BIOLOGICAL OVERVIEW

There are two Drosophila NSF genes encoding polypeptides with 84% amino acid identity to one another, designated comatose (dNSF1) and dNSF2 (Boulianne, 1995; Pallanck, 1995). dNSF1 is expressed at high levels in adult flies and at relatively lower levels earlier in development, while dNSF2 is expressed at similar levels during the larval and adult stages of development. Temperature-sensitive alleles of dNSF1, termed comatose (here designated dNSF1^comt), were identified in a classical genetic screen for mutations causing paralysis at elevated temperature (Siddiqi, 1976). Electrophysiological and ultrastructural studies of dNSF1^comt mutants have demonstrated that dNSF1 plays a priming role in preparing docked synaptic vesicles for fast calcium-triggered fusion (Kawasaki, 1998). Further, biochemical characterization of dNSF1^comt mutants indicates that the dNSF1 priming role involves disassembly or rearrangement of a neural SNARE complex at the plasma membrane (Tolar, 1998; Golby, 2001 and references therein).

Vesicle trafficking in the constitutive and regulated secretory pathways requires a set of core polypeptides that mediate interactions between the transport vesicle and the target membrane. Among these core polypeptides are vesicular membrane proteins known as v-SNAREs; target membrane proteins known as t-SNAREs, and a soluble ATPase known as the N-ethylmaleimide-sensitive fusion protein (NSF). Although the precise functional roles of these components in vesicle exocytosis remain unclear, a large body of work has demonstrated that v-SNAREs and t-SNAREs (see the t-SNARE Syntaxin 1A) can assemble to form a complex and that NSF can catalyze disassembly of this complex by coupling SNARE complex disassembly to ATP hydrolysis (Jahn, 1999; Klenchin, 2000; Lin, 2000; Wickner, 2000). These observations, together with other work on NSF and SNARE function, suggest several models by which these components may contribute to vesicle exocytosis. One possibility is that NSF is recruited to a SNARE complex anchoring a secretory vesicle to its target membrane and promotes membrane fusion by catalyzing complete or partial disassembly of the SNARE complex. Another model consistent with available data on NSF and SNARE function is that NSF is required after vesicle fusion to disassemble a SNARE complex formed prior to or during membrane fusion, thereby reactivating SNAREs for another round of exocytosis. Yet another possibility is that NSF plays both prefusion and postfusion roles in SNARE complex metabolism (Golby, 2001 and references therein).

More recent work suggests the possibility of novel functional roles for NSF. A mutationally altered version of NSF that apparently lacks both ATPase and SNARE complex disassembly activity is capable of promoting membrane fusion in an in vitro Golgi reassembly assay, suggesting, among other possibilities, a role for NSF in SNARE activation prior to membrane fusion (Muller, 1999). Another recent series of studies has shown that NSF may regulate the function or subcellular distribution of the AMPA class of postsynaptic ionotropic glutamate neurotransmitter receptors in hippocampal neurons (Nishimune, 1998; Osten, 1998; Song, 1998; Luscher, 1999; Luthi, 1999; Noel, 1999). This function of NSF appears distinct from its more familiar role in vesicle trafficking, as it requires direct binding of NSF to the glutamate receptor. Several models have been proposed to explain these results: NSF may regulate the insertion of glutamate receptors into the plasma membrane, anchor the receptors to prevent lateral diffusion away from release sites, or act as a chaperone to regulate receptor conformation or interactions with other proteins (Golby, 2001 and references therein).

While studies of the dNSF1^comt mutants have revealed much about the role that dNSF1 plays in neurotransmitter release, several features of dNSF1 function remain unexplored. For example, while it is well established that dNSF1 plays an important presynaptic role in the adult nervous system, it is not known whether it plays a similar functional role prior to the adult stage of development. Further, it remains unknown whether dNSF1 functions in a postsynaptic capacity to regulate glutamate receptor function or whether dNSF1 plays secretory roles outside of the nervous system. It also remains completely unclear what function is provided by the closely related dNSF2 gene. To investigate these issues, mutations were generated in dNSF2 and subjected dNSF2 mutants, along with novel loss-of-function dNSF1 mutants, to molecular and genetic analysis. Results of these analyses show that dNSF2 function is required for viability beginning at the first instar larval stage of development, while dNSF1 function is required for viability at the adult stage of development (Golby, 2001).

Transgenic rescue experiments using the GAL4 system indicate that the primary function of dNSF1 resides in the nervous system. Expression of dNSF1 in the nervous system suppresses the effects of dNSF1^comt mutants. Ectopic expression of dNSF2 protein in the nervous system is insufficient for significant rescue of the recessive lethal dNSF2 mutant phenotype. Instead, rescue of the dNSF2 mutants is produced by ectopic expression of dNSF2 protein in mesoderm. These results are supported by previous work establishing that dNSF2 is expressed in this tissue (Boulianne, 1995). Mesoderm is the precursor to several larval and adult tissues in Drosophila, principally visceral and somatic muscle, suggesting that dNSF2 may play an essential functional role in muscle. The fact that dNSF2 mutant larvae initially display normal locomotion suggests that muscle differentiation and early events in neuromuscular junction formation are not adversely affected by the dNSF2 mutations. A model more consistent with the dNSF2 lethal phase is that the dNSF2 mutants are defective in the synaptic growth and maturation that occurs during larval development, a process that requires proper trafficking of muscle-specific membrane proteins and secretion of a retrograde signal that acts to regulate presynaptic activity (Golby, 2001).

Alternatively, another possible role for dNSF2 in muscle is suggested by recent work demonstrating that postsynaptic glutamate receptor (see Drosophila Glutamate receptor IIA and Glutamate receptor IIB) function or subcellular localization is regulated by NSF in the vertebrate nervous system. Glutamatergic neurotransmitter receptors reside in the Drosophila larval body wall muscles in opposition to neurotransmitter release sites and are essential for fast synaptic transmission at the larval neuromuscular junction. Thus, similar interactions between dNSF2 and Drosophila glutamate receptors in muscle could explain the mesoderm-specific role of dNSF2. Although the vertebrate glutamate receptor sequences that mediate interactions with NSF are poorly conserved in the Drosophila glutamate receptors, the dNSF2 protein may have evolved compensatory changes to allow maintenance of this interaction. Another possibility is that dNSF2 interacts with other muscle proteins involved in synapse development or function that contain sequences similar to the NSF-binding sequence present in mammalian glutamate receptors. Further experiments are currently in progress to define the precise functional role of dNSF2 in muscle (Golby, 2001 and references therein).

In addition to the role of dNSF2 in mesoderm, the increased efficiency of rescue of dNSF2 mutations mediated by simultaneous expression of dNSF2 in the nervous system and mesoderm indicates that dNSF2 also functions in the nervous system. This is also supported by the finding that expression of dNSF2 in the nervous system alone can extend the lethal phase of the dNSF2 mutants. It is possible that dNSF2 participates in constitutive vesicle trafficking in the nervous system, and/or that dNSF2 collaborates with dNSF1 in presynaptic neurotransmitter release mechanisms, as suggested by experiments showing that dNSF2 can rescue the dNSF1 mutations and can participate in neural SNARE complex disassembly. Alternatively, dNSF2 may be involved in regulating postsynaptic membrane insertion or localization of glutamate receptors in the central nervous system. Future electrophysiological studies and glutamate receptor localization experiments involving the dNSF2 mutants should resolve these possibilities (Golby, 2001).

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).

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, which 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 has been 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 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., Syntaxinís 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).

PROTEIN STRUCTURE

Amino Acids - 745 (Comatose) and 752 (Nsf2)

Structural Domains

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. A second homolog of NSF, called dNSF-2, has been identified and it is a ubiquitous and widely utilized fusion protein belonging to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1); 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases (Boulianne, 1995).

date revised: 25 June 2001

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