases the dye contained within the SVs (unloading). Terminals loaded with FM1-43 for 5 min release most of this dye via the fast-cycling SV pool after a comparable 5 min unloading period. Under conditions of equal loading and unloading periods, the majority of dye in both wild-type (85.5 ± 1.0%) and stnC (83.7 ± 5.3%) NMJ synapses is released via Ca2+-dependent exocytosis. In contrast, shortening unloading times to 1 min of high-K+ saline application is still sufficient to unload the majority of dye in wild-type terminals (88.1 ± 1.8%), but the amount of dye released from stoned boutons is significantly reduced. These findings confirm that the readily releasable pool is smaller in stoned mutants, and although these vesicles are competent to fuse, they do so in a slower time course. The impairment of dye release is consistent with the defect in exocytosis observed in stoned mutants and may result from the aberrant vesicle trafficking observed in stoned terminals. These data further suggest that the aberrantly distributed SVs in stnC mutants are releasable and do not have the 'barrier' thought to spatially separate the reserve and ready SV pools (Fergestad, 2001).
Previously characterized defects in exocytosis and endocytosis have suggested that SV maturation in stoned mutants may be impaired. Elegant studies on rat hippocampal cultures have recently estimated the time course for SV maturation ('repriming') to be from 5 to 40 sec. To test whether the delay period from endocytosis to exocytosis is increased in stoned mutants, the lapsed time required before loaded dye could be released from NMJ boutons was examined. FM1-43 dye was loaded with high-K+ saline (30 sec and 1 and 5 min), and then the preparation was washed in calcium-free saline for a variable period before K+-evoked unloading. No significant change between controls and stoned mutant boutons in the amounts of FM1-43 release was detected in these assays. These studies suggest that although fewer SVs are recycled via the endo-exo pool in stoned mutants, no difference in the rate of SV maturation is detectable (Fergestad, 2001).
Time-lapse studies using FM1-43 dye uptake assays indicate the rates of membrane retrieval after the fusion event to be t1/2 of ~20 sec or even faster. To determine whether endocytosis is delayed after exocytosis in stoned mutants FM1-43 was applied either during a 30 sec high-K+ stimulus or for 30 sec immediately after the stimulus. In wild-type NMJ terminals, a 30 sec application of high-K+ saline with FM1-43 loads synaptic terminals to levels similar to those of longer loading times, indicating that endocytosis is tightly temporally coupled to exocytosis. FM1-43 application for 30 sec immediately after the stimulation results in much lower levels of dye uptake, indicating that reduced endocytosis continues after the stimulation period. In contrast, stoned mutant boutons display greatly reduced endocytosis during the initial time period, when exocytosis and endocytosis levels are normally tightly coupled, and substantial levels of dye uptake only after the depolarizing stimulation. The striking dye uptake difference normally seen between stoned mutants and control animals is no longer present when the dye is added to the preparation after a 30 sec delay. Because longer dye application times do not allow complete loading, the delay in loading the stoned SV pool cannot alone account for the decreases in overall dye uptake. Thus, stoned mutants show both a significantly delayed onset of endocytosis and a significantly smaller recycling SV pool (Fergestad, 2001).
The Stoned proteins and Synaptotagmin specifically interact in the presynaptic terminal. Both StnA and StnB have been shown to bind Synaptotagmin directly, and Synaptotagmin is specifically mislocalized and subsequently degraded in stoned mutants. Moreover, the stoned and synaptotagmin mutant phenotypes are strikingly similar; both show comparably decreased and nonsynchronous synaptic transmission, decreased synaptic vesicle density, and aberrant, enlarged synaptic vesicles. One hypothesis to explain these diverse findings is that the Stoned proteins and Synaptotagmin mediate the same endocytotic function and that Stoned is required to recruit and/or maintain Synaptotagmin during plasma membrane endocytosis (Fergestad, 2001).
A key prediction of this hypothesis is that elevated levels of Synaptotagmin should alleviate the severe phenotypes observed in stoned mutants. To test this hypothesis, neurally expressing GAL4 drivers were used to mediate expression of a UAS-Synaptotagmin transgene construct, thus elevating Synaptotagmin levels in synaptic boutons. Whether overexpression of Synaptotagmin in the embryonic lethal stoned mutant background would rescue viability was first tested. Homozygous lethal stn13-120 animals, containing the UAS-Synaptotagmin construct alone, remain embryonic lethal in the absence of a GAL4 driver. However, two temporally different neural GAL4 drivers both rescue the embryonic lethality of stn13-120 in a manner consistent with the onset of their expression. (1) The 1407-GAL4 driver expresses throughout the nervous system during embryogenesis but ceases expression after hatching. When the 1407 driver is crossed to UAS-Synaptotagmin; stn13-120 animals, mutant animals now hatch (~98%) at normal times but then proceed to die as L1 stage larva, consistent with the termination of the 1407 expression. (2) The 4G-GAL4 driver is expressed during later stages of embryogenesis but then remains expressed in the nervous system throughout the life of the animal. When driving Synaptotagmin expression in stn13-120 mutants with the 4G driver, embryos also now hatch (~96%), although this hatching is delayed (mutant animals now hatch between 21 and 35 hr AF), consistent with the later onset of expression. Furthermore, maintained Synaptotagmin overexpression with 4G now rescues the embryonic lethal allele stn13-120 to adult viability. These results show that elevated Synaptotagmin can rescue the stoned lethality and that persistent elevated Synaptotagmin is required to compensate for the loss of Stoned during maintained synaptic function (Fergestad, 2001).
The prediction from these studies is that elevated levels of Synaptotagmin can alleviate the endocytosis defects caused by stoned mutation. To test this prediction, FM1-43 dye uptake was assayed at the larval NMJ. Overexpression of Synaptotagmin in stnC mutants rescues the endocytotic functional defects observed in stnC. UAS-Synaptotagmin; stnC larvae without a GAL4 driver are similar to stnC mutants alone and no rescue of the dye uptake defect is seen. Strikingly, however, the introduction of the 4G-GAL4 driver almost completely rescues the defects in dye uptake; no longer significantly different from control. Interestingly, the overexpression of Synaptotagmin alone in a control background (4G/UAS-Synaptotagmin) shows a striking increase in the amount of dye loaded, as compared with that of controls. These findings show that the stoned mutant phenotypes can be directly rescued by elevation of Synaptotagmin levels in the presynaptic terminal. The similarity of the stoned and synaptotagmin mutant phenotypes and the data presented here suggest that the sole role for the Stoned proteins may be to maintain the presynaptic function of Synaptotagmin (Fergestad, 2001).
At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and StonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Possible roles of the stoned proteins in synaptic vesicle internalization were tested via analyses of third instar larval neuromuscular synapses in two Drosophila stoned mutants: stnts and stn8P1. Both mutations reduce presynaptic levels of StonedA and StonedB, although stnts has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn8P1 mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, new methods were implemented to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stnts, an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shits mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stnts is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which StonedA and StonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane (Stimson, 2001).
Both stoned proteins are enriched in presynaptic terminals of the embryonic and larval NMJ. The effects of stnts and stn8P1 on stonedA and stonedB levels were examined at the larval motor terminal. The stnts mutation is predicted to cause only a single amino acid change in stonedA. This mutation does not alter presynaptic levels of stonedA consistently, although reduced levels are frequently observed. This variability is seen among synapses in a particular preparation, not only between individual larvae. Surprisingly, even when stonedA levels appear relatively normal, stnts causes a marked reduction in stonedB levels. This observation suggests the intriguing possibility that an ORF1 mutation alters stonedB levels because stonedA has a function that affects the stability of stonedB at nerve terminals (Stimson, 2001).
The stn8P1 mutation has striking effects on the levels of both Stoned proteins. The stn8P1 allele is semi-lethal, and adult stn8P1 males survive at a frequency of ~1% relative to their control siblings. These survivors are extremely lethargic and can be immobilized for 1-2 min by mechanical disturbances (such as tapping or shaking the vial). Viability and behavior of stn8P1 males are not complemented by stoned lethal alleles (such as stn13-120) but are restored to wild-type by Dp(1,Y)y+Ymal+ (Dp), a modified Y chromosome containing region 20 of the X chromosome, which includes stoned. Both stonedA and stonedB are reduced to undetectable levels in the stn8P1 mutant, an observation consistent with the severe effects of stn8P1 on viability and behavior. Because the stn8P1 mutation removes all detectable stonedA and stonedB immunoreactivity from the larval NMJ, it was anticipated that analysis of stn8P1 mutants would allow the effects of nearly complete stoned loss-of-function in the third instar larval motor synapse to be assessed. This preparation has some advantages over the embryonic synapse in which the effects of other stn lethals have been analyzed previously (Stimson, 2001).
To determine the effects of stn8P1 on synaptic vesicle cycling, the efficacy of synaptic transmission at the larval NMJ was first assessed by performing intracellular recordings from postsynaptic muscle. The stnts mutant exhibits a threefold increase in the frequency of miniature excitatory junctional potentials (mejps), indicating an enhanced rate of spontaneous synaptic vesicle fusions. However, mejp frequency in stn8P1 is nearly identical to that of controls. This observation probably is explained by reduced vesicle number and altered ultrastructure of mutant presynaptic terminals (Stimson, 2001).
Excitatory junctional potentials (EJPs) evoked by stimulation of the motor nerve, normal in stnts, are reduced in stn8P1 mutants to ~10% of wild-type and stn8P1/Dp controls. This decreased EJP amplitude derives from a severe reduction in quantal content, the number of synaptic vesicles fusing during a single evoked event. Quantal content was calculated by dividing the EJP amplitude (corrected for nonlinear summation of individual quanta). Quantal content is only 4.7 ± 0.7 in stn8P1 mutants as compared with 133.9 ± 16.1 in stn8P1/Dp controls. Thus, compared with other stn mutants that survive to the larval third instar, stimulus-evoked synaptic vesicle fusion is limited severely at stn8P1 neuromuscular synapses (Stimson, 2001).
Previous studies have indicated that altered neurotransmitter release in stoned mutants probably arises from a depletion of functional synaptic vesicles. In stoned lethal mutants the boutons at the embryonic NMJ contain a relatively low density of synaptic vesicles, and many of these are morphologically abnormal. However, viable stnts mutants with normal evoked release show no decrease in synaptic vesicle density and no change in synaptic vesicle size at the larval NMJ (Stimson, 2001).
In stn8P1 mutants, the boutons of the larval NMJ exhibit a 2.6-fold decrease in synaptic vesicle density as compared with controls. Vesicles in stn8P1 boutons are larger and more irregular in size than controls. Vesicles in stn8P1 average 44.8 ± 1.1 nm in diameter as compared with 34.4 ± 0.6 nm in stn8P1/Dp controls, corresponding to approximately twofold increases in mean vesicle volume and in vesicle size variability. Despite evidence from other studies that mejp amplitude often is correlated with vesicle size, no increase in the average mejp amplitude or in the distribution of mejp amplitudes is found in stn8P1 mutants. This could indicate that the large vesicles of stn8P1 are incompetent for fusion at the mature larval synapse. These morphological observations at larval motor terminals corroborate previous studies performed at the embryonic motor synapse. They are consistent with stoned proteins being essential for the formation of synaptic vesicles either during biogenesis or during recycling from the plasma membrane (Stimson, 2001).
Light microscopic analysis of the distribution of synaptic vesicle proteins at embryonic motor terminals indicates that stoned lethal mutations specifically alter the distribution of Synaptotagmin (Syt). This suggests an intriguing hypothesis that Stoned proteins act as specific adaptors for Synaptotagmin. However, a preliminary examination of stonedts and stonedc mutants reported that both Synaptotagmin and Csp show altered distribution at the larval motor terminal. A weakness of this study was that Synaptotagmin and Csp distributions were not compared within the same terminal. Because a mechanistic hypothesis for how Stoned proteins function depends significantly on establishing how stoned mutations affect different vesicle proteins, this issue was reexamined in double-immunostained preparations. The large size of larval motor terminals permits substantial detail to be resolved by optical microscopy; specifically, plasma membrane and bouton interior may be discriminated clearly (Stimson, 2001).
In wild-type and control boutons, Synaptotagmin and Csp are restricted to doughnut-shaped patterns surrounded by plasma membrane. However, in stoned8P1 mutants, Synaptotagmin and Csp immunoreactivity is present diffusely over the boutons, colocalizes in the bouton periphery with plasma membrane staining, and invades interbouton regions of the motor terminal that usually are completely free of synaptic vesicle protein. Similar, although less pronounced, redistribution of Synaptotagmin and Csp is also seen in double-stained stonedts boutons. The observed distribution of these proteins in stoned mutants is consistent with increased retention of Synaptotagmin and Csp on presynaptic plasma membrane; the complex distribution pattern likely results from inefficient internalization and lateral movement of synaptic vesicle proteins along the axonal membrane. A particularly interesting observation is that, although both Csp and Synaptotagmin are enriched on the plasma membrane of stoned mutants, Synaptotagmin shows much stronger immunoreactivity in the interbouton intervals than Csp. It is conceivable that the lateral movement of Csp away from boutons is restricted by physical interactions with presynaptic Ca2+ channels or other membrane proteins anchored within boutons. The significant redistribution of both Csp and Synaptotagmin to the plasma membrane of larval motor terminals suggests that Stoned proteins facilitate the sorting and assembly of at least two synaptic vesicle proteins into functionally mature synaptic vesicles. An immediate, rather than indirect, role for Stoned proteins in these processes is argued by immunolocalization studies. Three known components of endocytosis -- alpha-adaptin, dynamin, and clathrin heavy chain -- are not reduced significantly in levels or altered in distribution in stonedts or stoned8P1 mutant motor terminals. Thus, Stoned proteins function downstream of the events required for expression and correct targeting of these endocytosis molecules, perhaps in the internalization process itself (Stimson, 2001).
Phenotypes of stoned mutants could arise formally from defective synaptic vesicle biogenesis at the cell body. Abnormally sized vesicles might have arisen easily from the Golgi complex, and not from plasma membrane. Altered sorting, budding, and transport of synaptic vesicle components from the Golgi complex also could cause synaptic vesicle proteins to be targeted to the plasma membrane by a default sorting pathway. Abnormal fusion of presynaptic vesicles also might be a source for large presynaptic vesicles. To establish more firmly a role for stoned proteins in vesicle recycling from the plasma membrane, a detailed analysis of phenotypes more directly associated with endocytosis at nerve terminals was performed (Stimson, 2001).
Genetic interactions of stonedts with shits mutations, that disrupt synaptic vesicle recycling, were further explored. Specifically, the effects of stonedts on rapid temperature-sensitive paralysis of shits, a phenotype believed to reflect synaptic failure directly, were examined. Although shits1-stonedts double mutants are lethal, combining stonedts with shi alleles weaker than shits1, including shits2 and shits4, produces viable shits-stonedts double mutants. Wild-type flies do not paralyze at sublethal temperatures (<42°C). Mutant stonedts flies are sluggish but do not show temperature-sensitive paralytic behavior. In contrast, shits2 and shits4 flies show tight and complete paralysis in 2 min at 28 and 29°C, respectively. Double mutant shits2-stonedts and shits4-stonedts flies undergo paralysis at 26°C, a temperature 2-3°C below the restrictive temperature for shits alone. The observation that stonedts lowers the temperature required to induce paralysis of shits mutants adds to the previous discovery of synthetic lethality between stonedts and shits1. It suggests that stonedts aggravates synaptic transmission defects in shits mutants rather than defects in the various nonsynaptic functions of shi. The specificity of the shits-stonedts genetic interactions is emphasized by control double mutant studies that show the absence of any interaction of stonedts with parats1 and comatosetp7 (comttp7), temperature-sensitive paralytic mutants defective for action potential propagation and synaptic vesicle fusion respectively. In addition, comtts alleles have no effect on the temperature of paralysis of shits (Stimson, 2001).
To confirm the cell biological interpretation of the behavioral interactions, the effects of stonedts on synaptic vesicle recycling in shits mutants were directly assessed. In shits mutants the physiological consequence of synaptic vesicle depletion is synaptic depression, an activity-dependent decline in quantal content over time. It was expected that, if stonedts inhibits synaptic vesicle recycling, it should enhance synaptic depression caused by a partial inhibition of recycling in shits2 mutants. These depression experiments are uniquely possible in shits-stonedts double mutants because, unlike all other characterized stoned alleles, stonedts does not alter EJP amplitude or vesicle number. Thus, effects on vesicle recycling may be assayed without confounding effects from altered vesicle number or probability of release (Stimson, 2001).
Because shits mutations have obvious effects on the behavior of adult flies, the effects of shits on synaptic physiology have been investigated most extensively at an adult fly NMJ on the dorsal longitudinal flight muscles (DLMs). To investigate the effects of stonedts on shits2 depression, conditions for inducing depression at the larval NMJ of shits2 mutants were optimized. With 10 Hz stimulation at 28°C the shits2 larval NMJ shows only a slight depression relative to the wild-type NMJ. Raising the temperature to 30°C causes a sharp distinction to emerge between shits2 and wild type. At 30°C, 10 Hz stimulation of the shits2 larval NMJ causes the EJP to decline from ~31 mV (~145 quanta) to ~9 mV (~30 quanta) after ~9 min. In contrast, this 2° temperature change has no effect on the wild-type NMJ, which continues to show relatively robust synaptic transmission at 30°C. Thus, these experiments establish that, at the larval NMJ, the shits2 mutation causes a weak inhibition of synaptic vesicle recycling at 28°C, but a strong inhibition at 30°C (Stimson, 2001).
Identical experiments performed on stonedts mutants provide only tentative support for the proposal that stonedts affects synaptic vesicle recycling. Like shits2, stonedts alone at 28°C causes a marginal increase in the rate of synaptic depression, as compared with wild type. However, unlike the case for shits2, the effect of stonedts on depression remains slight even at 30°C. Thus, in isolation, stonedts shows only a marginal temperature-insensitive effect on synaptic depression; this observation is consistent with a model in which stonedts causes a small reduction in the rate of synaptic vesicle recycling at larval NMJs (Stimson, 2001).
Whether this slight reduction in recycling rate would be made more obvious in a 'sensitized' shits background was investigated under conditions in which the vesicle recycling is slowed down already. Such analyses comparing shits2-stonedts double mutants with shits2 show that the stonedts mutation has obvious effects on vesicle depletion in response to 10 Hz stimulation. At 28°C, stonedts causes a marked enhancement of the weak depression produced by shits2 alone, an effect that parallels the enhancement of shits2 paralysis by stonedts. Genetic control experiments show that both effects are caused specifically by stonedts, and not by extragenic modifiers; thus, shits2 stonedts;shits2 stoned13-120 mutants show depression and paralysis profiles identical to those of shits2-stonedts. In principle, enhanced vesicle depletion may be caused by a smaller initial vesicle pool size (being depleted more quickly) or by slower vesicle recycling. Analyses performed at 30°C distinguish between these possibilities and show that stonedts slows down vesicle recycling. If stonedts accelerates synaptic vesicle depletion by limiting the initial pool of releasable synaptic vesicles, then even at 30°C, where shits2 strongly inhibits synaptic vesicle recycling, enhanced depression in shits2-stonedts would be expected. On the contrary, at 30°C, stonedts has no detectable effect on the rate of shits2 depression, probably because the strong effects of shits2 on synaptic vesicle recycling mask more subtle effects of stonedts. Because stonedts alone shows marginal temperature-independent depression, the best explanation for enhanced temperature-dependent depression in stonedts-shits2 double mutants is that Stoned proteins facilitate synaptic vesicle recycling. This role is made visible by an analysis of stoned function under sensitized conditions in which the recycling rate limits the efficiency of sustained transmitter release (Stimson, 2001).
Attempts were made to determine the specific stage of synaptic vesicle recycling that is affected by stnts. Because sequence analysis and genetic interactions with shi suggest that Stoned proteins act during membrane internalization, an assay was implemented that uses the fluorescent lipophilic dye FM1-43 to monitor the rate of synaptic vesicle internalization optically. Because FM1-43 has a weak affinity for lipid membranes, bath-applied FM1-43 associates with the exposed lumenal surfaces of vesicles and becomes internalized into synaptic terminals during endocytosis. After washing away noninternalized plasma membrane-associated FM1-43, the fluorescence intensity of internalized FM1-43 can be used to quantify the amount of endocytosis (Stimson, 2001).
To induce large-scale synaptic vesicle endocytosis, the larval NMJ was subjected to a 30 sec, 30 Hz stimulation 'buzz'. To measure endocytosis, FM1-43 was applied either just before the buzz or at incremental time points after the buzz and then at least 5 min was allowed for endocytosis to run to completion before washing away noninternalized FM1-43. Adding FM1-43 just before the stimulation labels those synaptic vesicles that have been released and recycled consequent to the stimulation ('max' staining). FM1-43 added after the stimulation labels only those synaptic vesicles that recycle relatively slowly from the plasma membrane, whereas vesicles that have internalized before the dye application escape labeling. By making quantitative fluorescence measurements at each time point, this assay can be used to determine the rate of synaptic vesicle endocytosis. The assay shows that synaptic vesicle internalization in stonedts mutants is delayed relative to wild type. In stonedts boutons, intense FM1-43 uptake persists after the 30 Hz stimulation has ended, whereas in wild-type boutons FM1-43 uptake rapidly wanes after stimulation. Normalized fluorescence intensities show that, whereas only ~40% of vesicle membrane in wild-type boutons remains to be internalized after stimulation, >60% of vesicle membrane in stonedts boutons is internalized after stimulation. At 1 min after stimulation, in which the FM1-43 uptake is barely detected in wild-type boutons, the difference between wild type and stonedts is especially pronounced. This phenomenon, obvious in stonedts, is even stronger in stonedts/stn13-120 heterozygotes, indicating that delayed vesicle internalization is caused by a mutation in stoned. Delayed vesicle internalization in the stonedts mutant indicates that Stoned proteins facilitate synaptic vesicle recycling by promoting endocytosis from the presynaptic plasma membrane (Stimson, 2001).
Although unrelated by sequence, the Drosophila stoned proteins are translated from a single dicistronic mRNA, transcribed under the control of a single genetic promoter (Andrews, 1996). This arrangement resembles the polycistronic RNAs commonly found in prokaryotes, which are known to facilitate the coexpression of gene products that act in a common pathway. Simply based on the molecular organization of stoned, it is a logical extension that StonedA and StonedB proteins are expressed coordinately because they share some overall function. A more speculative idea is that coordinate expression of StonedA and StonedB promotes physical interaction between them by increasing their local concentrations (Stimson, 2001).
Analysis of StonedA and StonedB immunoreactivity in stoned mutants provides some supportive evidence for both of these possibilities. stoned alleles either reduce levels of presynaptic StonedA and StonedB or else carry specific lesions in ORF2. These studies have suggested that mutations in ORF1 interfere with the translation of StonedA and StonedB (Fergestad, 1999). Contrary to this suggestion, it has been found that stonedts, a missense mutation in ORF1, severely reduces StonedB levels even when StonedA levels are only marginally affected, as judged by immunofluorescence analysis. Thus, stonedts appears to have a primary effect on StonedA function, not expression, and a secondary effect on the presence of StonedB in presynaptic boutons. This suggests that mutations of StonedA alter the abundance of StonedB because StonedA protein regulates the transport and/or stability of StonedB within presynaptic terminals. The interdependence of StonedA and StonedB observed in vivo reinforces the notion that StonedA and StonedB share common functions. Such a model is supported by sequence analysis, indicating the presence in StonedA of µ-adaptin binding sequences and in StonedB of a µ-adaptin homology domain as well as PEST sequences that target proteins for turnover in the absence of protective interactions. Recent biochemical studies also provide some support for a model in which StonedA and StonedB associate in a single macromolecular complex at some stage of synaptic vesicle traffic (Stimson, 2001).
In the context of sequence motifs present in Stoned proteins, phenotypes of stoned mutants, combined with the enrichment of StonedA and StonedB in presynaptic boutons, specifically suggest that the stoned proteins regulate the recycling of synaptic vesicles. Previously described stoned phenotypes, namely the mislocalization of synaptic vesicle proteins as well as the enlargement and depletion of synaptic vesicles, provide strong support for the proposal that StonedA and StonedB promote synaptic vesicle recycling. Similar phenotypes have been observed in the Drosophila and Caenorhabditis elegans mutant for AP180, an adaptor protein that regulates the assembly of clathrin cages and colocalizes with clathrin on budding vesicles. Although these data are consistent with a role for Stoned in regulating recycling, the data fall short of demonstrating such a function. Given the relative paucity of information on the biochemical activities of Stoned proteins, direct data are especially important to support the hypothesis that the proteins regulate vesicle formation (Stimson, 2001).
shits mutations were used as tools to probe the specific effects of the stonedts mutation on synaptic vesicle recycling. This analysis shows that stonedts can enhance paralysis (and the underlying synaptic depression) caused by shits inhibition of synaptic vesicle recycling. Further studies under conditions in which the recycling is blocked almost completely exclude the formal possibility that stonedts accelerates synaptic vesicle depletion by reducing the size of the initial vesicle pool. Thus, stonedts enhancement of shits depression, detectable when the inhibitory effects of shits are weak, is not apparent when the effects are strong. This constitutes the first direct evidence that Stoned proteins modulate synaptic vesicle recycling (Stimson, 2001).
Interpreted from a genetic standpoint, the finding that shits can mask the effect of stonedts (i.e., shits is epistatic to stonedts) suggests that the Stoned proteins function in the same cellular pathway as Dynamin, probably as novel components of endocytic vesicle formation (Stimson, 2001).
Previous studies have suggested a model in which Stoned proteins selectively recruit Synaptotagmin into synaptic vesicles either during endocytosis or during subsequent unidentified trafficking events in synaptic vesicle recycling (Fergestad, 1999). The findings that stoned mutations slow the internalization of synaptic vesicle membrane and disrupt the retrieval of at least two synaptic vesicle proteins allow this model of Stoned function to be refined. In this revised model it is suggested that Stoned proteins are novel components of endocytosis that promote the recovery of synaptic vesicle membrane and proteins from the presynaptic plasma membrane. In support of this, both StonedA and StonedB bind Synaptotagmin in vitro (Phillips, 2000); StonedA contains consensus binding sites for alpha-adaptin (Stimson, 1998, and StonedB contains consensus binding sites for Eps15 (Salcini, 1997). Although the biochemical properties of StonedA and StonedB are not firmly established, new analyses presented here show that Stoned proteins have the functional characteristics expected of molecules involved in synaptic vesicle internalization. Together, the available data suggest a model in which stoned proteins physically link synaptic vesicle proteins with components of the clathrin-associated endocytosis machinery during synaptic vesicle reformation (Stimson, 2001).
Dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. A highly specific synaptic mislocalization and degradation of Synaptotagmin I has been documented stoned mutants. Transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. It is concluded that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane (Fergestad, 2001).
StonedA protein is highly enriched at Drosophila nerve terminals. Mutant alleles that affect StonedA disrupt the normal regulation of synaptic vesicle exocytosis at neuromuscular synapses of Drosophila. Spontaneous neurotransmitter release is enhanced dramatically, and evoked release is reduced substantially in such stoned mutants. Ultrastructural studies reveal no evidence of major disorganization at stoned mutant nerve terminals. Thus, a direct role for StonedA in regulating synaptic vesicle exocytosis is indicated. However, genetic and morphological observations suggest additional, subtle effects of stoned mutations on synaptic vesicle recycling. Remarkably, almost all phenotypes of stoned mutants are similar to those previously described for mutants of Synaptotagmin, a protein postulated to regulate both exocytosis and the recycling of synaptic vesicles (Stinson, 1998).
The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mosaic analysis of a stoned lethal allele suggests that stoned functions either in the nervous system or in both the nervous system and musculature, but is not required for gross neural development. The behavioral alleles stnts and stnC appear to be defective in a diametrically opposite sense, show interallelic complementation, and indicate distinct roles for the stoned gene product in the visual system and in motor coordination. A number of other neurological mutations have been investigated for their possible interaction with the viable stoned alleles. Mutations at two loci, dunce and shibire, act synergistically with the stnts mutations to cause lethality, but fail to interact with stnC. A third variant (Suppressor of stoned) has been identified that can suppress the debilitation associated with the stnts mutations. These data, together with a previously identified interaction between the stnts and tan mutants (tan codes for beta-alanyl-dopamine hydrolase), indicate a central role for the stoned gene product in neuronal function, and suggest that the stoned gene product interacts, either directly or indirectly, with the neural cAMP second messenger system, with the synaptic membrane recycling pathway via dynamin, and with biogenic amine metabolism (Petrovich, 1993).
Using deletion mapping and complementation tests, five behavioral mutations (shaking-B2, small optic lobesKS58, sluggish-AEE85, stonedts1, and stress-sensitive-C1) have been localized to four genetic complementation groups at the base of the X-chromosome. shaking-B2 is an allele of the lethal complementation group R-9-29 near band 19E3; small optic lobesKS58 and sluggish-AEE85 belong to adjacent complementation groups, between lethals W2 and A112 near band 19F4; and stonedts1 and stress-sensitive-C1 are both alleles of the 8P1 lethal complementation group between lethals 114 and 13E3 near bands 20B-C (Miklos, 1987).
Protein IV from synaptosomal fractions of Drosophila heads was phosphorylated in vitro by an endogenous cyclic adenosine monophosphate (cAMP)-dependent protein kinase. The in vivo phosphorylation of this protein is affected by light. Two visual mutants, tan and stoned, exhibit altered levels of in vivo phosphorylation of protein IV. The tan strain shows depressed in vivo levels of phosphorylation of protein IV, whereas stoned shows an increase in the in vivo level of phosphorylation of this same protein. Protein D is phosphorylated in vitro by an endogenous Ca2+/calmodulin-dependent protein kinase and has a molecular weight identical to that of protein IV. The stoned mutant strain shows an increase in the in vivo level of phosphorylation of protein D. The data presented here suggest that the phosphorylation of protein IV, and perhaps D, may play a role in the early processing of visual information in the fly (Kelly, 1983a).
Mutations at the stoned locus of Drosophila produce a reversible temperature-sensitive debilitation. At permissive temperatures they also exhibit an unusual jump response to a light-off stimulus. An increase in the amplitude of the off-transient of the electroretinogram (ERG) is associated with the abnormal jump. Both the jump response and the increased amplitude of the off-transient are shown to be dependent on the duration of the light pulse prior to the light-off stimulus. In stoned flies that are light adapted, the jump response, as measured by recording from the indirect flight muscles, is seen to habituate with increasing light-off frequency. This habituation corresponds to the decrease in the amplitude of the off-transient that also occurs with high-frequency stimulation. Another visual mutant, tan, removes the off- and on-transients of the ERG. The combining of the stoned mutation with tan in the tan;stoned double mutant results in the loss of the jump behavior as well as the partial restoration of the off-transient to an otherwise tan-like ERG. Discussed is the relationship between the increase in the amplitude of the off-transient in stoned flies and the eliciting of the jump response (Kelly, 1993b).
The dicistronic Drosophila stoned mRNA produces two proteins, StonedA and StonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of StonedA and StonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. These data, although most consistent with a hypothesis in which StonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic StonedA by transgenically provided StonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic StonedA is dependent on StonedB. Together, these findings improve understanding of the functional relationship between StonedA and StonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved StonedB homologs that represent a new family of 'orphan' medium (µ) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA (Estes, 2003).
This analysis of StonedB function is particularly relevant since the analysis constitutes the first in vivo functional analysis of a member of the stonin family of proteins. The data predict that the stonins in general will be found to regulate endocytosis of synaptic-vesicle proteins and that stonin-deficient synapses will display phenotypes of stoned mutants. Indeed stonin genes may be good candidates for certain congential myasthenic syndromes, a class of human genetic diseases that interrupt neuromuscular transmission. Some of these have been associated with morphological defects at the NMJ that are similar to those of stoned mutants. The underlying mechanism of stonin function at synapses is likely to involve known molecular interactions of stonins with synaptotagmin, Eps15, and intersectin. A particularly attractive idea is that it serves as a 'pseudoadaptin' that, at a certain stage of vesicle formation, competes for the AP2-binding sites on vesicle proteins and, by displacing AP2, facilitates large-scale, sequential changes in the assembly state of endocytic proteins that underlie the ordered progression of events in the endocytic pathway. However, this model is not easily reconciled with the observation that stonedB remains associated with a vesicle fraction isolated from heads of shibire flies depleted of synaptic vesicles (Estes, 2003).
A major issue to be addressed is whether stonedB in particular and stonins in general participate in a wide range of endocytic events or only in the relatively rapid and specialized process of synaptic-vesicle endocytosis. The experiments described in this study address this issue in two ways. First, the observation that stonedB expression in the nervous system restores normal viability to otherwise lethal alleles of stoned argues for a neural, if not synapse-specific, function for the protein. Nonneuronal functions of stonedB, if any, must be dispensable. However, the second observation that stonedB is also required for regulating morphological changes in boutons associated with synaptic growth suggests a role for stonedB in events not limited to synaptic-vesicle recycling. Satellite boutons similar to those described in stn8P1 are found in synapses of Drosophila overexpressing the wild type, but not in an endocytosis-defective form of the Drosophila amyloid precursor protein homolog appl. Thus, it is possible that stonedB influences endocytosis of APPL or other growth-related cell surface molecules that are part of a normal pathway for structural synaptic change (Estes, 2003).
Given the reported ubiquitous expression of mammalian stonins in multiple cell types and the ability of an overexpressed dominant-negative stonin to interfere with endocytosis in nonneuronal cells, it is possible that mammalian stonins have wider functions. Perhaps stonins, initially selected for a specialized task like synaptic-vesicle recycling, have since evolved and diversified to be capable of broad, general functions in endocytosis. The concurrent proliferation of synaptotagmin-encoding genes in mammals may have contributed to diversification of stonin functions in mammalian species (Estes, 2003).
The stoned dicistronic mRNAs in eukaryotes are a genetic oddity whose functions and evolution are poorly understood. Unlike most polycistronic mRNAs that are processed to yield individual monocistronic mRNAs, the mature stoned transcript exists in a dicistronic form. Potential reasons suggested for this organization of the stoned mRNA include (1) maintainance of stoichiometry and (2) facilitation of dimer formation between the two proteins because of spatially associated translation of the two proteins. Biochemical experiments demonstrating that the two proteins may be found in a single complex provide some support for these hypotheses (Estes, 2003).
Neither of these hypotheses are supported by the current observations. (1) The experiments clearly demonstrate that stoichiometry is not an important factor in stoned function. Animals in which stonedA-stonedB stoichiometry is severely altered show completely normal viability and synaptic function. (2) It has been shown that splitting the two cistrons of stoned into the two constituent ORFs encoding stonedA and stonedB separately allows stonedB-dependent localization of stable stonedA at nerve terminals. This argues that selective pressure to maintain the dicistronic organization of stoned is not particularly strong and may not be driven by the two previously suggested mechanisms (Estes, 2003).
Additional data pertinent to the evolution of this dicistronic mRNA are provided by analyzing the conservation of stonedA and stonedB coding sequences in other species. While stonedB is conserved across metazoa, the only clear stonedA homolog known is found encoded in the genome of the mosquito Anopheles gambiae (~45% identical). Like its fruit fly counterpart, mosquito stonedA has five conserved DPF motifs plus a sixth DPF not found in the fruit fly. However, the potential leucine zipper motif of fruit fly stonedA is not conserved. In mosquito, the stonedA coding cistron lies no more than 39 bases upstream of an identically oriented stonedB coding cistron; thus, the data are consistent with the existence of a conserved dicistronic organization in insects. Because nematode and mammalian genomes have monocistronic orthologs for stonedB but not for stonedA, it is possible that the dicistronic stoned mRNA originated in arthropods some time after divergence from the vertebrate lineage, but before the divergence of Drosophila from Anopheles. Combined with the current data, these observations suggest that there may not be strong functional reasons for the evolutionary conservation of stonedA (Estes, 2003).
One remarkable conserved feature of stonedA sequence both in mosquitos and in Drosophila is the complete absence of internal methionine residues in the coding sequence. In a single 900-amino-acid protein the probability of such an absence occurring by chance alone is ~7 x 10-7, if one makes the simplistic assumption that all codons occur at an equal frequency (63/64). Given its conservation in mosquito, it appears likely that this unusual feature of stonedA coding sequences is relevant to the mechanism by which the dicistronic mRNA is translated into two different proteins. While the current experiments do not address this mechanism, the definition of a single dicistronic cDNA including intercistronic sequences sufficient to direct translation of the two stoned proteins should facilitate, in future, the detailed analysis of molecular mechanisms that allow the unusual translation of this mRNA (Estes, 2003).
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date revised: 20 November 2006
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