InteractiveFly: GeneBrief

Synaptobrevin: Biological Overview | References

BIOLOGICAL OVERVIEW

The SNARE proteins may be segregated into four major subfamilies based on the amino acid sequence homologies of the SNARE domains (see Phylogenetic tree of SNAREs) The Syn subfamily is also referred to as Qa subfamily, while the S25N and S25C are the Qb and Qc subfamilies, respectively. Members of the VAMP subfamily (the subject of this gene page of The Interactive fly) are all R-SNAREs. The classification of Qa, Qb, Qc, and R SNAREs is based on the residue at the position of the zero ionic layer of the 4-helical bundle. Analysis of SNARE complex formation in reconstituted lipid vesicles has provided another designation for t-SNAREs. Qa/Syn SNARE is considered to be the heavy chain, while Qb and Qc SNAREs (either from the same protein such as SNAP-25 or from two different proteins) are considered as the light chains (Hong, 2005).

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem a powerful, quantitative assay was developed for measuring secretion, and it was used in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed identification of at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/SNAP29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes (Gordon, 2017).

Constitutive secretion delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability. This pathway is required for the exocytosis of molecules such as antibodies, cytokines and extracellular matrix components so has both significant physiological and commercial importance. The majority of constitutive secreted proteins are synthesised at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles and tubules which fuse with the plasma membrane. Constitutive secretory vesicles are not stored within the cell and do not require a signal to trigger their fusion with the plasma membrane which is in contrast to dense core secretory granules or synaptic vesicles. In some cell types, such as MDCK cells and macrophages, there is evidence that constitutive secretory cargo passes through a endosomal intermediate on its way to the cell surface. However, in non-polarised cells endosomal intermediates do not appear to play a major role in this pathway (Gordon, 2017).

Vesicle fusion is driven by a family of molecules known as SNAREs. SNARE are generally small (14-42kDa), C-terminally anchored proteins that have a highly conserved region termed the SNARE motif that has the ability to interact with other SNAREs. For membrane fusion to occur, SNAREs on opposing membranes must come together and their SNARE motifs zipper up to form a SNARE complex. Detailed characterisation of the neuronal SNARE complex (syntaxin 1A/VAMP2/SNAP25) required for synaptic vesicle fusion has provided a mechanistic framework for understanding the function of SNAREs. There are 38 SNAREs encoded in the human genome and they can be classified as either R or Q-SNAREs depending on the presence of a conserved arginine or glutamine in their SNARE motif. Q-SNAREs can be further subdivided into Qa, Qb and Qc SNAREs based on their homology to syntaxin and SNAP25. A typical fusogenic SNARE complex will contain four SNARE motifs (Qa, Qb, Qc and R). Qbc-SNAREs such as SNAP23, 25, 29 and 47 contribute two SNARE motifs to the SNARE complex. R-SNAREs can also be further classified as either longin or brevin type SNAREs. Longin type R-SNAREs contain a longin type fold and are found in all eukaryotes and while brevin type SNAREs are less widely conserved across species (Gordon, 2017).

Over the past twenty years significant progress has been made defining the SNARE complexes required for the majority of intracellular transport steps within eukaryotic cells. In addition, there are an increasing number of examples where the SNARE complexes required for the secretion of specific cargo such as Wnt, TNF and IL-6 have been identified. However, these proteins are not delivered directly to the cell surface from the TGN but pass through an endosomal compartment. Many labs have attempted to identify the machinery which drive the fusion of constitutive secretory vesicles with the plasma membrane and on the whole very little progress has been made. This in part may be due to the fact that there are multiple routes to the cell surface from the Golgi and redundancy in the fusion machinery. If just the R-SNAREs are considered, the human genome encodes seven post-Golgi SNAREs and a typical mammalian cell line can express at least five R-SNAREs so disruption of just one R-SNARE is unlikely to block secretion if they are functionally redundant. To overcome this problem SNARE function was characterized in Drosophila cells , as they have a simpler genome with less redundancy. The Drosophila genome encodes 26 SNAREs with 16 of them predicted to be localised to post-Golgi membranes based on their homology to mammalian SNAREs. The complexity is reduced even further as Drosophila cell lines just express two post-Golgi R-SNAREs, Syb and VAMP7 (based on publically available microarray data generated by the modENCODE project) (Gordon, 2017).

This study has developed a novel, quantitative assay for measuring constitutive secretion based on a reporter cell line that can be effectively used to monitor secretion by flow cytometry, immunoblotting and fluorescence microscopy. Depletion of known components of the secretory pathway in Drosophila cells (STX5, SLH and ROP) causes robust blocks in ER to Golgi and Golgi to plasma membrane transport, therefore validating this approach. As predicted, there is redundancy in the post-Golgi SNAREs and multiple SNAREs must be depleted to obtain robust blocks in secretion. This study has detected strong negative genetic interactions between Drosophila STX1 and STX4, SNAP24 and SNAP29, STX1 and Syb, and SNAP24 and Syb. A novel and unexpected genetic interaction was detected between Syb and YKT6. Depletion of YKT6 and VAMP3 in mammalian cells also causes a robust block in secretion indicating that this negative genetic interaction is conserved across species and provides evidence that these two R-SNAREs function in the late secretory pathway (Gordon, 2017).

Using well characterised targets (STX5, SLY1 and ROP) this study has validated the system and the assay was shown to be capable of differentiating blocks in ER to Golgi and Golgi to plasma membrane transport based on proteolytic processing and accumulation of the secretory cargo in post-Golgi transport vesicles. The experimental data suggests that there are at least three fusion complexes operating at the Drosophila PM. The first complex consists of STX1, SNAP24/29 and Syb. The second complex consists of STX4, SNAP24/29 and Syb. The third complex consists of STX1, SNAP24 and YKT6. The reason the possibility of a STX4, SNAP24/29, YKT6 complex was excluded is because depletion of both STX1 and Syb led to a complete block in secretion. Indicating that STX4 and YKT6 are unable to form a SNARE complex that can substitute for the loss of STX1 and Syb. Genetic interaction data also suggests that SNAP29 is unable to substitute for the loss of SNAP24 under conditions when both SNAP24 and Syb are depleted. This data suggests that the third SNARE complex specifically consists of STX1, SNAP24 and YKT6. At present it is unclear whether these SNARE complexes define parallel pathways to the plasma membrane or simply reflect the ability of these SNAREs to substitute with each other (Gordon, 2017).

The most striking observation in this study is that an unexpected role for YKT6 in the fusion of secretory carriers with the plasma membrane was uncovered. Depletion of YKT6 and Syb/VAMP3 in combination causes a complete block in secretion and leads to an accumulation of post-Golgi transport vesicles within Drosophila cells. YKT6 is a lipid anchored R-SNARE that has been shown to function on many pathways including ER to Golgi transport, intra-Golgi transport, endosome-vacuole fusion, endosome to Golgi transport and exosome fusion with the plasma membrane. YKT6 actively cycles on and off membranes in a palmitoylation dependant manner so potentially it is well suited to function on a wide variety of intracellular pathways. Due to the promiscuous nature of YKT6 some caution must be taken when interpreting rhe functional data. It is possible that loss of YKT6 may be indirectly affecting post-Golgi transport and fusion at the plasma membrane. However, the simplest interpretation of this data is YKT6 is directly involved in this process as this study was able to biochemically detect an interaction between YKT6 and STX1 (Gordon, 2017).

Using the knowledge obtained from the Drosophila system, the role of R-SNAREs in constitutive secretion in mammalian cells was reexamined. As previously reported, depletion of VAMP3 and other post-Golgi R-SNAREs did not perturb secretion in HeLa cells. However, depletion of VAMP3 and YKT6 in combination caused a complete block in secretion. This data suggests that YKT6 and VAMP3 may be functioning in the fusion of secretory carriers with the plasma membrane in mammalian cells. Significant efforts were made to localise endogenous YKT6 and VAMP3 on post-Golgi secretory carriers. However, the attempts have been hampered by the fact the endogenus YKT6 is expressed at very low levels and over expressed YKT6 does not target correctly to membranes and remains cytoplasmic (Gordon, 2017).

As expected, there is redundancy in the Q-SNAREs required for the fusion of secretory carriers with the plasma membrane. However, it is clear that certain SNAREs have a more prominent role in this process. The main Q-SNAREs at the Drosophila plasma membrane are STX1 and STX4. Depletion of STX1 causes a partial block in secretion while depletion of STX4 does not. It is unclear why STX1 is the favoured Qa-SNARE. It could simply be that STX1 is more abundant than STX4 or has a higher affinity for the R-SNARE on the vesicle. It may also reflect the route by which the synthetic cargo traffics to the cell surface. This study also observed redundancy between the Qbc-SNAREs SNAP24 and SNAP29 (orthologues of Sec9). A complete block in secretion is detected when both are depleted. It has previously been shown that SNAP29 interacts with STX1. However, the complexes it forms are not SDS-resistant suggesting that they may not be fusogenic (Gordon, 2017).

A potential problem with gene disruption and RNAi mediated depletion studies is compensation by other genes in the same family. For example, VAMP2 and 3 are upregulated in certain tissues of the VAMP8 knockout mouse and VAMP3 is upregulated in VAMP2 deficient chromafin cells isolated from VAMP2 null mice. Based on immunoblotting data this study did not observe any compensation between R-SNAREs when they are depleted using RNAi in Drosophila cells. No evidence was seen of this in previous work performed in HeLa cells. It was initially thought that STX1 and STX4 were being upregulated in STX5 and Syb depleted cells based on immunoblotting. However, when the samples were directly prepared in Laemmli sample buffer, rather than a TX100 based extraction buffer, no difference in the levels of these SNAREs was observed. It is possible that the change in extractability may be caused by an alteration in the localisation of the Q-SNAREs from TX100 insoluble micro-domains at the plasma membrane. However, this hypothesis was not tested. To directly assess changes in gene expression during the RNAi experiments the mRNA levels were measured of several SNAREs using RT-PCR. Depletion of STX1 leads to an upregulation of STX4 and Syb. However, no significant change was observed in the protein level of these SNAREs by immunoblotting. Thus it is unclear how significant these changes are. In the future, it will be interesting to determine how the expression levels of SNAREs, which function on the same pathway, are co-ordinated and regulated (Gordon, 2017).

To validate the genetic interaction data a published S. cerevisiae proliferation-based genetic interaction map was have interrogated to determine if the yeast homologues share similar genetic interactions to those observed in Drosophila cells (under the assumption that constitutive secretion is essential for growth). Negative genetic interactions were observed between Drosophila STX1 and STX4, STX1 and Syb, Syb and SNAP24, SNAP24 and SNAP29, YKT6 and Sec22b and Syb and YKT6. Similar genetic interactions were also observed in S. cerevisiae indicating that the data generated from Drosophila cells is physiologically relevant and the genetic interactions are evolutionary conserved. Importantly the homologues of YKT6 and Syb/VAMP3 were also found to genetically interact in yeast (YKT6 and SNC2) (Gordon, 2017).

In summary, this study has identified the SNARE complexes required for the fusion of constitutive secretory vesicles with the plasma membrane in Drosophila cells. This study has uncovered a novel role for YKT6 in the fusion of secretory vesicles with the plasma membrane which is conserved from yeast to man. This observation may in part explain why RNAi and gene disruption studies in higher eukaryotes have failed to yield the expected phenotypes. In the future, it should be possible to use the secretion assay in combination with SNARE depletion as a tool to further dissect the post-Golgi pathways involved in secretion and generate post-Golgi secretory carriers for proteomic profiling (Gordon, 2017).

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, the expression of each gene was driven in the absence of the other to look for phenotypic rescue. Expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, this study finds that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms (Bhattacharya, 2002).

SNARE-dependent signaling at the Drosophila wing margin

The wing of Drosophila has long been used as a model system to characterize intermolecular interactions important in development. Implicit in an understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. The Drosophila wing was used to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and a mutant form of NSF was constructed whose expression was directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, this phenotype was used to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. It is concluded that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways can act as a sensitive reporter of partial membrane-trafficking disruption (Stewart, 2001).

The Syntaxin, VAMP, and SNAP-25 families of proteins are proposed to target and fuse transport vesicles with specific membrane compartments. The SNARE complex is a parallel four-helix bundle with one helix contributed by each of Syntaxin and VAMP and two contributed by SNAP-25. The formation of a trans-membrane complex, with VAMP on the transport vesicle and Syntaxin and SNAP-25 on the target membrane, is thought to lead to the fusion of the two membranes, resulting in a cis-membrane complex. It follows that the cis-residing protein complexes need to be broken apart to make those proteins available for further trans-complex formation. This complex breakdown occurs under the action of N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase. NSF contains two nucleotide binding domains and demonstrable ATPase activity. Structural analyses have shown that NSF forms a hexamer in vivo. NSF is a homolog of the yeast gene SEC18 and analysis of SEC18 function also reveals its requirement for intracellular membrane transport. NSF-dependent ATP hydrolysis is required to disassemble SNARE complexes, although it is not required for the fusion step. Thus the role of NSF in vesicular transport appears to be primarily one of priming vesicles for fusion and dissociation of SNARE complexes to permit their recycling (Stewart, 2001).

In Drosophila there are two homologs of NSF: dNSF1 and dNSF2 (NEM-sensitive fusion protein 2). dNSF1 is the gene product of comatose and is primarily found in neurons, whereas dNSF2, in addition to being neuronally expressed, is broadly expressed within imaginal discs, salivary glands, and the ring gland. Thus, dNSF2 is the most likely isoform to contribute to intracellular trafficking in nonneuronal tissue. Despite their proposed role in most intracellular trafficking events, in vivo studies of SNARE proteins have concentrated on two main systems: the budding yeast and calcium-triggered exocytosis in neurons. Relatively little attention has been given to other in vivo contexts in which the SNARE proteins are likely to have important roles. For example, in signaling pathways it is self-evident that transmembrane receptors and ligands need to be delivered to the plasma membrane, although few studies have been devoted to specifically studying the role of SNARE proteins in this process and their potential influence on the strength of intracellular signaling (Stewart, 2001).

To investigate the role of SNARE proteins within a defined developmental process, advantage was taken of the key role of NSF in membrane-transport processes. Specifically, a dominant negative form of dNSF2 was expressed in wing imaginal discs and this was shown to disrupt proper wing margin formation. This phenotype is enhanced in trans-heterozygous combinations of mutant alleles of the SNARE proteins syntaxin or synaptobrevin, further supporting a role for SNAREs in this process. Using genetic and immunocytochemical analysis it has been shown that this phenotype can be attributed to a failure in the signaling pathways that normally govern wing margin development. Thus, SNARE-dependent transport mechanisms are critical to wing formation and their manipulation may provide new insights into the mechanisms controlling developmentally important signaling pathways (Stewart, 2001).

To investigate the function of SNARE-dependent transport mechanisms in Drosophila point mutants in the ATP-binding region of the D1 domain of dNSF2 were constructed. Each nucleotide-binding subdomain of NSF contains consensus ATP-binding domains known as the Walker A and Walker B motifs. The DEAD box of the Walker B motif is conserved in a large number of ATP-dependent enzymes and was first identified in RNA helicases that use ATP hydrolysis to unwind RNA prior to translation. This motif binds the Mg2⁺ ion that coordinates the phosphates of ATP for hydrolysis. In RNA helicases replacement of the glutamate residue within the modified DEAD box (DEID) eliminates ATP hydrolysis without affecting ATP binding). In mammalian NSF a similar substitution within that protein's DEID box, E329Q, reduces ATPase activity and NSF-dependent Golgi transport activity. NSF has been shown to form hexamers and, when mixed with wild-type protein NSF^E329Q, forms hexamers that also lack ATPase activity, leading to a dominant negative effect. Drosophila NSF2 shows 59% overall amino acid identity with CHO NSF and nearly 100% conservation within the ATP-binding p-loop and DEID box of the D1 domain. Thus the structural and functional properties of the dNSF2 ATPase domains are very likely to be identical to those previously defined in RNA helicases and mammalian NSF, and mutation of the glutamate residue with the Drosophila DEID box motif should also impair the ATPase activity of the protein (Stewart, 2001).

A NSF^E/Q construct was created with a glutamate-to-glutamine substitution at position 326 of the dNSF2 D1 domain. In two separate ATPase assays it was found that the NEM-sensitive ATPase activity of NSF^E/Q is 47.5% and 57.1% that of dNSF2^WT. The mean ATPase activity is 15.2 nmol Pi/microg/h for the wild-type protein and 7.8 nmol Pi/microg/h for the mutant protein. The remaining ATPase activity in NSF^E/Q may be attributable to the second ATPase site within the D2 domain of the protein (Stewart, 2001).

To express the mutant dNSF2 transgenic flies were created carrying UAS-NSF^E/Q and UAS-NSF^WT constructs for use in the Gal4-UAS expression system. C96-Gal4 is expressed in developing wing discs in a pattern that is similar to, though slightly broader than, wing margin proteins such as Wingless. When UAS-NSF^E/Q is driven by C96-Gal4, loss of wing margin is observed. The expression of NSF^WT does not cause any visible phenotype, indicating that simple overexpression of dNSF2 in the wing margin is not a cause of the phenotype (Stewart, 2001).

The observation that NSF^E/Q causes loss of wing margin implies that SNARE-dependent transport is important for wing margin formation. To test this further mutant alleles of synaptobrevin and syntaxin, two well-characterized SNARE proteins, were used to determine whether they would enhance the wing phenotype. Indeed, all trans-heterozygous combinations of NSF^E/QC96 with synaptobrevin or syntaxin loss-of-function alleles enhance the wing margin phenotype, thus providing further evidence of the involvement of SNARE proteins in wing margin development (Stewart, 2001).

The wing phenotype observed is similar to that observed with mutant alleles of Notch and Wingless signaling pathway genes. To determine whether components of these pathways could be contributing to the NSF^E/QC96 wing phenotype the protein pattern of Wingless in third-instar imaginal wing discs was first examined and a striking effect on the distribution of Wingless was observed. In control discs Wingless appears as a three- to four-cell-wide stripe across the wing disc, whereas in discs expressing the mutant dNSF2 Wingless appears very narrow and patchy. Wg expression was then examined using a Wg-lacZ reporter construct and an incomplete pattern of Wingless expression was found, as was observed for the Wingless protein (Stewart, 2001).

Because Wg is a secreted protein Wg was examined under higher magnification using confocal microscopy to determine directly whether Wg secretion was impaired. In control discs there is punctate Wg staining, indicative of Wg secretion, in the tissue surrounding the narrow stripe of wing margin cells. In the regions of the mutant discs that are immunoreactive for Wg, punctate staining is seen surrounding the positive cells. However, the Wg signal is much stronger in those cells and confocal sectioning of the cells has revealed the accumulation of Wg at the apical region of the wing margin cells. These data indicate that mutant NSF^E/Q impairs, but does not eliminate, Wingless secretion. Because Wingless expression is impaired and its activation is under the control of Notch signaling, the distribution patterns of other proteins involved in the Notch pathway were examined. Notch protein distribution was examined directly using a monoclonal antibody that recognizes the extracellular domain of Notch. At low magnification there is no major difference between mutant and control samples, with the antibody labeling the cell membranes in the wing pouch. However, at higher magnification, in addition to the membrane staining, immunoreactive puncta were also observed within the cells of the mutant wing disc that were not readily observed in the control discs. These puncta likely represent improperly sorted Notch proteins (Stewart, 2001).

The distribution of Cut, Delta, and Achaete, coded for by genes that are downstream of Notch activation in the wing margin signaling pathway, was examined -- all of these markers were disrupted in NSF^E/QC96 larval wing discs. Cut is normally found in a pattern that overlaps with Wg along the presumptive wing margin, whereas in the mutant discs it appears in a broken pattern similar to that of Wg. Delta is normally expressed in two parallel bands along the D/V boundary and this pattern is thought to be the result of the downregulation of Delta in boundary cells by Cut and the upregulation of Delta in flanking cells by Wingless. In NSF^E/QC96 wing discs the expression of Delta is reduced and the two parallel bands appear to be collapsed into a single band along the boundary. Achaete is normally expressed in two broad bands parallel to the D/V boundary in the anterior compartment of the wing disc defining a proneural cluster. In the NSF^E/QC96 discs this pattern is severely disrupted: the number of Achaete-expressing cells is reduced and there is complete absence of Achaete in some areas (Stewart, 2001).

A similar pattern of disruption was found when lacZ reporter constructs were used to examine the expression of neuralized and vestigial, two other genes in the Notch pathway. neu^A101-lacZ is normally detected in sensory organ precursors (SOPs) located in two rows of single cells parallel to the D/V boundary in the anterior compartment of late third-instar wing discs. In the mutant discs this pattern is disrupted and lacking in some areas along the wing margin, while SOPs elsewhere in the disc are unaffected. Similarly, vg^BE-lacZ expression is disrupted. In wild-type discs vg^BE-lacZ expression is seen in the D/V and anterior/posterior (A/P) boundaries, whereas in the mutant discs the expression in the D/V boundary is disrupted (Stewart, 2001).

Interestingly, expression in the A/P boundary remains, although the C96-Gal4 expression pattern overlaps this region. Taken together these results demonstrate that NSF^E/Q affects the distribution and expression of several downstream components of the Notch signaling pathway. To confirm the effect of NSF^E/Q on Notch signaling loss-of-function alleles of several genes in the Notch and Wingless pathways were examined for their ability to enhance the adult wing phenotype caused by NSF^E/Q expression. In that Notch signaling is known to be highly sensitive to haploinsufficiency of interacting gene products, it was reasoned that these loss-of-function alleles should show genetic interaction. Two alleles of Notch and one each of Delta, Serrate, wingless, and fringe were examined and it was found that they all enhanced the wing phenotype in transheterozygous combination with NSF^E/QC96. The severity of the phenotype produced by each allele was similar, although Df(1)N8, a null allele of Notch, did produce a more severe phenotype than did N^nd-3, a hypomorphic allele. With the exception of Df(1)N8, none of these mutants produces a wing-nicking phenotype when examined alone as heterozygotes. Thus, the enhancement of the adult wing phenotype by mutants in the Notch pathway supports the conclusion that NSF^E/Q expression causes a defect in wing margin signaling pathways (Stewart, 2001).

Finally, the ability of UAS constructs of Notch, Delta, and Serrate to rescue the wing phenotype generated by NSF^E/QC96 were tested. Complete rescue could be obtained with both Notch and Delta constructs. Serrate generally appears to rescue less well than do the other constructs because minor nicks in the distal wing persist. Furthermore, no rescue effect was seen when crosses were made to UAS-lacZ lines, indicating that competition for Gal4 protein is not responsible for rescue of the phenotype. The observation that UAS-Notch and UAS-Delta can completely rescue the NSF^E/Q wing phenotype further indicates that the mutation affects intracellular transport and does not create a cell-lethal phenotype because cell lethality should not be rescued by Notch or Delta (Stewart, 2001).

Having established that NSF^E/Q disrupts signaling at the wing margin in a SNARE-dependent manner, and that enhancement of the phenotype can be attributed to haploinsufficiency of known genes, it was asked whether the wings of the NSF^E/QC96 flies could be used as a sensitized background to find novel genes involved in wing margin formation. To this end a small-scale screen was conducted for enhancers and suppressors of the phenotype. In the first set of experiments specific alleles of two genes were tested: big brain and porcupine. These have been shown to be important in Notch and Wingless signaling in other developmental contexts but have not previously been known to be important for wing margin development. In the NSF^E/QC96 background it was found that both mutant alleles of these genes enhance the NSF^E/QC96 wing margin phenotype. This result is the first report of the involvement of these two genes in wing margin development and suggests that NSF^E/QC96 wings provide an ideal sensitized background for conducting forward genetic screens to identify novel genes involved in wing margin development (Stewart, 2001).

In the second set of experiments a test was performed for genetic interactions with deficiencies that uncover most of the Drosophila genome. Of the deficiencies tested, 33 interacting lines were identified that enhanced or suppressed the wing margin phenotype. The further characterization of these loci may reveal novel components of the SNARE or Notch and Wg signaling pathways (Stewart, 2001).

In view of current membrane-trafficking models, it is expected that expressing NSF^E/Q impairs the ability of NSF to dissociate cis-SNARE complexes, making fewer SNARE proteins available for functional transmembrane complex formation and thus reducing intracellular transport. These results provide solid evidence that SNARE proteins are important in wing margin formation. This implies that the mutant NSF must suppress but not block all membrane traffic. The disruption of molecular markers, such as Wg, Delta, Achaete, Cut, Vestigial, and Neuralized, indicates that the NSF^E/Q wing phenotype observed is the result of impaired signaling at the developing wing margin. This is consistent with data presented in other studies that manipulated the signaling pathway directly. For example, reduction of Notch activity with N^ts alleles can lead to reduced and patchy Wingless expression. Wingless and Cut expression is also reduced and patchy in Notch mutant wing discs. Stripes of Delta and Serrate that normally flank the D/V boundary collapse into a single stripe along the margin in N^ts alleles exposed to restrictive temperature. In NSF^E/QC96 wing discs changes in Wingless, Cut, and Delta patterns were observed that are similar to those that occur when Notch activity is directly manipulated; therefore, it seems that NSF^E/Q expression phenocopies genetic mutants of Notch (Stewart, 2001).

Because the Notch and Wingless signaling pathways are so intertwined in controlling wing margin development it is difficult to determine whether the dNSF2 mutants cause a primary defect in one or the other of these proteins, although it seems likely that there are parallel effects on both. The experiments show not only a direct impairment of Wingless trafficking but also that Wg-lacZ expression is disrupted. The latter suggests that an upstream activator of Wingless expression is impaired (although this could be Wingless itself). It is found that Notch subcellular localization is disrupted and that a Wg-independent target of Notch signaling, the vestigial boundary enhancer, is also disrupted. Because this vestigial enhancer element is thought to be under the sole control of Notch this supports the idea that NSF^E/Q has a direct effect on Notch signaling. Thus vg^BE-lacZ expression the data strongly suggest direct effects on both Wg and Notch. Moreover, because these molecules are at the top of the hierarchy controlling signaling at the wing margin this provides the likely explanation for the disruption of downstream targets of these genes (Stewart, 2001).

The molecular and genetic interactions that regulate developmentally important signaling pathways are important for defining the final outcome of the signaling cascade. For example, previous studies have identified several molecules, including Fringe, Big Brain, and Numb, that are proposed to influence Notch signals. Because the SNARE proteins interact with many protein partners, some of which are proposed to regulate their availability (e.g., Syntaxin's interaction with rop/nsec-1), these data indicate that regulation of SNARE-dependent transport steps may represent an additional mechanism by which signal transduction pathways can be modulated during development (Stewart, 2001).

A synaptic vesicle membrane protein is conserved from mammals to Drosophila

The structure of synaptobrevin, an intrinsic membrane protein of small synaptic vesicles from mammalian brain, was studied by purification and molecular cloning. Its message in bovine brain encodes a 116 amino acid protein whose sequence reveals it to be the mammalian homolog of the ray Torpedo VAMP-1. Antibody probing demonstrates that the protein is also present in Drosophila, and its Drosophila homolog was cloned. Alignment of the sequences of synaptobrevin/VAMP-1 from the three species shows it to contain four domains, including a highly conserved central region of 63 amino acids that contains 75% invariant residues. The finding that a membrane protein from vertebrate synaptic vesicles is conserved in Drosophila points toward a central role of this protein in neurotransmission and should allow a genetic approach to neurotransmitter release (Sudhof, 1989).

Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells

VAMP (synaptobrevin) is a highly conserved membrane protein originally described as a component of brain synaptic vesicles. The Drosophila melanogaster VAMP-encoding gene (syb) comprises five exons. Splicing exons 1,2,3,4,5 (syb-b) results in a protein with a C-terminal hydrophobic domain and a negligible intraluminal domain. Splicing exons 1,2,3,5 (syb-a) predicts a protein with a 20-amino-acid luminal domain at the C terminus. The ratio of syb-a to syb-b transcripts is highly regulated during development. The syb transcripts show no enrichment in the nervous system and are present in very early embryos, well before neurogenesis. The greatest concentration of syb transcripts was found in cells of the gut and Malpighian tubules. Thus, syb may have a general role in membrane trafficking and, perhaps, a role in the secretion of digestive enzymes (Chin, 1993).

Search PubMed for articles about Drosophila Synaptobrevin

Bhattacharya, S., Stewart, B. A., Niemeyer, B. A., Burgess, R. W., McCabe, B. D., Lin, P., Boulianne, G., O'Kane, C. J. and Schwarz, T. L. (2002). Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability. Proc Natl Acad Sci U S A 99(21): 13867-13872. PubMed ID: 12364587

Chin, A. C., et al. (1993). Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells. Gene 131(2): 175-81. PubMed ID: 8406010

Gordon, D. E., Chia, J., Jayawardena, K., Antrobus, R., Bard, F. and Peden, A. A. (2017). VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane. PLoS Genet 13(4): e1006698. PubMed ID: 28403141

Hong, W. (2005). SNAREs and traffic. Biochim Biophys Acta 1744(2): 120-144. PubMed ID: 15893389

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date revised: 2 January 2023

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