InteractiveFly: GeneBrief

stoned A and stoned B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionarly Homologs | References


Gene names - stoned A and stoned B

Synonyms -

Cytological map position - 20A4--5

Function - synaptic vesicle endocytosis

Keywords - neuromuscular junction, synapse, synaptic vesicle recycling

Symbol - stnA and stnB

FlyBase ID: FBgn0016976 and FBgn0016975

Genetic map position -

Classification - novel protein (StnA) and clathrin adaptor protein-like (StnB)

Cellular location - cytoplasmic



NCBI links for StnA: | Entrez Gene
NCBI links for StnB: | Entrez Gene
BIOLOGICAL OVERVIEW

After a nerve fires, proteins encoded by the stoned locus help ready the synapse for the next round of firing. stoned was identified several decades ago on the basis of stress-sensitive behavioral mutants (Grigliatti, 1973). The Drosophila stoned locus is dicistronic, that is, the locus encodes two distinctive presynaptic proteins, Stoned A (StnA) and Stoned B (StnB) from a single mRNA polymer. StnA is a novel protein without homology to known synaptic proteins, and StnB contains a domain with homology to the endocytotic protein AP50. After synaptic vesicle exocytosis during neural firing, the coordinated endocytosis of synaptic vesicle membrane proteins is mediated by adaptor proteins. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the 'lattice network' characteristic of endocytotic domains in Drosophila presynaptic terminals. Dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the 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 (Fergestad, 2001; Stimson, 2001; Phillips, 2000).

A highly specific synaptic mislocalization and degradation of Synaptotagmin occurs in stoned mutants. Overexpression of Synaptotagmin rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin directly regulates the cycling synaptic vesicle pool size. In vitro interaction studies indicate a physical interaction between Stoned B and Synaptotagmin. The Stoned A protein is also found in association with vesicles, and it too exhibits an in vitro association with Synaptotagmin. However, the bulk of Stoned A is in a nonmembranous fraction. These studies suggest that Stoned proteins regulate the AP2-Synaptotagmin interaction during synaptic vesicle endocytosis. It has been concluded that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin from the plasma membrane (Fergestad, 2001; Stimson, 2001, and Phillips, 2000).

Since the evidence for physical interaction between Stoned proteins and Synaptotagmin has now gained acceptence, it is interesting to look back at the genetic evidence for an involvement of Stoned proteins in the recycling of Synaptogamin. This genetic evidence consists of an examination of the effects of stoned mutation on the localization of Synaptogamin, carried out by Fergestad (1999), and described in detail below. These results suggest that the Stoned proteins are essential for the recycling of synaptic vesicle membrane and are required for the proper sorting of Synaptotagmin during endocytosis.

Several lethal stoned alleles have been identified, including two transposable element insertions, stn13-120 and stnPH1, that lie in the StnA and StnB reading frames, respectively, and an EMS-induced allele, stnR9-10, that has not been characterized at the molecular level. All of these lethal stoned mutants die as mature embryos after a failure to hatch from the egg case, apparently from lack of coordinated movement. This defect is not a result of alterations in gross embryonic morphology; mutant embryos show normal segmental patterning of the epidermis, muscles, and nervous system. Thus, the mutant embryos appear morphologically normal but are impaired in the ability to move in a coordinated manner (Fergestad, 1999 and references therein).

Wild-type and mutant embryos (22-24 hr) were labeled with each of the Stoned antibodies to determine the effect of each mutation on protein expression and localization. At the embryonic wild-type NMJ, StnA and StnB proteins are highly concentrated in presynaptic boutons. None of the stoned mutant alleles display detectable StnB staining in the synaptic terminal, including the viable stnC embryo and the stnC third instar larva. In contrast, the mutants show variable levels of StnA expression. Both the stn13-120 and stnR9-10 alleles have severely reduced or undetectable levels of StnA expression, whereas the stnPH1 allele appears to have only moderately reduced levels of StnA. The viable stnC mutant NMJ also shows strongly reduced or undetectable StnA staining at 22 hr AF; however these animals do display very weak StnA expression at the third instar NMJ. These results suggest that a mutation in the first open reading frame of the dicistronic stoned locus (StnA) renders the second reading frame (StnB) unreadable. Thus, the stnPH1 allele primarily removes the StnB product, consistent with the transposable element insertion in the second reading frame (StnB), whereas all other alleles strongly effect the expression of both StnA and StnB (Fergestad, 1999).

Using a number of immunological markers, stoned mutant NMJs appear morphologically and molecularly similar to those of wild-type, although the terminals appear slightly smaller than normal. Despite this slight structural difference, the mutant NMJs display clear, punctate expression of several vesicle-associated proteins in the synaptic boutons, including CSP and Syb. In addition, the expression of other synaptic markers, including a neuronal membrane marker (HRP), syntaxin (Syx), and Rab3, appear normal in the mutant terminals. The quantified expression level and bouton localization of the synaptic proteins are similar to those of wild type (Fergestad, 1999).

In contrast, Synaptotagmin (Syt) protein appears to be strikingly mislocalized in all four stoned mutant alleles. Instead of the punctate bouton localization of Syt observed in wild type, the stoned mutants display reduced Syt expression in the boutons and the protein aberrantly dispersed throughout the presynaptic terminal. This mislocalization is not resolved with development in the viable stnC allele because Syt expression remains mislocalized at the third instar NMJ. Double-labeling assays with other presynaptic markers indicate this mislocalization is specific to Syt, because CSP, the neuronal membrane marker HRP, another synaptic vesicle protein (Syb), and the membrane protein Syx display normal patterns of expression in all mutants. These results suggest that stoned mutants specifically mislocalize the SV (synaptic vesicle) protein Syt in the synaptic terminal and retain other synaptic proteins properly (Fergestad, 1999).

For all four mutant alleles, the intensity of the Syt and CSP expression in individual, double-labeled boutons is quantified using digital confocal imaging and compared with wild-type expression levels in parallel trials. All mutants show an equal and similar ~40%-60% loss of Syt synaptic localization, compared with the normal expression and localization of CSP. This reduction in Syt expression is significant in all four alleles, and the alleles are not significantly different from each other. Syt expression in whole embryos was analyzed to determine further the nature of the synaptic loss of Syt staining. Quantified Western blots were performed to determine Syt expression levels in the different stoned alleles. Mutants display a significant decrease in Syt levels, with protein levels in the range of 20%-60% of those found in wild-type embryos. The decrease in in situ synaptic localization is consistent with the loss of Syt displayed on Western blots. These findings suggest that Syt is not only mislocalized in stoned mutants but may also be subject to rapid degradation when not properly localized (Fergestad, 1999).

To determine whether the striking synaptic staining pattern for the stoned proteins is consistent with a physiological function at the synapse, transmission properties were assayed with electrophysiological recordings at the embryonic NMJ. In all stoned mutant alleles, nerve stimulation produces muscle contraction, demonstrating that presynaptic depolarization evokes transmitter release and that the muscle excitation-secretion response is intact. However, evoked excitatory junctional currents (EJC) peak amplitudes are significantly reduced below wild-type levels, typically by 30%-50%, for all stoned alleles. Furthermore, the release of neurotransmitter at mutant synapses is markedly asynchronous. The asynchronous mutant transmission seems to result from delayed presynaptic vesicle fusion, similar to that observed for the previously identified synaptotagmin mutant (Fergestad, 1999).

The reduced and erratic evoked transmission of stoned mutants is further increased after prolonged, repetitive stimulation at moderate or high frequencies (5-20 Hz). To determine the nature of this debilitation, animals were subjected to a high-frequency stimulus protocol (10 Hz) sustained over a 5 min period. The average EJC amplitude at wild-type NMJs decreases by an absolute amount similar to that of mutant animals (~500 pA), suggesting that the NMJ of both genotypes is fatiguing comparably. However, the stoned mutants start with significantly impaired performance and fatigue by an average of 50-75% during the stimulus train, whereas wild-type amplitude decreases by only ~20%-25%. The decrease in mean EJC amplitude is accompanied by a large increase in transmission failure in the stoned synapses. Wild-type synapses maintain high-amplitude, high-fidelity transmission over a sustained stimulation period of 5 min and show no failures even at the end of the stimulus train. In contrast, the failure frequency of the mutant synapses is initially significant (~5%-10%) and increases rapidly during sustained stimulation to a level of 25%-80% at the end of the stimulus train. The three alleles that more strongly affect both StnA and StnB expression (stnR9-10, stn13-120, and stnC) show a more marked transmission failure rate (50%-80%) than does the primarily StnB mutant (stnPH1; 25%). The striking increase in failure rate during a prolonged stimuli train suggests that repetitive stimulation results in a severe depletion of SVs in the stoned mutants (Fergestad, 1999).

The Drosophila embryonic NMJ is characterized by the presence of presynaptic varicosities (boutons) containing specialized, densely staining, T-shaped structures (t-bars) at the presynaptic active zones, the putative SV fusion sites. The pre- and post-synaptic membranes surrounding the t-bars are densely stained and are separated by a cleft ~15 nm wide. Clear SVs of 30-40 nm diameter are observed clustered around the t-bars in a semicircular area with a radius of ~250 nm. Synaptic vesicles dock with the membrane immediately adjacent to t-bars in preparation for evoked fusion. For analytical purposes, vesicles are considered docked if distributed less than one vesicle diameter (<30 nm) from the plasma membrane at active sites. Synaptic vesicles also appear outside the clusters surrounding t-bars, although at a much lower density than that of the clustered vesicles. Other membrane structures, such as large dense core vesicles and translucent vesicles larger than typical SVs (presumed to be endosomes), are also occasionally observed in sections through boutons containing t-bars (Fergestad, 1999 and references therein).

Striking differences in synaptic ultrastructure are observed for all of the stoned mutant alleles relative to wild-type controls. Presynaptic boutons containing t-bars are present in all stoned alleles, and the association of presynaptic tissue with muscle cells is similar to that in wild type, indicating that development of NMJs in the mutant embryos occurs normally. However, a striking reduction in the number and density of SVs present in boutons is observed in stoned mutants. All four stoned alleles have ~50% fewer SVs clustered around the active zone t-bars, and SV density outside of the clustered radius surrounding t-bars is also reduced by ~50%. Analysis of stnPH1/Df(1)HM430 animals showed that they displayed features identical to those of animals hemizygous for stnPH1. A similar 50% reduction in the number of docked vesicles per t-bar is observed at stn13-120, stnR9-10, and stnPH1 synapses, whereas the viable stnC allele has ~30% fewer docked vesicles per t-bar than does wild type. Thus, SV density is severely reduced in all mutant alleles, throughout the presynaptic bouton, at active zones, and in the number of docked vesicles (Fergestad, 1999).

An additional difference observed in stoned bouton ultrastructure is an increase in intermediates of the SV cycle. In wild-type embryos, SVs are the most prominent membrane structures in sections through boutons containing t-bars. A much smaller number of translucent vesicles (cisternae) noticeably larger than SVs and early endosomes are also present in these sections, and, rarely, multivesicular bodies (MVBs) are seen. The early endosomes represent a step in the normal synthetic pathway for SVs in these terminals. Increased numbers of MVBs result from increased synaptic activity. These MVBs are usually removed from the region of the active zone and are targeted to somatic lysosomes. Because MVBs have been shown to contain SV proteins, they represent the normal degradative route for synaptic proteins (Fergestad, 1999).

In stoned mutants, sections through boutons containing t-bars have a significantly greater number of large vesicles (>60 nm; endosomes and cisternae) than do wild-type synapses. In addition, a greater number of these large vesicles appear within the SV cluster surrounding active zones in mutant embryos. The number of large vesicles present within the clustered radius of t-bars is at least three times greater in all of the mutant strains than in wild type. Furthermore, a significant increase in the number of MVBs is observed in sections through boutons containing t-bars in stoned mutants. stnPH1 and stnC have an approximate fourfold increase in the number of MVBs compared with that in wild type (Fergestad, 1999).

These results indicate that the stoned mutants have normal gross synaptic morphology; however, these mutants display a severe reduction in synaptic vesicle number and an increase in recycling intermediates including large cisternae and MVBs. This ultrastructural analysis combined with the abnormal labeling of synaptotagmin and debilitated synaptic transmission strongly suggests a role for the stoned proteins in regulating the synaptic vesicle-recycling pathways.

Early models of synaptic membrane recycling suggested that newly endocytosed vesicles join a sorting endosome compartment before subsequent budding and maturation. In addition, under periods of sustained or high-frequency transmission, large patches of membrane may be retrieved from the plasma membrane to form an early endosome from which new vesicles may be generated. Recent studies provide evidence that synapses may also recycle synaptic vesicle membrane directly, without first fusing with an endosomal-sorting compartment. Evidence from Drosophila argues that these two recycling pathways may act in parallel, corresponding to functionally and spatially distinct vesicle pools. The stoned mutants are clearly defective in one or more of these recycling pathways. All alleles show a significant decrease in SVs at active zones and throughout the presynaptic terminal and a correspondingly increased sequestering of membrane in enlarged membranous compartments. These defects may result from a direct defect in endocytosis leading to improper regulation of vesicle size, or alternatively, the large vesicles may be endosomal compartments that accumulate owing to an inability to bud and segregate new SVs during endosome-mediated recycling. The existence of a population of very large-amplitude spontaneous fusion events (200-500 pA) at stoned synapses suggests that these large vesicles can function to release neurotransmitter in a constitutive manner (similar to large dense-core vesicles). However, the apparent functional competence of these vesicles does not allow for a determination of whether they represent abnormally large SVs or endosomes that have spilled over into the active zone. The accumulation of MVBs may be more informative. These structures clearly derive from endosomes and represent a normal degradative pathway in which SV proteins and membrane are targeted to somatic lysosomes. In parallel with MVB accumulation, Syt levels at the terminal and throughout the embryo decrease by ~50% in stoned mutants. It is therefore suggested that the Stoned proteins are specifically involved in the recruitment and localization of Syt during SV recycling and, in the absence of correct targeting, that Syt is degraded via a default degradative pathway involving MVBs (Fergestad, 1999).

Mature synaptic vesicles have a specific complement of proteins required for a variety of functions. Each protein must be selectively recruited to the maturing SV during endocytosis. The mislocalization of Syt in stoned synapses is not accompanied by a loss of other SV proteins (e.g., synaptobrevin and CSP), suggesting that the stoned proteins may be involved in the specific recycling of Syt from endocytosed membrane. It is hypothesized that the Stoned proteins normally function at a choice point segregating recycled Syt protein into maturing synaptic SVs and away from the MVB degradative pathway. Such a role has been suggested for the AP3 complex, shown recently to be required for localization of a SV transporter protein and to be required for the generation of SVs. Similarly, the Drosophila gene LAP, which encodes AP180 (associated with clathrin-dependent endocytosis with the AP2 complex), has been shown recently to be involved in regulating SV size and the proper recruitment of the vesicle coat protein clathrin (Zhang, 1998). In the C. elegans AP180 mutant (UNC-11), the protein also seems to have a specific role in recruiting synaptobrevin to the recycled vesicle (E. Jorgensen, personal communication to Fergestad, 1999). These AP180 data, combined with the observation that SV size is not altered in mutant animals lacking synaptobrevin, suggest that AP180 has two distinct functions: structural budding of membrane and the specific recycling of synaptobrevin (Fergestad, 1999).

The similarities between these studies led to the hypothesis that there may be separate mechanisms required for the recycling of each distinct SV protein and that these mechanisms may be intimately integrated into the membrane-budding machinery. Such a coupled mechanism would guarantee that newly generated SVs have the correct functional complement of SV proteins. Clearly, within this general mechanism, the Stoned proteins couple the specific recruitment of Syt to proper SV biogenesis. The Stoned proteins may participate in the AP2-mediated plasma membrane mechanism or, alternatively, act in a separate and/or later site such as AP3-mediated endosomal sorting to direct the recruitment and/or localization of Syt into mature SVs (Fergestad, 1999).

Retrieval of SV components from the plasma membrane is tightly temporally coupled to exocytosis. Mutation of stoned disrupts this temporal coupling and delays the onset and rate of endocytosis after exocytosis. Delayed application of FM1-43 to stoned synapses after stimulation reveals that the delayed component of endocytosis is comparable with wild-type levels. There are several possible explanations for these altered endocytosis kinetics in stoned mutants. A likely rationale is that global membrane retrieval is delayed in stoned mutants, suggesting that Stoned proteins may play a role in either initiating endocytosis or facilitating the speed of endocytosis. An alternative scenario may be that there are multiple pathways for SV endocytosis, which differ in temporal kinetics, and that the rapid endocytosis mechanism is specifically impaired in stoned mutants. Although it is formally possible that delays in exocytosis might also explain the delayed membrane retrieval, recordings of synaptic transmission show that exocytosis speed is only very minimally impaired in stoned mutants (Fergestad, 2001).

How are the numerous different components of the SV recognized and recombined in the precise stoichiometric ratios required for synaptic function? There is an increasing body of evidence that a cast of specific recycling proteins is required to recognize and retrieve specific components of the SV during plasma membrane endocytosis. At center stage, the AP2 complex plays a prominent role in clathrin-mediated SV endocytosis. Genetic removal of the alpha-Adaptin subunit in Drosophila shows that the AP2 complex is absolutely required for the SV endocytotic process in this system (Gonzalez-Gaitan, 1997). Moreover, the AP2 complex has been shown to bind Synaptotagmin (Zhang, 1994), and it thus seems likely that this complex mediates the endocytotic recovery of Synaptotagmin, likely in addition to other integral SV proteins (Fergestad, 2001).

It is proposed that the role of the Stoned proteins may be to recycle Synaptotagmin by mediating the association with the AP2 complex. It has been shown recently that both StnA and StnB bind with high specificity to Synaptotagmin (Phillips, 2000) and therefore likely act in a cooperative manner in Synaptotagmin retrieval. Studies reported by Haucke and De Camilli (1999) have shown that the AP2-Synaptotagmin interaction can be stimulated by the presence of Yxxphi-containing peptides, which also enhance the recruitment of AP2 to the plasma membrane. Because both Stoned proteins contain multiple copies of these tyrosine-based motifs, as well as other AP2 and Clathrin binding domains, it is hypothesized that the Stoned proteins specifically promote the retrieval of Synaptotagmin from the plasma membrane by mediating the AP2-Synaptotagmin binding. Because the Stoned proteins are encoded by a dicistronic locus, polarity constraints have to date prevented an independent dissection of the roles of StnA and StnB in Synaptotagmin endocytosis. Why does the process require two proteins, and what does each contribute to the recycling mechanism? Targeted homologous knock-out techniques are being used to explore these questions (Fergestad, 2001).

As the proposed calcium sensor for SV fusion, Synaptotagmin is likely to function as a key regulator of transmission strength. The number of Synaptotagmin proteins in an SV membrane may play an important role in regulating the response of the presynaptic terminal to depolarizing stimuli. Overexpression of Synaptotagmin alone is capable of substantially increasing the size of the endo-exo SV pool. Therefore, the Stoned proteins, by regulating Synaptotagmin recycling, also act as key regulators of neurotransmission strength. Future experiments are focusing on the specific regulation of Synaptotagmin levels by each of the Stoned proteins (Fergestad, 2001 and references therein).


REGULATION

Protein Interactions

Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicate a presynaptic function for Stoned and suggest an involvement in vesicle cycling. Immunological studies have revealed colocalization of the Stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein Synaptotagmin (Syt). Stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The StnB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of StnB and the C2B domain of the SytI isoform. The StnA protein is also found in association with vesicles, and it too exhibits an in vitro association with SytI. However, the bulk of StnA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, StnB has been shown to be present on some synaptic vesicles before exocytosis. However, StnB is not associated with all synaptic vesicles. It is hypothesized that StnB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that has yet to be determined (Phillips, 2000).

Hypomorphic mutations at the Drosophila synaptotagmin locus result in behavioral, electrophysiological, and morphological phenotypes similar to those seen in stn mutants. Synaptotagmin (Syt) is a synaptic vesicle protein with a proposed role in both exocytotic and endocytotic functions in both mammals. The aim of these experiments was to determine whether either of the viable stn alleles, when in combination with mutations at the synaptotagmin locus, enhances or suppresses the syt phenotype. The stnC and stnts alleles are homozygous viable as adult flies and were isolated in the same genetic background, and both possess the same insertional polymorphism as the original Oregon-R strain. The flies used were heterozygous for two stn null mutations, sytAD4 and sytD27. The P-element-mediated transposition of a stn minigene to the third chromosome provides 10% of wild-type Syt protein levels and allows the survival of flies with lethal null mutations on both chromosomes at the syt locus to produce fertile adults. Double mutant combinations were constructed, and the viability and behavior of the resulting flies were investigated. The data clearly indicate a genetic interaction between the stnts mutations and syt. Because synaptotagmins are an integral component of synaptic vesicle membranes, this data also suggests an interaction between the stoned protein(s) and synaptic vesicles (Phillips, 2000).

Hydrophobicity analysis of both the StnA and StnB proteins suggests that they should be soluble proteins. Wild-type fly head extracts, homogenized in both the presence and absence of calcium, were subjected to differential centrifugation to produce P1 (1000 × g), P2 (25,000 × g), and P3 (125,000 × g) pellets and a final supernatant fraction, S3. Western blots prepared from these fractions were probed with anti-StnA, anti-StnB, and anti-Syt antibodies. The anti-Syt antibodies were raised against the recombinant cytoplasmic region of the Syt protein and recognize a number of isoforms of Synaptotagmin (54-69 kDa). Neither the StnA nor StnB proteins could be visualized in the supernatant fraction. The StnB protein co-sediments with the synaptic vesicle protein marker Syt, primarily in the P2 and P3 fractions. StnA, in contrast, preferentially partitions into the P1 fraction, although some StnA was found in both the P2 and P3 fractions. This indicates that both stoned proteins preferentially partition into either membrane fractions or fractions containing large protein complexes. The association of StnA with the P1 fraction was investigated further. Solublization of StnA from P1 was not achieved with Triton X-100, deoxycholate, or high NaCl concentrations; however, the chaotropic agent KI effectively solubilizes all of the StnA protein from the P1 fraction. This indicates that StnA in the P1 fraction is not associated with heavy membranes but is more likely to be associated with a large protein complex (Phillips, 2000).

As expected, the Syt isoforms were associated with both the P2 and P3 fractions, plasma membrane, and vesicle-enriched fractions, respectively. Also observed was a coincidental shift of Syt and StnB from the P3 to the P2 fraction when homogenization was performed in the presence of Ca2+. The supernatant fraction from the P1 centrifugation (S1) was applied to a glycerol gradient and centrifuged to separate membrane components. A peak of Syt, corresponding to the synaptic vesicle fraction, was observed. StnB protein co-sediments with the Syt peak, whereas StnA, although entering the gradient, peaks in fractions 3-6. These two results, the coincident redistribution of StnB and Syt in the presence of Ca2+ and their co-sedimentation in glycerol gradients, are consistent with an association of the StnB protein with synaptic vesicles. The plasma membrane marker syntaxin was also present in the gradients, probably indicating fragmentation of plasma membrane during homogenization, although its distribution does not mirror that of synaptotagmin/StnB or StnA (Phillips, 2000).

To determine whether the StnB present in the P3 fraction is associated with synaptic vesicles, anti-StnB antibodies were attached to Protein A-coated magnetic beads, and incubated with a P3 fraction prepared from Drosophila head homogenates in the absence of calcium. These beads were then analyzed for the presence of StnB and the synaptic vesicle protein markers Syt, Csp, and Syb as well as the plasma membrane marker Syx. The results indicate that a major proportion of the StnB protein in the P3 fraction is immunoprecipitated. The StnB antibodies coprecipitate all three synaptic vesicle markers (Syt, Csp, and Syb), but not Syx. Although multiple species of Syt can be identified in P3 fractions, only the 69 kDa Syt isoform is present in these precipitates (Phillips, 2000).

On the basis of its deduced amino acid sequence, StnB does not contain any putative transmembrane segments and is unlikely to be an integral membrane protein. What then is the molecular nature of the StnB/vesicle association? To address this question, fractions of the StnB immunoprecipitations were washed extensively with 1% Triton X-100. The presence of the detergent entirely removes Csp from the precipitates and considerably reduces the amount of Syb present. However, Triton X-100 has no effect on the amount of Syt bound to the beads. This result suggests that StnB is not associating with the lipid components of the vesicle membrane and that the interaction may be via Syt (Phillips, 2000).

There was relatively little StnA seen in the P3 fraction on the Western blots. However, StnA protein was observed on the glycerol gradients, and although there was no coincidence of the peak fractions, there was overlap between StnA and the synaptic vesicle peak. The immunoprecipitations were therefore further probed for the presence of StnA. The StnA protein was found to be associated with the immunoprecipitates and to be insensitive to the Triton X-100 washes. When immunoprecipitations were performed using anti-StnA antibodies attached to beads, again Syt and Csp were coprecipitated. Therefore, both StnB and StnA can be found associated with synaptic vesicles in the P3 fraction (Phillips, 2000).

The continued association of Syt with immunoprecipitated StnB even after Triton X-100 treatment suggests a direct interaction between StnB and Syt. It appeared likely that this interaction would be via the µ2-like domain of StnB. To investigate this, a 621 residue protein containing the µ2-like region, residues 883-1089, and adjacent sequence up to residue 1261 was expressed in the pGEX expression vector to produce a GST fusion. The resin-bound GST/µ2-like fusion protein was incubated overnight with a crude extract of bacterial cells expressing the cytoplasmic portion of Drosophila Syt as a 6x-His (pET) fusion protein of 39 kDa. When proteins eluted from the resin were Western-blotted and the blot was exposed to anti-Syt antibody, the expected 39 kDa Syt fusion protein was identified. The GST protein itself was unable to bind Syt in this assay. A series of fusion constructs with different regions of the µ2-like domain of StnB were similarly assayed. In repeated experiments, fusion proteins terminating at amino acid residue 930 were unable to bind Syt. However, Syt binds to all fusion proteins containing amino acid residues 847-1108 of the StnB protein. Because residues 883-1138 of StnB constitute the µ2-like domain, this domain is sufficient for Syt binding in vitro. It is possible, however, that residues outside the µ2-like domain influence the strength of this interaction in vivo (Phillips, 2000).

StnA can be found associated with synaptic vesicles immunoabsorbed by the StnB antibodies. To determine whether the StnA protein might be associated directly with synaptotagmin, Syt binding to StnA fusion proteins was analyzed. Three constructs were used. The first was a fusion protein that included the first 290 residues of StnA (GST/5'StnA), the second included residues 26-350 (GST/StnA Xho-p33), which removes most of the N-terminal region that would be missing if the methionine in the stnts mutant acted as a novel translation initiation site, and the third was identical to GST/5'StnA but contained the sequence encoding the K to M substitution found in the stnts flies. The results indicate that residues 26-290 of the amino terminal region of StnA can bind Syt. This region includes the sequence altered in the stnts mutation. Binding of Syt to the GST/5'StnA construct containing the stnts mutation shows that this mutant protein also binda Syt. The affinity of StnA for Syt appears less than that seen with the StnB constructs; however, the binding of StnA to Syt in vitro is observed consistently (Phillips, 2000).

There are two C2 domains, C2A and C2B, in the Syt monomer, and the two domains have been found to interact with different intracellular components. Two Syt protein constructs were produced, each containing one of the C2 domains, and the ability of these protein constructs to bind to both the StnA/GST and StnB/GST fusions was investigated. The protein containing the cytoplasmic sequences including the C2A domain (residues 137-327) but excluding the C2B domain failed to bind to either StnA or StnB. However, the protein containing only the C2B domain (residues 317-473) was found to bind to both StnA and StnB (Phillips, 2000).

The immunoprecipitation studies indicate that only the 69 kDa Syt isoform is coprecipitated with StnB. However, there are other synaptotagmin species in Drosophila, including a SytV homolog of 55 kDa that are associated with synaptic vesicles. The C2B domain (residues 325-374) of SytV was expressed as a pMAL fusion protein and shown to cross-react with the polyclonal anti-Syt antibodies. Therefore, it was asked whether StnB could interact with SytV in vitro. The SytV fusions were then assayed for their ability to bind to StnB. The SytV C2B domain indeed interacts with the µ2-homology region of StnB (Phillips, 2000).

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the micro-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. This study report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the micro-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles (Soekmadji, 2012).

This study has investigated the effect of Ca2+ upon Stoned proteins binding to SYT-1 C2B domain. Previous study has show the Drosophila μHD of STNB binds to C2B domain of SYT-1, which is in agreement with a study that showed the μHD of the stonin-2, the mammalian homologue of the Drosophila STNB protein, could also bind SYT-1. It has also been reported that stonin-2 is able to bind the C2A domain, even though the role of Ca2+ in this binding was not explored. This study also observed that STNB is able to bind to the C2A domain in vitro, albeit at a much lower level as compared to the binding to the C2B domain. The data show that the binding of Drosophila STNB to Drosophila SYT-1 is different from that of the murine μ2 subunit of AP-2. While μ2 binding to C2B domain requires phosphatidylserine (PS) and Ca2+, the binding of the STNA and STNB proteins did not require PS. In fact PS-containing phospholipids nearly abolished the STNB binding to SYT-1 C2B. Deletion of the polyK region of the C2B domain also did not abolished STNB binding to SYT-1, indicating that this region cannot be the binding site for either STNA or STNB. The data support a recent publication that showed that, in Drosophila, μ2 cannot replace the function of the μHD of STNB in vivo, and suggest that STNB and AP-2 might represent alternative mechanisms for synaptic vesicle recycling (Soekmadji, 2012).

AP-2 is ubiquitously expressed and implicated in general mechanisms of endocytosis from the plasma membrane, while in Drosophila, STNB is expressed and functions only in the nervous system. Differences in the μHD of STNB and the μ2 domain of AP-2 may reflect the need for flexibility of μ2 subunit of AP-2 to act in a number of endocytic pathways, while the function of STNB may be specific for synaptic vesicle retrieval. The data, in conjunction with a report that STNB may specifically regulate the sorting of a subset of SV, suggest the Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound SYT-1 associated SV (Soekmadji, 2012).

Upon Ca2+ binding, SYT-1 was reported to undergo a conformational change that protects SYT-1 against trypsin and chymotrypsin digestion in vitro. Ca2+ has also promoted homo-oligomerization of SYT-1 and/or hetero-oligomerization with other synaptotagmins. Studies using mutations that affect Ca2+ dependent oligomerization, such as Y311N, showed a partial inhibition for internalization of SYT-1 in PC12 cells while the corresponding mutation in Drosophila (AD3) alters the rate of exocytosis. The AD3 SYT-1 is still capable of binding to SNARE complexes, synprint and AP-2, which implies that the mutation does not cause a complete loss of function. Hence the docked vesicles in AD3 flies (a mutation that affects Ca2+ dependent oligomerization) require higher Ca2+ concentration to undergo exocytosis, suggesting that defects in Ca2+ dependent oligomerization renders C2B SYT-1 inefficient for exocytosis. It was reported that Ca2+-triggered SYT-1 clustering is via the C2B domain and is required for exocytosis. In a potential endocytic model that includes STNB, the oligomerized SYT-1 that has led to exocytosis, could then be a target for STNB binding, and STNB, in turn, could recruit dynamin via the intersectin DAP-160. This would create what amounts to a Ca2+-dependent endocytic complex. However, a previous study has shown that STNB can be found bound to synaptic vesicles, via SYT-1, prior to exocytosis, that is, prior to the Ca2+ influx that might trigger SYT-1 oligomerization and the coupling of excitation and vesicle fusion. Either this bound STNB reflects the low level of oligomerization of SYT-1 in the absence of Ca2+, or that other factors, such as those that might alter the structure of the C-terminal region of SYT-I C2B, are playing a role in potentiating the binding of STNB to SYT-1 even in the absence of Ca2+. Certainly there is no STNB protein in the soluble fraction from fly head extracts, suggesting that all STNB is in a bound form, and although some is certainly is, perhaps not all is bound to SYT-1 (Soekmadji, 2012).

The D3,4N mutation in transgenic flies results in a reduction in the rate of endocytosis of synaptic vesicles, this may be due to a failure of this mutant SYT-I to interact with Stoned proteins. Another mutation which gives constitutive dimerization, the D3,4N mutation, was incapable to restore internalization in CHO cells. The D3,4N mutation did not only show a reduction in the rate of endocytosis in Drosophila, but also exocytosis defect by decrease evoked transmitter release and reduce in apparent Ca2+ affinity for synaptic transmission. Indeed, similar to these synaptotagmin mutant flies, the stoned mutants showed alterations in both spontaneous and evoked release at larval NMJ and severe neurotransmission defect that may lead to embryonic lethality, as well as depletion of synaptic vesicle and increase of membrane recycling intermediate that might be due to mislocalization of synaptotagmin during endocytosis. In contrast with μ2 and SYT-1 interaction, the binding of Stoned proteins do not require phospholipids. Studies using Folch liposome has shown that D3,4N mutant are not able to induce a close proximity membrane curvature, which may be a prerequisite for SNARE mediated membrane fusion. A synthesized peptide consist of the 3rd loop of C2B SYT-1 can outcompete the μ-homology domain of STNB, suggesting the interaction of STNB to SYT-1 is mediated by a region in loop 3 of C2B domain. Thus, it is an attractive hypothesis that STNB may act as an inhibitor for membrane fusion. In a recent paper, it is shown that SYT-1 bound to PtdIns at the same level as PS and that this binding is also required Ca2+; while the binding of PIP2 with SYT-1 is less affected by Ca2+. It would be interesting to investigate whether these lipids will have similar effect as PS in affecting Stoned and C2B SYT-1 binding (Soekmadji, 2012).

The role of STNA in the synaptic vesicle cycle remains elusive. The presence of STNA at the larval NMJ appears nonessential. However STNA is certainly associated with synaptic vesicles and it is clear that STNA has a strong affinity for SYT-1. This study has shown that the oligomerization of SYT-1 dramatically increases the binding of STNA and presumably STNA will bind under similar in vivo conditions as STNB. The presence of putative AP-2 binding motifs in STNA, may make vesicles bound by STNA targets for AP-2 mediated endocytosis. This is in contrast to STNB that lacks such AP-2 binding motifs. Whatever the specific mechanism of action of the STNA protein, it is clear from this study that the action of Ca2+ has a marked effect on STNA and STNB association with SYT-1 and confirms them as important proteins in the mechanism(s) of synaptic vesicle recycling in Drosophila (Soekmadji, 2012).


DEVELOPMENTAL BIOLOGY

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).

Stoned proteins, Synaptotagmin I and plasma membrane endocytosis

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).


EFFECTS OF MUTATION

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).

Functional dissection of a eukaryotic dicistronic gene: Transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants

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).

Stoned B mediates sorting of integral synaptic vesicle proteins

A continuous supply of fusion-competent synaptic vesicles is essential for sustainable neurotransmission. Mutations of the dicistronic stoned locus disrupt normal vesicle cycling and cause functional deficits in synaptic transmission. Although both Stoned A and B proteins putatively participate in reconstituting synaptic vesicles, their precise function is still unclear. This study investigated the effects of progressive depletion of Stoned B (STNB) on the release properties of neuromuscular synapses using a novel set of synthetic STNB hypomorphic alleles. Decreasing neuronal STNB expression to ~35% of wildtype level causes a strong reduction in EJC amplitude at low stimulation frequencies and a marked slowing in synaptic depression during high-frequency stimulation, suggesting vesicle depletion is attenuated by decreased release probability. Recovery from synaptic depression after prolonged stimulation is also decelerated in mutants, indicating a delayed recovery of fusion-ready vesicles. These phenotypes appear not to be due to a diminished vesicle population, since the docked vesicle pool is ultrastructurally unaffected, and the total number of vesicles is only slightly reduced in these hypomorphs, unlike lethal stoned mutants. Therefore, it is concluded that STNB not only functions as an essential component of the endocytic complex for vesicle reconstitution, as previously proposed, but also regulates the competence of recycled vesicles to undergo fusion. In support of such role of STNB, synaptic levels of the vesicular glutamate transporter (vGLUT) and synaptotagmin-1 are strongly reduced with diminishing STNB function, while other synaptic proteins are largely unaffected. It is concluded that STNB organizes the endocytic sorting of a subset of integral synaptic vesicle proteins thereby regulating the fusion-competence of the recycled vesicle (Mohrmann, 2008).

The Drosophila stoned locus was identified 35 years ago based on severe behavioral impairments. It is one of the few dicistronic loci characterized in Drosophila, encoding Stoned A and B proteins; however, the Stoned A protein appears totally dispensable for known functions. In contrast, the crucial importance of the Stoned B protein for synaptic transmission has been well established. Nevertheless, the exact mechanistic function of STNB remains enigmatic. This study has generated and characterized a graded set of STNB hypomorphic animals to provide evidence that STNB has a dose-dependent limiting function regulating neurotransmission strength as a potent sorting factor governing a specific set of integral vesicular proteins during synaptic vesicle reconstitution. The key findings supporting this interpretation of STNB function are (I) the occurrence of diminished and altered release in a progressive series of STNB hypomorphic alleles, in the absence of significant ultrastructural defects of the vesicle pools, thereby indicating a changed fusion competence of synaptic vesicles, and (II) the differential loss of integral synaptic vesicle proteins dependent on the level of STNB activity (Mohrmann, 2008).

In previous studies, severely compromised basal synaptic transmission has been reported as a prominent feature of the physiological phenotype of various classical stoned mutants. Simplistically, defective synaptic release might be entirely a secondary effect of a primary impairment in vesicle pool maintenance, due to disrupted vesicle endocytosis. However, recent studies of endophilin and synaptojanin endocytic mutants suggest that only a surprisingly small number of synaptic vesicles is actually required to support normal synaptic function at basal stimulation frequencies: In both endophilin and synaptojanin mutants, basal synaptic transmission is completely normal despite the near elimination of the presynaptic vesicle population, and the loss of synaptic uptake of FM1-43. This raises the question that STNB may be involved in other processes that affect exocytosis, apart from limiting the availability of vesicles. The observation that viable stnC mutants exhibit release defects without major alterations in vesicle pool size seems to support such notion. However, recent findings show that the stnC mutation induces the expression of a C-terminally truncated STNB variant, together with low levels of the full-length product, thereby possibly involving dominant-negative effects or a partial functionality of the truncated STNB variant. Thus, the physiological phenotype in stnC mutants cannot be clearly interpreted. To clarify whether a hypomorphic condition exists that would allow for a segregation of the putative release defect and vesicle pool depletion, a new collection of graded STNB hypomorphic alleles was generated by transgenic expression of STNB in the stn13-120 mutant background. In the hypomorphic condition, STNB levels should be sufficient to support vesicle resupply and to maintain normal vesicle pools, but the shortage would still compromise basal synaptic transmission (Mohrmann, 2008).

This study demonstrates that physiological defects first emerge when STNB expression is reduced to less than 40% of wildtype level. Below this threshold, the decrease in basal EJC amplitude correlates closely with the expression level of STNB. Hypomorphic stn-vl mutants expressing ~35% of the wildtype level are of particular interest for this analysis, because their expression is only slightly lower than this threshold, and yet this condition causes a large 40% drop in basal amplitude. Strikingly, the ultrastructural analysis of stn-vl synapses showed that clustered and docked vesicle pools at the presynaptic active zone were completely unaffected in this hypomorphic condition, although there is a slight reduction in overall vesicle density. At the Drosophila NMJ, an 'exo/endo cycling vesicle pool' (ECP) has been demonstrated at the bouton periphery, which probably corresponds to the electrophysiologically defined readily releasable pool (RRP), and a reserve pool (RP) at the bouton center. According to this study the ECP/RRP alone is sufficient to allow for full-scale basal synaptic transmission after pharmacological depletion of the RP, and a loss of RP vesicles mainly affects synaptic fatigue during high-frequency stimulation. Since ultrastructural data indicate the integrity of the ECP/RRP in stn-vl hypomorphs, it must be concluded that the reduction in average amplitude during low frequency stimulation cannot be simply due to a small vesicle depletion restricted to the RP. Rather, defective basal synaptic transmission must be caused by the reluctant fusion of existing vesicles, correlating with the loss of STNB (Mohrmann, 2008).

In order to fully characterize this potential adverse effect on exocytosis, synaptic response patterns evoked by different stimulation paradigms were analyzed. Strikingly, stn-vl mutants exhibited significantly less pronounced synaptic depression during short stimulus trains applied at different frequencies. Interestingly, altered depression was found only in those low STNB-expressing hypomorphs that also showed defective basal transmission, suggesting a linkage relationship between these phenotypes. A simplistic model of synaptic depression could be satisfied by a progressive depletion of fusion-ready synaptic vesicles during phases of increased activity. Using shibire mutants to study depression in the absence of compensating endocytosis, it has been demonstrated that short trains (5-20Hz) that selectively deplete the readily-releasable pool caused a depression profile whose shape is reminiscent of the de-staining kinetics of FM1-43 labeled RRP in presynaptic boutons. Hence, the initial phase of depression at NMJ synapses presumably reflects the depletion of the RRP. Based on this interpretation, the altered depression profile in STNB hypomorphs is most likely due to an alteration in mobilization and/or fusion-rate of RRP vesicles. Since abnormal response patterns are also observed for simple paired-pulse stimuli, a shortage of STNB might generally change release properties. It is widely accepted that basal release probability is a determinant factor for short-term plasticity. Indeed, the presence of a depletion-based mechanism of synaptic depression readily implies a dependence of depression kinetics on initial release probability. Therefore, the reduction in basal EJC amplitude and the slowing of depression kinetics represent concurring indicators of an underlying decrease of release probability caused by removal of STNB (Mohrmann, 2008).

Though STNB could in principle play a dual role by independently functioning in exocytosis and endocytosis, no evidence was found to support such hypothesis. In fact, stn-vl hypomorphs, which exhibited less pronounced synaptic depression, also demonstrated a delayed recovery after prolonged stimulation indicating an accompanying defect in vesicle recovery. More likely, STNB serves functions on two different levels during vesicle reconstitution: Apart from simply being an essential component constituting functional endocytic complexes, STNB potentially also acts on a governing stage ensuring proper recovery of fusion-competent vesicles. Since a compromised complement of synaptic proteins on recovered vesicles could readily account for the decreased release probability in stnB hypomorphs, assays were performed for possible alterations in presynaptic expression levels and localization of synaptic vesicle proteins. While the expression of all tested proteins was at least slightly reduced in stn-vl hypomorphs, a definite subset of integral vesicle proteins was clearly most affected by STNB depletion. Synaptobrevin and synapsin expression levels were only slightly (≤30%) reduced, while the abundance of synaptotagmin and vGLUT were more severely decreased (≤50%). Synapsin adheres to available vesicular membranes in a dynamic fashion based on its phosphorylation state and presumably without obligatory endocytic sorting. Therefore, the reduction in synapsin levels likely represents a decrease in vesicle number within presynaptic terminals. Indeed, this conclusion is well supported by the comparable level of reduction in vesicle density observed at the ultrastructural level. However, the more pronounced effects on synaptotagmin and vGLUT cannot be attributed simply to a physical depletion of vesicles, suggesting a specifically reduced presence in vesicular membranes, consistent with trafficking defects (Mohrmann, 2008).

A mislocalization of synaptotagmin-1 was already reported in lethal stoned mutants, spawning discussions of a synaptotagmin-focused function of stoned proteins. New data shows that presynaptic expression of different vesicle proteins is differentially affected by reduced STNB levels, excluding the possibility that defective endocytosis causes an nonselective loss of vesicle proteins from synaptic boutons. The new data also suggest that STNB function might be important for the correct localization of a specified set of vesicle proteins, which questions whether the loss of synaptotagmin-1 alone is primarily responsible for the physiological phenotypes observed in stoned mutants. Based on the finding that stnB hypomorphs exhibit impaired neurotransmitter release, and accompanying alterations in vesicle protein configuration, it is considered most likely that STNB acts as a stabilizing and/or sorting factor for several synaptic vesicle proteins supporting this function. Similarly, a recent study (Diril, 2006) postulated that the mammalian STNB homolog stonin2 acts as an endocytic sorting adapter for synaptotagmin-1. Confusingly, however, STNB lacks several N-terminal WVxF motifs of its ortholog, which supposedly mediate a stonin2-AP2 interaction crucial for the reported stonin2-induced acceleration of synaptotagmin endocytosis. Nevertheless, association with other AP2-interacting proteins might also enable the recruitment of STNB to appropriate sites (Mohrmann, 2008).

Synaptotagmin sorting is predicted to critically depend on the ability to interact with the MHD of STNB. To examine the role of the MHD-synaptotagmin interaction, targeted mutations were generated to abolish this binding capability. Most interestingly, the complete removal of the MHD, and similarly the introduction of two point mutations within the putative binding interface for synaptotagmin, prevented the expressed STNB protein from restoring viability in lethal stoned mutants. Epitope-tagged mutant variant proteins could be neuronally expressed in wildtype animals, but completely failed to localize at synaptic sites. This suggests a crucial role for MHD-based interactions in presynaptic trafficking or anchorage of STNB. It is noteworthy that the STNB Y1125G, R1135A variant could still enter proximal parts of the axon, possibly indicating defective active transport of the protein. In contrast, the C-terminally truncated STNB variant is fully retained in discrete, punctate accumulations within the soma. Surprisingly, a similar, truncated variant of stonin2 seems to distribute uniformly in mammalian neurons (Walther, 2004). This might be due to higher expression levels, or different interactions of the remaining N-terminal portion of stonin2. A STNB-AP50 chimera, which contains corresponding sequences of AP50 instead of its original MHD, was also completely missing from synaptic sites, and exhibited punctate somatic localization similar to the truncated STNB variant. Thus, the MHD and the μ2-subunit are not functionally equivalent, even though the transplanted sequences contain the putative synaptotagmin-binding interface, and are predicted to confer the ability to bind synaptotagmin. Hence, STNB localization is not dependent on an association with synaptotagmin. This conclusion is independently confirmed by showing that synaptotagmin null mutants sytAD4 exhibit only relatively minor changes in STNB localization (Mohrmann, 2008).

Unlike the well-established SYT-STNB interaction, a direct binding activity between vGLUT and STNB has not been tested. Though the molecular mechanisms of STNB dependent vGLUT localization/stabilization are unclear, several recent studies in mammals report a direct interaction between its vertebrate homolog, VGLUT1, and endophilin, thereby establishing a connection to the endocytic protein network. It will be very interesting to examine the exact relationship between STNB and vGLUT in the future (Mohrmann, 2008).


EVOLUTIONARY HOMOLOGS

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery, have been identified. The human stonins are homologous to the Drosophila melanogaster Stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery (Martina, 2001).

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 has been shown to localize to axonal vesicle clusters through its micro-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to micro2. Mutation of the tyrosine motif-binding pocket of micro2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2001).

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 localizes to axonal vesicle clusters through its mu-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to mu2. Mutation of the tyrosine motif-binding pocket of mu2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2004).

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. This study shows that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. These data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling (Diril, 2006).


REFERENCES

Search PubMed for articles about Drosophila stoned A and stoned B

Andrews, J., et al. (1996). The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides. Genetics. 143(4): 1699-711. 8844157

Cremona, O. and De Camilli, P. (1997). Synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 7: 323-330. 9232811

De Camilli, P. and Takei, K. (1996). Molecular mechanisms in synaptic vesicle endocytosis and recycling. Neuron 16: 481-486. 8785046

Diril, M. K., Wienisch, M., Jung, N., Klingauf, J. and Haucke, V. (2006). Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling. Dev. Cell. 10(2): 233-44. 16459302

Estes, P. S., et al. (2003). Functional dissection of a eukaryotic dicistronic gene: Transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants. Genetics: 165: 185-196. 14504226

Fergestad, T., Davis, W. S. and Broadie, K. (1999). The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal. J. Neurosci. 19(14): 5847-60. 10407025

Fergestad, T. and Broadie, K. (2001). Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis. J. Neurosci. 21(4): 1218-27. 11160392

Gonzalez-Gaitan, M. and Jackle, H. (1997). Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. Cell 88: 767-776. 9118220

Grigliatti, T. A., et al. (1973). Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults. Molec. gen. Genet. 120: 107-114. 4631264

Haucke, V. and De Camilli, P. (1999). AP-2 recruitment to synaptotagmin stimulated by tyrosine-based endocytic motifs. Science 285: 1268-1271. 10455054

Kelly, L. E. (1983a). The regulation of phosphorylation of a specific protein in synaptosomal fractions from Drosophila heads: the effects of light and two visual mutants. Cell. Mol. Neurobiol. 3(2): 127-41. 6317178

Kelly, L. E. (1983b). An altered electroretinogram transient associated with an unusual jump response in a mutant of Drosophila. Cell. Mol. Neurobiol. (2): 143-9. 6418384

Martina, J. A., et al. (2001). Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery. J. Cell Biol. 153: 1111-1120. 11381094

Miklos, G. L., et al. (1987). Localization of the genes shaking-B, small optic lobes, sluggish-A, stoned and stress-sensitive-C to a well-defined region on the X-chromosome of Drosophila melanogaster. J. Neurogenet. 4(1):1-19. 3104567

Mohrmann, R., et al. (2008). Stoned B mediates sorting of integral synaptic vesicle proteins. Neuroscience 153(4): 1048-1063. PubMed Citation: 18436388

Petrovich, T. Z., Merakovsky, J. and Kelly, L. E. (1993). A genetic analysis of the stoned locus and its interaction with dunce, shibire and Suppressor of stoned variants of Drosophila melanogaster. Genetics 133(4): 955-65. 8462853

Phillips, A. M., et al. (2000). The products of the Drosophila stoned locus interact with synaptic vesicles via synaptotagmin. J. Neurosci. 20(22): 8254-61. 11069931

Roos, J. and Kelly, R. B (1998). Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J. Biol. Chem. 273: 19108-19119. 9668096

Salcini A. E., et al. (1997). Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev 11: 2239-2249. 9303539

Soekmadji, C., Angkawidjaja, C. and Kelly, L. E. (2012). Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1. PLoS One 7: e38822. PubMed ID: 22701718

Stimson, D. T., et al. (1998). A product of the Drosophila stoned locus regulates neurotransmitter release. J. Neurosci. 18(23): 9638-49. 9822725

Stimson, D. T., et al. (2001). Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization. J. Neurosci. 21(9): 3034-44. 11312288

Tebar, F., et al. (1996). Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J. Biol. Chem. 271: 28727-28730. 8910509

Walther, K. et al., (2001). Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating. EMBO Rep. 2: 634-640. 11454741

Walther, K., et al. (2004). Functional dissection of the interactions of stonin 2 with the adaptor complex AP-2 and synaptotagmin. Proc. Natl. Acad. Sci. 101: 964-969. 14726597

Zhang, J. Z., et al. (1994). Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78: 751-760. 8087843

Zhang, B., et al. (1998). Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron 21: 1465-1475. 9883738

Zhang, Y. Q. and Broadie, K. (1999); Cloning, mapping and tissue-specific expression of Drosophila clathrin-associated protein AP50 gene. Gene 233(1-2): 171-9. 10375633


Biological Overview

date revised: 25 May 2013

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.