InteractiveFly: GeneBrief

Nsf1 and Nsf2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - comatose and N-ethylmaleimide-sensitive factor 2

Synonyms - dNSF1 and dNSF2, NEM-sensitive fusion protein 2

Cytological map positions - 11E1 and 87F14-88A1

Function - vesicle fusion

Keywords - SNARE complex, vesicle fusion, mesoderm, CNS, synapse, neuromuscular junction

Symbol - comt and Nsf2

FlyBase IDs: FBgn0000346 and FBgn0266464

Genetic map position - 1- and 3-

Classification - AAA ATPase superfamily

Cellular location - cytoplasmic



NCBI link: Entrez Gene

comt orthologs: Biolitmine
Nsf2 orthologs: Biolitmine

Recent literature

Gokhale, A., et al. (2015). The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity. J Neurosci 35: 7643-7653. PubMed ID: 25972187.
Summary:
Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. This study consisted of a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. Components of the vesicle fusion machinery were identified as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, the role for NSF was studied in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, this study found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. These results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity.

Yamakawa, T., Atsumi, Y., Kubo, S., Yamagishi, A., Morita, I. and Matsuno, K. (2018). Insight into Notch signaling steps that involve pecanex from dominant-modifier screens in Drosophila. Genetics. Pubmed ID: 29853475
Summary:
Notch signaling plays crucial roles in intercellular communications. In Drosophila, the pecanex (pcx) gene, which encodes an evolutionarily conserved multi-pass transmembrane protein, appears to be required to activate Notch signaling in some contexts, especially during neuroblast segregation in the neuroectoderm. Although Pcx has been suggested to contribute to endoplasmic reticulum homeostasis, its functions remain unknown. This study performed a genetic modifier screens of pcx. This study found that pcx heterozygotes lacking its maternal contribution exhibit cold-sensitive lethality, which is attributed to a reduction in Notch signaling at decreased temperatures. Using sets of deletions that uncover most of the second and third chromosomes, four enhancers and two suppressors of the pcx cold-sensitive lethality were uncovered. Among these, five genes encode known Notch-signaling components: big brain, Delta (Dl), neuralized (neur), Brother of Bearded A (BobA), a member of the Bearded (Brd) family, and N-ethylmaleimide-sensitive factor 2 (Nsf2). BobA suppresses Dl endocytosis during neuroblast segregation in the neuroectoderm, as Brd family genes reportedly do in the mesoderm for mesoectoderm specification. Analyses of Nsf2, a key regulator of vesicular fusion, suggested a novel role in neuroblast segregation, which is distinct from Nsf2's previously reported role in imaginal tissues. Finally, jim lovell, which encodes a potential transcription factor, may play a role in Notch signaling during neuroblast segregation. These resalts reveal new research avenues for Pcx functions and Notch signaling.
Saadin, A. and Starz-Gaiano, M. (2018). Cytokine exocytosis and JAK/STAT activation in the Drosophila ovary requires the vesicle trafficking regulator alpha-Snap. J Cell Sci. PubMed ID: 30404830
Summary:
How vesicle trafficking components actively contribute to regulation of paracrine signaling is unclear. This study has genetically uncovered a requirement for alpha-Soluble NSF Attachment Protein (alpha-Snap) in the activation of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway during Drosophila egg development. alpha-Snap, a well-conserved vesicle trafficking regulator, mediates association of N-ethylmaleimide-Sensitive Factor (NSF) and SNAREs to promote vesicle fusion. Depletion of alpha-Snap or the SNARE family member Syntaxin1A in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in alpha-Snap-depleted egg chambers, indicating that the lack of border cells in mutants is due to impaired signaling. Genetic experiments implicate alpha-Snap and NSF in secretion of a STAT-activating cytokine. Live imaging suggests that changes in intracellular calcium may be linked to this event. These data suggest a cell-type specific requirement for particular vesicle trafficking components in regulated exocytosis during development. Given the central role for STAT signaling in immunity, this work may shed light on regulation of cytokine release in humans.
Warecki, B., Ling, X., Bast, I. and Sullivan, W. (2020). ESCRT-III-mediated membrane fusion drives chromosome fragments through nuclear envelope channels. J Cell Biol 219(3). PubMed ID: 32032426
Summary:
Mitotic cells must form a single nucleus during telophase or exclude part of their genome as damage-prone micronuclei. While research has detailed how micronuclei arise from cells entering anaphase with lagging chromosomes, cellular mechanisms allowing late-segregating chromosomes to rejoin daughter nuclei remain underexplored. This study finds that late-segregating acentric chromosome fragments that rejoin daughter nuclei are associated with nuclear membrane but devoid of lamin and nuclear pore complexes in Drosophila melanogaster. Acentrics pass through membrane-, lamin-, and nuclear pore-based channels in the nuclear envelope that extend and retract as acentrics enter nuclei. Membrane encompassing the acentrics fuses with the nuclear membrane, facilitating integration of the acentrics into newly formed nuclei. Fusion, mediated by the membrane fusion protein Comt/NSF and ESCRT-III components Shrub/CHMP4B and CHMP2B, facilitates reintegration of acentrics into nuclei. These results suggest a previously unsuspected role for membrane fusion, similar to nuclear repair, in the formation of a single nucleus during mitotic exit and the maintenance of genomic integrity.
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 dNSF1comt), were identified in a classical genetic screen for mutations causing paralysis at elevated temperature (Siddiqi, 1976). Electrophysiological and ultrastructural studies of dNSF1comt 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 dNSF1comt 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 dNSF1comt 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 dNSF1comt 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 NSFE329Q, 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 NSFE/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 NSFE/Q is 47.5% and 57.1% that of dNSF2WT. 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 NSFE/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-NSFE/Q and UAS-NSFWT 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-NSFE/Q is driven by C96-Gal4, loss of wing margin is observed. The expression of NSFWT 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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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. neuA101-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, vgBE-lacZ expression is disrupted. In wild-type discs vgBE-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 NSFE/Q affects the distribution and expression of several downstream components of the Notch signaling pathway. To confirm the effect of NSFE/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 NSFE/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 NSFE/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 Nnd-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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/QC96 background it was found that both mutant alleles of these genes enhance the NSFE/QC96 wing margin phenotype. This result is the first report of the involvement of these two genes in wing margin development and suggests that NSFE/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 NSFE/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 NSFE/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 Nts 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 Nts alleles exposed to restrictive temperature. In NSFE/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 NSFE/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 NSFE/Q has a direct effect on Notch signaling. Thus vgBE-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., 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).

The Neurexin-NSF interaction regulates short-term synaptic depression

Although Neurexins, which are cell adhesion molecules localized predominately to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their role in the regulation of synaptic vesicle release during repetitive nerve stimulation is unknown. This study shows that Drosophila neurexin mutant synapses exhibit rapid short-term synaptic depression upon tetanic nerve stimulation. Moreover, the intracellular region of Neurexin was demonstrated to be essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, it was found that the intracellular region of Neurexin interacts with N-ethylmaleimide sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. The binding sites of each molecule were mapped, and it was demonstrated that the Neurexin-NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. These results reveal a previously unknown role of Neurexin in the regulation of short-term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of Neurexin in synaptic vesicle release (Li, 2015).

Previous studies have shown that the α-NRXs functionally couple Ca2+ channels to the presynaptic machinery to mediate synaptic vesicle exocytosis and that defective synaptic vesicle release can be restored with 1 mm Ca2+ (Li, 2007). In the current experiments, 1.8 mm Ca2+ was used to eliminate the effects of abnormal Ca2+ sensitivity in neurotransmitter release. Under this condition, it was shown that nrx mutant synapses exhibit rapid short term synaptic depression and reduced quantal content of nerve-evoked synaptic currents at the steady state during tetanic stimulation. These observations suggest that NRX regulates activity-dependent synaptic plasticity. Similar observations have been reported in the fast-twitch diaphragm muscle of the α-NRX double knock-out mouse, which indicates that presynaptic efficacy but not presynaptic homeostatic plasticity is normal under basal conditions (Li, 2015).

Deficits in both the presynaptic neurotransmitter release machinery and the postsynaptic neurotransmitter receptors may cause rapid short term synaptic depression. As a presynaptic adhesion molecule, NRX interacts with the postsynaptic adhesion molecule, Neuroligin, and bridges the synaptic cleft that aligns the presynaptic neurotransmitter release machinery with the postsynaptic neurotransmitter receptors. It has been suggested that the activity-dependent regulation of the NRX/Neuroligin interaction mediates learning-related synaptic remodeling and long term facilitation. A recent study reveals that the alternative splicing of presynaptic NRX-3 controls postsynaptic AMPA receptor trafficking and long term plasticity in mice (Aoto, 2013). This study showed that the kinetics of the individual synaptic currents were stable during tetanic stimulation in the nrx mutant synapses. Moreover, NRX was shown to mediate synaptic plasticity through the presynaptic machinery, which was supported by rescue experiments (Li, 2015).

The intracellular sequence of NRX, including the C-terminal PDZ-binding motif, is identical across several vertebrate and invertebrate species. Mammalian NRXs only possesses a short cytoplasmic tail (55 amino acids), including a PDZ-binding motif. To identify the potential binding partners, several laboratories have conducted yeast two-hybrid screening by using the cytosolic tail of mammalian NRX as a bait. Two PDZ-containing proteins, CASK and syntenin, have been identified in the previous screening, and further studies show that the binding between CASK and NRX is abolished by deletion of the last three amino acids of the intracellular C-terminal region of NRX. However, it seems that the previous screenings are not saturated, as the NRX-interacted protein Mints was not identified in these screenings (Li, 2015).

Drosophila NRX contains a long cytoplasmic tail (122 amino acids), including a functional PDZ-binding motif. Thus, it is possible that the long cytoplasmic tail may associate with some partners in a PDZ-independent manner. In this study, normalized Drosophila cDNA libraries were used in the screening, which helps to identify the binding partner with low abundance. In the screening, 23 cDNAs clones that encode the fragments of 18 proteins were recovered. Previous study has demonstrated that the cytoplasmic tail of NRX associates with RFABG and facilitates the retinol transport. This study identified the intracellular region of NRX binds with NSF through the amino acid sequence 1788-1813 but not the PDZ-binding motif of NRX. This NRX/NSF interaction was found to be essential for the NSF recruitment to the presynaptic terminals and plays an important role in synaptic vesicle release. In addition, multiple lines of evidence that NRX associates with NSF at the presynaptic terminals. An alignment analysis of the NSF-binding site of NRX revealed that this sequence is conserved across different species. Moreover, this sequence is present across many proteins that serve different functions, which implies that this sequence may be essential for protein interactions (Li, 2015).

Each NSF molecule contains an N-terminal domain that is responsible for the interaction with α-SNAP and the SNARE complex, a low affinity ATP-binding domain (D1 domain) whose hydrolytic activity is associated with NSF-driven SNARE complex disassembly and a C terminal high affinity ATP-binding domain (D2 domain). This study mapped the NRX-binding sites of NSF to the D2 domain, which mediates the ATP-dependent oligomerization of NSF. An alignment analysis showed that both the D2 domain in NSF and the NSF-binding site in NRX are highly conserved. This result suggests that the NRX/NSF interaction may occur in other species, a possibility that needs to be investigated further (Li, 2015).

It has been documented that the D2 domain of NSF is essential for its hexamerization. This study shows that NRX exhibits a declined NSF binding capacity in high Ca2+ concentration. These observations suggest that NSF can be released from NRX under stimulation. Although NRX and NSF do not bind with Ca2+ directly, it is possible that they undergo Ca2+ signaling-dependent modifications or bind with some Ca2+-binding proteins. In the yeast two-hybrid screening, one potential Ca2+-binding protein (CG33978) has been identified. However, the mechanism of how NRX releases NSF under high Ca2+ concentration need to be further investigated (Li, 2015).

Previous studies have shown that the PDZ-binding motif of NRX associates with several molecules involved in the synaptic vesicle exocytosis machinery, including synaptotagmin and the PDZ domain-containing proteins CASK and Mints. Synaptotagmin functions as a Ca2+ sensor and controls synaptic membrane fusion machinery. Adaptor protein Mints regulates presynaptic vesicle release (Ho, 2006), whereas the other adaptor protein CASK is not essential for the Ca2+-triggered presynaptic release. In contrast, CASK interacts with NRX and protein 4.1 to form a trimeric complex and regulates synapse formation. The process of synaptic vesicle release includes several consecutive steps, docking, priming, and fusion. Depending on the stimulation given to synapses, different synaptic vesicle trafficking steps become rate-limiting for synaptic vesicle release. This causes short term changes in synaptic transmission that determine many higher brain functions such as sound localization, sensory adaptation, or even working memory. Thus, the PDZ-binding motif-linked synaptic vesicle exocytotic machinery might regulate the different steps during synaptic vesicle release. Indeed, nrx mutant synapses that express NRXΔ4 exhibit a rapid current decline over the first several dozen stimulations. In contrast, the expression of NRXΔ4 largely restores the reduced steady-state mean EJCs and quantal content in neurexin mutant synapses. Together with the C-terminally truncated NRX rescue experiments, these data suggest that intracellular regions of NRX other than the PDZ-binding motif also regulate short term synaptic depression. The existing literature extensively documents the roles of NSF in SNARE complex disassembly and short term synaptic depression. This study extended these findings to show that the NRX/NSF interaction facilitates the recruitment of NSF to the presynaptic terminals and promotes the subsequent SNARE complex disassembly (Li, 2015).

Previous studies have established that the NSF hexamer serves as the only active form for SNARE complex disassembly. In this study, the binding assay showed that purified NRX binds to NSF in a concentration-dependent and saturable manner. NRXs appear to serve as scaffold proteins to recruit NSF, and the NRX/NSF interaction may promote SNARE complex disassembly in vivo. Live image studies have shown that NSF mutant (i.e. comt) synapses exhibit defective NSF re-distribution during tetanic nerve stimulation. In this study, immunocytochemical and sedimentation analyses revealed that a lack of the NRX/NSF interaction results in an altered distribution of NSF and an accumulation of 7S SNARE complexes. These results imply that NRX/NSF interaction serves as a potential mechanism to restrict the mobilization of NSF (Li, 2015).

Electrophysiological recordings revealed that synaptic depression was comparable between nrx mutant synapses and wild-type synapses in response to low frequency stimulations. The long intervals between low frequency stimulations may allow the remaining NSF to disassemble the SNARE complexes and generate enough free t-SNARE for the subsequent synaptic vesicle fusion events. Another possibility that needs to be investigated further is that tetanic stimulation may have other effects on the re-distribution of NSF (Li, 2015).

In summary, this study provides evidence that the NRX/NSF interaction recruits NSF to the presynaptic terminals and promotes SNARE complex disassembly. These findings have revealed a previously unknown role of NRX in the regulation of neurotransmitter release and provide a linkage between the presynaptic adhesion molecules and the presynaptic plasticity machinery (Li, 2015).


REGULATION

Transcriptional Regulation

One of the most significant morphogenic events in the development of Drosophila is the elongation of imaginal discs during puparium formation. This macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. The fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks. Using mRNA in situ hybridisation, it has been shown that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, Syx5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). The transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. The ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing understanding of the relationship between gene expression and organelle biogenesis (Dunne, 2002).

Protein Interactions

Syntaxin 5 is a Golgi-localized SNARE protein that has been shown to be required for ER-Golgi traffic in yeast (Dascher, 1994) and Golgi reassembly following cell division in mammalian cells (Rabouille, 1998). The Drosophila ortholog, Syx5, like its mammalian and yeast counterparts is localized to the Drosophila Golgi and binds to alpha-SNAP. Null mutations in Syx5 are larval lethal and demonstrate impaired transport of vesicles through the secretory pathway. A hypomorphic allele of Syx5 results in impenetrant lethality, and escaping adult flies are male sterile. The male sterility results both from failure of germ cells to complete cytokinesis and from defects in spermatid elongation and maturation. Together, these results show that Syx5 is required for the proper function of the Golgi apparatus and that an efficiently functioning Golgi apparatus is required for the steps leading to the completion of cytokinesis and formation of mature sperm (Xu, 2002).

A predicted function of a SNARE protein is its ability to interact with other members of the SNARE pathway. The phenotype that results from the overexpression of a dominant-negative form of Drosophila NSF2 at the developing wing margin has been described. To determine whether Syx5 interacts genetically with NSF2, a single copy of a null mutation in Syx5 was introduced into the flies expressing dominant-negative NSF2 along the wing margin; a significant enhancement of wing notching was seen. This enhancement was as strong as any of the other known interactors identified previously and provides further evidence that Syx5 functions as a SNARE in vivo (Xu, 2002).


DEVELOPMENTAL BIOLOGY

Embryonic

In situ hybridization techniques reveal expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis (Boulianne, 1995).


EFFECTS OF MUTATION

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. A Drosophila temperature-sensitive paralytic mutation in syntaxin has been isolated that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses (Littleton, 1998).

The SNARE hypothesis has been proposed to explain both constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this model, a vesicle targeting/docking complex consisting primarily of vesicle- and target-membrane proteins, known as SNAREs, serves as a receptor for the cytosolic N-ethylmaleimide-sensitive fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While features of this model remain tenable, recent studies have challenged fundamental aspects of the SNARE hypothesis, indicating that further analysis of these components is needed to fully understand their roles in neurotransmitter release. This issue was addressed by using the temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of NSF in neurotransmitter release in vivo. Synaptic electrophysiology and ultrastructure in comt mutants have recently defined a role for NSF after docking in the priming of synaptic vesicles for fast calcium-triggered fusion. An SDS-resistant neural SNARE complex, composed of the SNARE polypeptides Syntaxin, n-Synaptobrevin, and SNAP-25, accumulates in comt mutants at restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes are distributed predominantly in fractions containing plasma membrane and docked synaptic vesicles. Together with the electrophysiological and ultrastructural analyses of comt mutants, these results indicate that NSF functions to disassemble or otherwise rearrange a SNARE complex after vesicle docking and that this rearrangement is required to maintain the readily releasable pool of synaptic vesicles (Tolar, 1998).

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, electrophysiological and ultrastructural analyses were performed in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses reveal that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures reveals a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, transmission electron microscopy was used to examine the distribution of vesicles within synaptic terminals, and a marked accumulation of docked vesicles at restrictive temperatures was observed in comt. Together, the results define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion (Kawasaki, 1998).

The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Work at neuromuscular synapses in the temperature-sensitive NSF mutant comatose (comt) has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway was investigated. These central synapses include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produces wild-type responses at both 20°C and 36°C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the 'long latency pathway') reveals a striking difference between wild type and comt at 36°C. Repetitive stimulation of the long latency pathway leads to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase that are observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release observed at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1 (Kawasaki, 1999).

The temperature-induced paralysis of comatose mutants of Drosophila is suggestive of a function for NSF in the CNS. Mutations in the para gene encoding the subunit of the voltage-gated sodium channel also result in a similar phenotype. Paralysis in comt flies is activity-dependent, and in the doubly mutant comt;para flies comt-like paralysis does not set in until the effects of para are reversed by shifting to permissive temperatures. During recording from the thoracic flight muscles, it was observed that comt flies show a burst of spontaneous activity at restrictive temperature. This has been reported earlier as a unique characteristic of comt paralysis. The comt;para double mutant shows this burst of activity not at restrictive but only on shifting back to permissive temperature. The unusual behavior and electrophysiology of the doubly mutant flies reported here indicates a role for NSF in synaptic vesicle cycling (Sanyal, 1999).

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden the analysis of synaptic transmission to other gene products, a genetic screen was performed for mutations that interact with comt. Four mutations that modify TS paralysis in comt are described, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel 1-subunit gene, cacophony (Dellinger, 2000).

Identification of cacTS2 as a modifier of comt raises a number of interesting issues. The original cacophony mutant (now known as cacS) was named on the basis of an aberrant male courtship song. The courtship song is produced by a patterned beating of the wings, and this pattern as well as the wing beat amplitude are altered in cacS mutants. The finding that cac-encoded calcium channels function in neurotransmitter release suggests that impairment of central synapses may contribute to altered song patterning in cacS. Given that cac-encoded alpha1-subunits function at flight muscle neuromuscular synapses, peripheral synaptic defects may contribute to the song phenotype as well. A second issue is whether the genetic interaction of cacTS2 and comt reflects direct or indirect interactions of the encoded gene products. Electrophysiological analysis indicates that the cac-encoded alpha1-subunit mediates fast neurotransmitter release and that dNSF1 functions in maintaining the readily releasable pool of synaptic vesicles. Thus the observed genetic interaction may reflect simply that both the comt and cac gene products function in neurotransmitter release. Alternatively, the genetic interaction may result from the well-characterized biochemical interactions of SNAREs with both NSF and calcium channels. While this issue remains unresolved, two observations favor the former possibility. (1) No sequence homology has been detected between the cac-encoded alpha1-subunit and SYNPRINT sequences thought to mediate direct interactions with other synaptic proteins. (2) Preliminary synaptic electrophysiology in cacTS2 comtST17 double mutants is consistent with independent actions of comt and cac mutations in neurotransmitter release (Dellinger, 2000).

Evidence that cac functions in neurotransmitter release arises from its genetic interactions with comatose (comt), coding for a homolog of the N-ethylmaleimide-sensitive fusion protein that functions in priming synaptic vesicles for fast, calcium-triggered fusion. comt mutants exhibit rapid temperature sensitive paralysis. These mutants typically develop and function normally at permissive temperature and can be shifted to restrictive temperature to examine the acute functional consequences of perturbing a specific gene product. To broaden the analysis of the biology of neurotransmitter release to other gene products functioning in synaptic vesicle trafficking, a genetic screen was conducted to identify mutations exhibiting functional interactions with comt. One enhancer of comt was determined to be a TS allele of cac and has been designated cacTS2. Electrophysiological analysis at neuromuscular synapses has revealed that neurotransmitter release in cacTS2 is markedly reduced at elevated temperatures, indicating that cac functions in synaptic transmission (Kawasaki, 2000). Notably, rescue of rapid, calcium-triggered neurotransmitter release can be achieved in comatose mutants by neural expression of a single cDNA containing a subset of alternative exons and lacking any conserved synaptic-protein interaction sequence (Kawasaki, 2002).

Drosophila Nsf1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation has revealed that dNSF-1 is enriched in a vesicle fraction along with the synaptic vesicle protein Synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlates with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comtTP7 larvae, there is no associated neurophysiological phenotype at the neuromuscular junction or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but in the larval NMJ, this function may be maintained by other isoforms (Mohtashami, 2001).

NSF is a cytosolic ATPase implicated in a variety of cellular functions including synaptic vesicle exocytosis. A lethal mutation in the Drosophila homolog of NSF (dNSF1), is reported in this study. Lethality staging and rescue experiments with the wild type dNSF1 transgene show that NSF1 is critically required during early larval stages and during late pupariation. Lethality in larval stages is associated with defects in neurogenesis as evidenced by an overall reduction in synapse size and synapse branching. Moreover, escaper adults, though showing abnormal seizure-like paralytic behavior, are normal in terms of synaptic transmission as assayed by electroretinograms. Taken together, these data indicate a role for NSF in neural growth and branching in addition to its documented role in synaptic transmission (Sanyal, 2001).

Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, a genetic analysis was undertaken of comatose (dNSF-1) in Drosophila. Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire;comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles (Littleton, 2001).

To characterize the role of NSF-1 in synaptic transmission, a series of new comt mutations were isloated using two approaches. The first approach was to screen for X-linked temperature-sensitive paralytic mutants. Approximately 150,000 males containing an ethyl methanesulfonate (EMS)-mutagenized X-chromosome were tested for reversible temperature-dependent paralysis at 38°C. Three mutations failed to complement comt and thus represented new alleles. Three additional temperature-sensitive alleles of comt were identified in independent screens. Together with three previously known comt mutations (ST17, TP7, and ST53), nine conditional alleles of NSF-1 were characterized. A second screen that permitted isolation of new comt alleles -- even if they were associated with a lethal phenotype -- also was carried out. This screen was based on the observation that flies heterozygous for a conditional comt allele and a deletion that removes the gene are viable and manifest temperature-sensitive paralysis. Males were mutagenized with either EMS or gamma-rays and crossed to homozygous comtST53 virgin females. Approximately 10,000 heterozygous F1 females were collected from these crosses and tested for temperature-sensitive paralysis at 38°C. Four new lethal alleles and one viable allele of comt were identified in this screen. Two additional independent recessive lethal comt alleles were included in this analysis (Littleton, 2001).

To determine the underlying amino acid changes that cause the comt mutant phenotypes, the ORF of each allele was sequenced. All but one of the temperature-sensitive paralytic mutants altered highly conserved amino acids within the D1 domain, a region required for NSF-mediated disassembly of the SNARE complex. comt alleles in this category include ST17 (G274E), JN17 (G387S), DN47 (E394K), TP7 (P398S), X238 (N418I), DN48 (A447V), X91 (A448T), and ST53 (S483L). One lethal mutation, LED, maps to the D1 domain and causes a G to S substitution at amino acid 443. The remaining comt temperature-sensitive paralytic mutation was DN34, which alters a highly conserved Gly residue in the N-domain of NSF (G58D), a region required for SNAP-mediated binding to the SNARE complex. Analysis of the crystal structure of the N terminus of NSF reveals that Gly-58 lies in the first loop of the double-psi ß-barrel (DPBB) motif. The DPBB motif is common in many proteins and is often important for substrate binding. A third class of comt alleles alters the D2 domain of NSF, a region required for assembly of NSF into functional hexamers. Three lethal comt mutants lie in this domain, including 1121 (P494L), 133 (E606K), and CLP, which has a 1-bp deletion that shifts the reading frame 36 aa upstream of the carboxyl terminus and leads to a premature stop codon. One viable allele, comtG4, also disrupts the D2 domain and results in a 3-aa deletion/2-aa substitution (VQQ to GS at amino acids 525-527) near the ATP-binding site in the D2 domain. comtG4 mutants are only weakly temperature sensitive, but show a substantial reduction in lifespan with progressive weakness and lack of coordination. Because the three-dimensional structures of the D1 and D2 domains of NSF are likely to be highly conserved, the sites of the comt D1 and D2 mutants were modelled on the crystal structure of NSF-D2. A large number of residues mutated in the temperature-sensitive mutants reside in a loop region that has been postulated to serve as a lever that imparts force to the SNARE complex during ATP hydrolysis. This region of the AAA domain also has been shown to undergo conformational shifts during the ATP-binding/hydrolysis cycle in the NSF-related protein, p97. The remaining conditional alleles cluster near the ATP-binding/hydrolysis site. Of the D2 domain alleles, comt133 alters a conserved glutamate in the ATP-hydrolysis domain, indicating that this mutation is likely to abolish hexamerization of NSF (Littleton, 2001).

To characterize further the requirements for NSF-1 in vesicle trafficking, both the temperature-sensitive and lethal comt mutants were analyzed in more detail. Most comt temperature-sensitive mutations paralyze within several minutes at 38°C, similar to alleles described. Although the comtDN34 allele displays temperature-sensitive paralysis at 38°C, it requires a longer incubation time (6-7 min) and shows residual locomoter activity during longer 38°C incubations. The delayed paralysis in comtDN34 mutants correlates with its unique molecular defect in the N-terminal domain. All temperature-sensitive comt alleles show a dramatic accumulation of SDS-resistant SNARE complexes compared with wild type, suggesting an essential role for NSF in the disassembly of SNARE complexes. Each comt temperature-sensitive mutant also blocked vesicle-trafficking in Drosophila photoreceptors at 38°C, as indicated by a loss of the on/off synaptic events in electroretinogram recordings from the retina. In contrast to the temperature-sensitive paralysis of the conditional alleles, complete removal of NSF-1 results in lethality. Two lethal alleles identified result in premature stop codons that cause truncations of NSF-1. comt191 truncates in the middle of the D1 domain and is likely to be a null allele. comtG8 truncates at the end of D1. These mutations can be rescued with a free duplication carrying a wild-type comt allele or with a heat-shock driven NSF-1 transgene, indicating the lethal phenotypes are specifically associated with loss of NSF-1 activity. Null mutants died during embryonic and larval stages, although a small percentage survived to pupae (Littleton, 2001).

The accumulation of SNARE complexes in the temperature-sensitive paralytic mutants suggests these mutations disrupt the ATPase activity or subsequent force generation required for SNARE-mediated disassembly. Consistent with this model, the altered NSF protein in comtST17 mutants has been biochemically demonstrated to have a temperature-dependent block in SNARE disassembly in vitro. However, it is unknown where NSF-mediated SNARE disassembly is required during the synaptic-vesicle cycle: before docking, after docking, during fusion, or after exocytosis. To determine the stage in the synaptic-vesicle cycle where NSF is required, the comtST17 mutation was studied in more detail. Advantage was taken of two other mutations that allow specific stages in vesicle trafficking to be blocked. shiTS1 is a temperature-sensitive mutation in dynamin that blocks synaptic-vesicle endocytosis. paraTS1 is a temperature-sensitive mutation in the alpha-subunit of the sodium channel that blocks action potential propagation and subsequent evoked-vesicle fusion. Double mutants containing shiTS1 comtST17 or comtST17 paraTS1 were generated by recombination. If NSF is required for exocytosis of the nerve terminal pool of synaptic vesicles, it would be expected that NSF should be epistatic to dynamin in nerve terminals and lead to a block in vesicle fusion before the terminals are depleted of vesicles. If the inactivation of NSF does not block a step on the exocytotic side of the synaptic-vesicle pathway, then all of the vesicles would be capable of translocating to active zones, and of docking, priming, and fusing. Then, a depletion of synaptic vesicles in shiTS1 comtST17 double mutants would be expected similar to what is observed in shiTS1 mutants. In shiTS1 mutants incubated at 38°C, synaptic-vesicle proteins such as synaptotagmin, CSP, and synaptobrevin redistribute to the plasma-membrane fraction at 38°C when assayed on sucrose velocity sedimentation gradients. In wild type or comtST17 mutations incubated at 38°C, there is no shift in the distribution of synaptic-vesicle proteins. However, in shiTS1 comtST17 double mutants and in shiTS1 mutants incubated at 38°C, synaptic-vesicle proteins redistribute to the plasma membrane, indicating the synaptic-vesicle pool is capable of completing all stages of exocytosis, including fusion in comt mutants. To extend this biochemical analysis, an ultrastructural analysis was performed on photoreceptor nerve terminals. comtST17 mutants accumulate docked and undocked synaptic vesicles when incubated for 10 min at 38°C under constant light stimulation. In contrast, shiTS1 photoreceptor terminals are depleted of synaptic vesicles. Photoreceptor terminals of shiTS1 comtST17 double mutants show a depletion of synaptic vesicles similar to shiTS1, confirming that both the docked and reserve pool of vesicles can fuse in comt mutants (Littleton, 2001).

Next, how SNARE-complex accumulation in comtST17 mutants is affected by blocking vesicle recycling in shiTS1;comtST17 double mutants was investigated. If the large accumulation of SNARE complexes represents those complexes that assembled during one round of vesicle fusion at nerve terminals, SNARE-complex accumulation in shiTS1;comtST17 double mutants should be the same as in comtST17 alone. However, if multiple rounds of exocytosis of the vesicle pool can occur in the absence of NSF activity, and if these rounds of fusion are necessary to generate the large excess of unresolved SNARE complexes, a decrease in 7S accumulation should be seen in the double mutant, because shiTS1 blocks multiple rounds of vesicular cycling. SNARE complexes were isolated from comtST17 and shiTS1;comtST17 mutants. SNARE-complex accumulation is greatly reduced in the double mutant compared with comtST17 mutants alone, suggesting that multiple rounds of vesicle cycling do occur in the absence of NSF. As expected, there is still more SNARE complex present in the double mutant than in wild type, which is consistent with the accumulation of unresolved SNARE complexes formed during a single round of exocytosis. If endocytosis could be blocked without disrupting NSF activity, it was reasoned that the accumulation of SNARE complexes should not occur in shiTS1;comtST17 mutants. This possibility was tested by taking advantage of the differential sensitivity of shiTS1 and comtST17 to temperature. Whereas shiTS1 mutants become paralyzed at 29°C, comtST17 mutants do not paralyze until 35°C. Therefore, double mutants were incubated at 29°C for 10 min to block endocytosis and to deplete the vesicle pool with NSF still active and then shifted to 38°C for 10 min to block NSF activity. Analysis of SNARE-complex accumulation with this protocol demonstrates that there is no SNARE-complex accumulation in the double mutant when the vesicle pool is depleted with NSF still active. These results suggest that NSF disassembles cis-SNARE complexes that reside in the plasma membrane as a consequence of fusion. In addition, free SNAREs that are acted upon by NSF and that are trapped in the plasma membrane in shi mutants do not spontaneously reassemble into cis-SNARE complexes once NSF activity is blocked (Littleton, 2001).

A prediction of the model for NSF function is that SNARE complexes accumulate in comt as a direct consequence of vesicle fusion and a subsequent failure in disassembly of the complex postfusion. Manipulations that block release would be predicted to block the accumulation of SNARE complexes. This hypothesis was tested by using comtST17;paraTS1 double mutants. It has been noted that paraTS1 does not block synaptic transmission in the fly retina (photoreceptors do not use para-dependent action potentials). Because the eye constitutes a significant portion of the Drosophila head and would, therefore, compromise assays in comtST17;paraTS1 double mutants, the eye, specifically the contribution of photoreceptor synapses to the SNARE-complex assays, was eliminated by expression of GMR-hid (which drives the cell-death gene hid in photoreceptor primordial cells). To avoid the cumulative effect of spontaneous fusion events contributing to the build up of SNARE complexes in this assay, SNARE accumulation was assayed at 38°C for 5 min. In flies lacking photoreceptors, paraTS1 suppresses the accumulation of SNARE complex that otherwise would occur in comt mutations incubated at 38°C. This result confirms that SNARE complexes accumulate in comt mutants as a consequence of vesicle fusion. In addition, paraTS1 also eliminates the pool of SNARE complexes normally present in wild-type flies, suggesting that assembly of SNARE complexes requires nerve activity. This finding argues against stable SDS-resistant SNARE complexes forming during synaptic-vesicle docking or priming, because paraTS1 specifically blocks action potentials. An alternative explanation for the experimental findings in comt double mutants is that loss of NSF is not rapid at the restrictive temperature but occurs slowly. Several lines of evidence argue against this interpretation. Initially, eight conditional comt paralytic mutants alter distinct amino acids in the D1 domain, yet all show delayed paralysis, suggesting that this result is a general feature of the comt phenotype. Most convincingly, however, is the behavior of comt;para double mutants. Double mutants exposed to 38°C for 5 min show a rapid recovery like para mutants alone when returned to the permissive temperature. After rapid recovery and normal behavior, these flies then suddenly reparalyze several minutes later at the permissive temperature. Because comt mutants would be completely paralyzed after 5 min at 38°C and require greater than 30 min to recover at permissive temperature, it is concluded that NSF would be inactivated in the double mutant at 38°C (Littleton, 2001).

Together, these results suggest that NSF does not function in fusion, recruitment of vesicles to active zones, docking, or priming. Rather, it is proposed that NSF specifically disassembles SNARE complexes after fusion and before endocytosis. In the absence of synaptic transmission in comtST17;paraTS1 mutants, SNARE complexes do not accumulate, and behavioral paralysis induced by loss of NSF function is delayed. Thus, in the absence of vesicle fusion, NSF is not required to prime or maintain synaptic vesicles in a fusion-competent state. Once NSF has been inactivated, vesicle release and activity continues for several minutes before paralysis occurs. In shi;comt double mutants, inactivating both dynamin and NSF results in a shi phenotype, with synapses devoid of synaptic vesicles, confirming that vesicles continue to fuse in the absence of NSF activity. Thus, a large pool of excess v-SNAREs are present and can support fusion in the absence of NSF. After these pools are exhausted, a postdocking defect in fusion occurs because of an inability to form trans-SNARE pairs. These findings complement and extend the models of NSF function provided by previous analyses of comt mutants in Drosophila. Dorsal longitudinal flight muscle recordings from temperature-sensitive comt mutants have demonstrated a reduction in synaptic current during repetitive stimulation that is accompanied by an increase in the number of docked synaptic vesicles. In the absence of repetitive stimulation, no defects in fusion are detected, confirming that vesicle fusion itself does not require NSF activity. Rather, NSF is required to restore the pool of vesicles that are readily released, likely through its ability to break apart cis-SNARE complexes and reactivate individual SNAREs for additional rounds of fusion. Thus, multiple lines of evidence support the conclusion that several rounds of vesicle fusion can continue in comt mutants before synaptic transmission is finally poisoned by a lack of fusion-competent SNAREs (Littleton, 2001).

Additional studies will be required to determine the pool size of fusion-competent SNAREs maintained by NSF activity at different synapses. This pool size will have important implications for presynaptic plasticity, as it would determine whether modulation of SNARE assembly/disassembly rates plays a role in controlling presynaptic output during stimulation. In fly photoreceptors and central synapses, it is estimated that this pool size is sufficient to mediate the fusion of several rounds of the entire nerve-terminal synaptic-vesicle population. This conclusion is based on the observation that in shi;comt double mutants, there is a substantial reduction (>2-fold) in the amount of SNARE complexes that accumulate, compared with comt mutants alone. This observation suggests that the large increase in assembled SNARE complexes in comt mutants results from recycling of the vesicle pool more than once. At adult Drosophila neuromuscular junctions, the pool of fusion-competent SNAREs seems to be smaller, because nerve-stimulation rates as low as 5 Hz can rapidly decrease exocytosis in comt conditional mutants. Thus, the size of the fusion-competent pool of SNAREs can be differentially controlled at distinct synapses. Although it is unknown how many SNARE complexes are required for the fusion of a synaptic vesicle, the results argue that there are sufficient v-SNAREs present on each vesicle to suffice for several rounds of exocytosis without being regenerated through NSF action. Likewise, there seems to be a large pool of synaptic t-SNAREs that can function in many cycles of fusion without requiring regeneration through NSF action. Overexpression of the NSF adapter alpha-SNAP at crayfish neuromuscular junctions or at the squid giant synapse results in an increase in the pool of synaptic vesicles readily released. These findings suggest that modification of the functional pool size of fusion-competent SNAREs over time can alter synaptic function and may be an important mechanism for modulating synaptic plasticity. The use of fast FM dyes recently has defined an additional synaptic-vesicle pathway of vesicle fusion and immediate reuse. In such cases, the ability to rapidly dissociate SNARE complexes may be essential, and NSF activity may be rate-limiting in this rapid reuse pathway. The recent finding that patients with schizophrenia show a substantial reduction in NSF expression in the prefrontal cortex suggests that disruption of SNARE-mediated disassembly rates also may be a contributing factor in psychiatric dysfunction. Further studies into the mechanisms that maintain fusion-competent SNAREs and accelerate NSF activity should provide insights into presynaptic plasticity and the machinery that modulates synaptic-vesicle fusion (Littleton, 2001).

A genetic screen for suppressors of Drosophila NSF2 neuromuscular junction overgrowth

The Drosophila larval neuromuscular system serves as a valuable model for studying the genes required for synaptic development and function. N-Ethylmaleimide sensitive factor (NSF) is a molecule known to be important in vesicular trafficking; neural expression of a dominant negative form of NSF2 induces an unexpected overgrowth of the Drosophila larval neuromuscular synapse. A genetic approach was taken to study this phenotype by conducting a gain-of-function modifier screen to isolate genes that interact with the overgrowth phenotype. The approach was to directly visualize the neuromuscular junction (NMJ) using a GFP transgene and screen for suppressors of NMJ overgrowth using the Gene Search (GS) collection of P-element insertions. Of the 3000 lines screened, 99 lines were identified that can partially restore the normal phenotype. Analysis of the GS element insertion sites by inverse PCR and comparison of the flanking DNA sequence to the Drosophila genome sequence revealed nearby genes for all but 10 of the 99 lines. The recovered genes, both known and predicted, include transcription factors, cytoskeletal elements, components of the ubiquitin pathway, and several signaling molecules. This collection of genes that suppress the NSF2 neuromuscular junction overgrowth phenotype is a valuable resource in efforts to further understand the role of NSF at the synapse (Laviolette, 2005).

Eighteen GS lines were discovered whose candidate genes can be broadly categorized as transcriptional regulators. Several of these candidate genes are expressed in the nervous system or in some cases loss-of-function alleles are known to produce neural phenotypes, e.g., longitudinals lacking, buttonless, couch potato, hoi polloi, E2f, brain tumor, nejire, and retinoblastoma-family protein (Laviolette, 2005).

Interestingly, cyclic AMP response element-binding protein (CREB) (nejire) function has been analyzed at larval neuromuscular synapses and while the presynaptic overexpression of CREB was found to impair neurotransmitter release, it has no effect on presynaptic morphology. This study found that a GS line in position to overexpress CREB in the NSF2E/Q mutant background rescues the overgrowth phenotype, suggesting that this gene may have a role in presynaptic development (Laviolette, 2005 and references therein).

Three of the 18 transcriptional regulators, buttonless, couch potato, and hoi polloi, have been recovered in loss-of-function screens designed to uncover genes important for peripheral nervous system development. The results indicate the potential for these genes to also be involved in motor neuron development (Laviolette, 2005).

One gene that was recovered, ovo, is not normally associated with neural function. ovo is normally expressed in the male and female germline cells, where it controls F-actin extension. When expressed in the eye under UAS/Gal4 control, eye bristle formation is impaired and induces ectopic extensions from the ommatadia, suggesting that it has a strong influence on cytoskeletal remodeling (Laviolette, 2005).

It is likely that overgrowth of the neuromuscular junction ultimately affects the underlying cytoskeleton but it was somewhat surprising to identify a large number of genes composing structural components of the cytoskeleton or enzymes with the potential to regulate cytoskeletal dynamics. The structural genes are Actin5c, moesin, fimbrin, syntrophin-like 2, Myosin binding subunit, Ptpmeg, and CG5740. Two genes that encode proteins with enzymatic activity that have the potential to regulate the cytoskeleton, RhoBTB and RhoGap18B, were also found (Laviolette, 2005).

Actin5C is one of two cytoplasmic actins in Drosophila and it is highly expressed in many tissues throughout development. It has been studied extensively in many processes, including spermatogenesis and dorsal closure. In the nervous system, Act5c has been recovered in a screen for changes in bristle number -- presumably a reporter of peripheral nervous system development -- and its role has been studied in glial cell function (Laviolette, 2005).

Moesin is a member of the ezrin-radixin-moesin (ERM) family of actin-binding proteins that link actin to the plasma membrane. While a neural role for Drosophila moesin has not previously been reported, it does have a role in growth cone motility in other neural systems, and a recent report demonstrates the role of Drosophila moesin in photoreceptor rhabdomere development (Laviolette, 2005).

Four genes involved in the ubiquitin protein degradation pathway were identified: bendless, Bruce, thread, and Ufd1-like. The gene products of bendless, Bruce, and thread (also known as DIAP1) are thought to act as E2 or E3 ubiquitin-conjugating enzymes. The Ufd1-like gene encodes a transcript that is involved in ubiquitin-dependent protein degradation. Thus, the four candidates in this category would all have the potential to enhance ubiquitin-dependent protein degradation if their expression were to be increased (Laviolette, 2005).

Identification of these genes is an important finding in light of previous developmental NMJ studies, which revealed that loss-of-function mutants of highwire, whose gene product contains a RING finger domain implicated in E3 ubiquitin ligase activity, leads to NMJ overgrowth that is very similar to NSF2E/Q-induced overgrowth. Furthermore, transgenic overexpression of fat facets also causes excessive NMJ overgrowth. faf encodes a deubiquitinating protease and is thought to protect ubiquitinated proteins from degradation (Laviolette, 2005).

Therefore, hiw loss of function or faf overexpression should lead to reduced ubiquitin-dependent protein degradation. Since the NSF2E/Q phenotype is rescued by expression of genes that should increase the ubiquitin degradation pathway, it is inferred that reduced ubiquitin pathway function may also be one of the molecular dysfunctions underlying the NSF2E/Q phenotype (Laviolette, 2005).

Three genes involved in G-protein-receptor-coupled signaling were identified: methuseleh (mth), Frizzled 4, and AlstR. methuseleh is a gene first identified for its effects on life span. Prior analysis of mth at the NMJ, using hypomorphic loss-of-function mutants, revealed no effect on the number of synaptic boutons or length of NMJ branches. The current results suggest that mth may have a previously undetected developmental role at the synapse (Laviolette, 2005).

Frizzled 4 is a Wnt receptor that is expressed in the CNS, but so far there has been no genetic analysis of this receptor. However, a recent study has demonstrated the involvement of Wnt signaling at the Drosophila NMJ, showing that wingless (wg) loss of function results in fewer boutons while presynaptic wg overexpression produces more boutons; these effects are thought to be mediated through the Wnt receptor Frizzled 2. While it is currently unknown if the CNS expression of fz4 is presynaptic or not, the current result imply that there may be an autocrine component to Wnt signaling at the synapse, as there is during wing development (Laviolette, 2005).

There is no previous neural expression or mutant data for Galpha73B, a component of a trimeric G-protein complex, and it is not presently clear how this gene aids the restoration of NSF2E/Q neuromuscular overgrowth (Laviolette, 2005).

Four genes with known or predicted kinase activity were also recovered: Pka-C1, grapes, Pak3, and CG6386. Prominent in this group is Pka-C1, which encodes cAMP-dependent protein kinase 1. Previous studies of rutabaga, the adenylyl cyclase that produces cAMP, and dunce, the phosphodiesterase that hydrolyzes cAMP, have shown dramatic effects on NMJ morphology and physiology. Loss of function of either rut or dnc leads to NMJ overgrowth, although the extent of that effect is not as large as is seen with the NSF2E/Q allele. Interestingly, GS9123 is inserted 252 bp upstream of the rutabaga 5'-UTR; however, the orientation of the UAS sequence appears to be in the wrong direction to drive expression of rut (Laviolette, 2005).

PAK3 is a member of the p21-activated kinase family that has been shown in other systems to strongly influence the cytoskeleton by way of its interactions with the small GTPases Rho and cdc42 that directly regulate actin biochemistry. Thus, the finding of PAK3 also potentially fits with observations of the cytoskeletal components interacting with NSF2 (Laviolette, 2005).

Three genes with activity in the Ras signaling pathway were found: C3G, PTP-ER, and CG32560. C3G is a RAS family guanine nucleotide exchange factor that activates Ras by catalyzing exchange of GDP for GTP. PTP-ER encodes a tyrosine phosphatase that dephosphorylates Drosophila MAPK, thereby downregulating its kinase activity. CG32560 is predicted to be a RAS GTPase activator, which would activate GTP hydrolysis and downregulate RAS signaling. Thus, the result of C3G appears to be in contradiction to the results of PTP-ER and CG32560 (Laviolette, 2005).

The Spitz protein is the EGF receptor (EGFR) ligand that has been implicated in many developmental processes, including photoreceptor axon guidance. Rolled is the Drosophila homolog of MAP kinase and a downstream target of EGFR. MAPK has been linked to Netrin-dependent growth cone attraction through the Netrin receptor DCC (Laviolette, 2005).

Finally, another important signaling gene identified was NetrinB. This is one of two Netrin isoforms in Drosophila that are well known for their role in axon guidance, both in the periphery and in central commissural axons that cross the midline. NetrinB is a secreted molecule that can interact with two receptors: frazzled, the DCC receptor that exerts attractive cues, and unc-5, which can mediate repulsive cues in axon guidance. Although Drosophila Netrins are widely known to be expressed in midline glial cells and in muscles, NetrinB RNA and protein are also found in a number of ventral-lateral neurons in the Drosophila embryo. Therefore, the rescue of the NSF2E/Q overgrowth phenotype by presynaptic expression of NetrinB may reveal an autocrine component to this signaling pathway whereby Netrin is released from the growth cone or mature nerve terminal and acts upon Netrin receptors there (Laviolette, 2005).

Two other interesting lines were recovered that do not fit into the above classifications. (1) GS3062 is positioned 0.4 kb upstream of polo, which acts as a serine/threonine kinase and hypomorphic alleles of this gene affect larval brain development through cytokinesis defects. However, this GS element is also 3.5 kb upstream of the soluble NSF attachment protein (snap) gene. The SNAP protein is the cofactor that links NSF to the SNARE complex and to AMPA-type glutamate receptors. Therefore, while snap is a very attractive candidate, it remains to be determined whether GS3062 drives expression of polo, snap, or both (Laviolette, 2005).

(2) GS8030 was identified as a line with the potential to activate Rab5 expression. This gene is a member of the Rab small GTPase family, and Rab5 in particular is thought to be an important component in the endocytic pathway. Recently, Rab5 has also been shown to be critical for receptor tyrosine-kinase-induced actin remodeling in mouse fibroblasts and thus there is a potentially interesting link between this gene and the cytoskeleton (Laviolette, 2005).

Synaptic vesicle mobility and presynaptic F-actin are disrupted in a N-ethylmaleimide-sensitive factor allele of Drosophila

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity Drosophila has two NSF genes, comatose (which encodes the NSF1 protein) and NSF2, whose proteins are 80% identical and are functionally redundant. NSF1 is the predominant functional isoform in the adult fly nervous system, although NSF2 is functionally important in other tissues and throughout development. Flies that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. This study tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging a substantial reduction was found in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. It is concluded that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. These data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin (Nunes, 2006; full text of article).

Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants

Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (an acronym derived from 'SNAP (Soluble NSF Attachment Protein) REceptor', consisting of proteins that mediated vesicular fusion with the target membrane) in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. The findings establish an in vivo role for the target-Snare (t-Snare) protein SNAP-25, one part of the four helix bundle of proteins referred to as SNAPs, in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for wild-type PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, Syntaxin and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported in this study establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression (Kawasaki, 2009).

The present study further defines the in vivo roles of NSF and SNAP-25 in synaptic vesicle trafficking and their contributions to conserved properties of short-term synaptic depression. Insight is also gained into the spatial organization of activity-dependent SNARE protein cycling with respect to active zones. The findings support a model incorporating in vivo molecular mechanisms of synaptic vesicle priming as important determinants of short-term depression and maintenance of neurotransmitter release during synaptic activity. These priming mechanisms include a direct role for SNAP-25 and an indirect contribution involving NSF-dependent replenishment of active zone free t-SNAREs for subsequent vesicle priming and fusion (see Working model of SNARE protein cycling during synaptic activity). In this model, recovery after a high frequence stimulation, in a measurement called paired pulse depression (PPD), depends on a limited pool of free active zone t-SNAREs (A), which is sufficient to support formation of trans-SNARE complexes (B) and refilling of the release-ready vesicle pool. This priming process is impaired in the SNAP-25TS mutant with respect to WT. The location of the SNAP-25TS mutation within the N-terminal region of the SNARE four helix bundle is consistent with a zippering model of SNARE complex function. On train stimulation, cis-SNARE complexes accumulate in the periactive zone (PAZ), resulting in depletion of the free t-SNARE pool at the active zone (AZ). Disassembly of cis-SNARE complexes by NSF and SNAP restores active zone t-SNARES for subsequent vesicle priming and fusion events. This aspect of synaptic vesicle priming is impaired in comatose, which encodes an enzyme that facilitates priming (Kawasaki, 2009).

The findings of this study reveal a role for SNAP-25 in determining the properties of recovery from short-term depression. An underlying function for SNAP-25 in synaptic vesicle priming was inferred from the following aspects of the SNAP-25TS synaptic phenotype: (1) activity dependence as indicated by a WT initial EPSC amplitude and release-ready synaptic vesicle pool size, (2) preservation of the WT EPSC waveform, (3) loss of fast recovery in PPD, which is associated with fast refilling of the release-ready pool and exhibits a time course comparable with that of synaptic vesicle priming, (4) dependence on previous synaptic vesicle fusion, and (5) slowed refilling of the release-ready vesicle pool. Thus, although SNAP-25 is required for evoked synaptic vesicle fusion (Washbourne, 2002), the SNAP-25TS mutation can selectively affect synaptic vesicle priming. This finding is consistent with systematic structure-function analysis of SNAP-25 in adrenal chromaffin cells, which provided strong support for the zippering model of SNARE function (Sorensen, 2004). In this model, vesicle priming involves initial assembly at the N-terminal end of the SNARE four helix bundle to form a loose trans-SNARE complex. Final assembly of its C-terminal end, which may be triggered by calcium influx, is critical for vesicle fusion. Notably, the SNAP-25TS mutation is positioned within the N-terminal region of the four helix bundle, consistent with a TS impairment of trans-SNARE complex formation associated with synaptic vesicle priming. Previous studies of SNAP-25TS indicate that synaptic vesicle docking at active zones is not disrupted at larval neuromuscular synapses, and that in vivo levels of SDS-resistant (likely cis) SNARE complexes are not altered in this mutant. Finally, it will be of great interest to further investigate the relationship of SNAP-25-dependent mechanisms underlying fast and slow components of recovery. These components are thought to involve calcium-dependent regulation of kinetically distinct synaptic vesicle pools exhibiting different release probabilities (Kawasaki, 2009).

The close resemblance of comatose (NSF) and WT synapses with respect to the initial EPSC amplitude and PPD suggests that mutant comatose (NSF) initially exhibits a WT release-ready vesicle pool and release probability. The strictly activity-dependent reduction in neurotransmitter release observed in comatose (NSF) mutants requires a brief period of synaptic activity as indicated by delayed onset of the synaptic phenotype during train stimulation. The underlying mechanism appears to involve activity-dependent redistribution of plasma membrane t-SNAREs away from the active zone. Together with systematic biochemical studies of comatose (NSF) demonstrating TS accumulation of (SDS-resistant) ternary SNARE complexes on the plasma membrane, these findings suggest that SNAREs accumulate in plasma membrane cis-SNARE complexes that are not retained within the active zone (see the working model). NSF disassembly of post-fusion plasma membrane cis-SNARE complexes has been directly demonstrated in studies of yeast exocytosis and the underlying heterotypic fusion of secretory vesicles with the plasma membrane. Previous and present efforts to address this issue at Drosophila synapses, including analysis of v-SNARE distribution in the presynaptic plasma membrane. Finally, an alternative view of NSF function favoring prefusion disassembly of SNARE complexes was reported in a study employing caged NSF peptides to acutely disrupt NSF function at the squid giant synapse. However, the activity-dependent effects of the peptide on EPSC amplitude are largely consistent with the working model presented in this study, in which the timing of the NSF requirement is not constrained by the total vesicle cycling time. Although the NSF peptide was also found to slow EPSC rise and decay times, no analogous effects were observed in the present study (Kawasaki, 2009).

The adult DLM neuromuscular synapse has had an important role in analysis of TS mutations in comatose (NSF), the presynaptic calcium channel α1 subunit gene, cacophony, and the Dynamin gene, shibire. This work has revealed that several functional characteristics of adult neuromuscular synapses are distinct from those described in the larva. In the present study, comparison of glutamatergic DLM neuromuscular synapses and cerebellar CF-PC synapses revealed a surprising degree of conservation in the detailed properties of synaptic function. These synapses exhibit morphological similarities as well, including extensive branching of axons and spatial isolation of active zones. Initial characterization of short-term depression at CF-PC synapses has been followed by a series of studies progressively defining the underlying factors in greater detail. The present study provides a basis for analysis of DLM neuromuscular synapses to characterize analogous factors and their molecular determinants. Finally, the results reported in this study complement previous and ongoing studies at DLM neuromuscular synapses of the Dynamin TS mutant, shibire, which exhibits a rapid enhancement of short-term depression as observed in SNAP-25TS. Such similarities may reflect interactions of the in vivo molecular mechanisms governing synaptic vesicle endocytosis and exocytosis (Kawasaki, 2009).

A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. This study demonstrates activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (Calcium ATPase at 60A: a SERCA mutant), and presents an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. All of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from the genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila (Freeman, 2010).

This study describes a new model for activity-dependent pre-synaptic plasticity in Drosophila. In the double mutant combination of comt and Kum, sustained elevation of neural activity (potentially including seizure-like motor neuron firing under normal rearing conditions) results in the expansion of motor synapses with a concomitant increase in transmitter release. These synaptic changes are mediated by the Ras/ERK signaling cascade and the activity of at least two key transcription factors, CREB and Fos. In vivo reporter assays also directly demonstrate Ras activation and enhanced transcription of Fos in the nervous system. CK is the only genetic model of synaptic plasticity in Drosophila in which pre-synaptic plasticity has been correlated with the Ras/ERK signaling cascade. This result is especially relevant given the wide conservation of the Ras/ERK signaling cascade in plasticity and recent demonstrations of the involvement of this signaling cascade in learning behavior in flies (Godenschwege, 2004; Moressis, 2009). Significant insights into Ras mediated regulation of both synapse growth and transmitter release are also presented (Freeman, 2010).

Non-invasive methods to manipulate neural activity in select neurons continue to be an important experimental target in plasticity research. In Drosophila, combinations of the eag and Shaker potassium channel mutants have long been used to chronically alter neural activity and study downstream cellular events. In recent years, transgenic expression of modified Shaker channels has also been generated and used to alter excitability in both neurons and muscles. However, the CK model of activity-dependent plasticity was developed since in synaptic changes in CK were consistently more robust than eag Sh and core plasticity-related signaling components were activated in a predictable manner in CK mutants. Another advantage with CK is the option of acutely inducing seizures as has been used to identify activity-regulated genes. CK thus combines advantages of both eag Sh and seizure mutants, and as is shown in this study, leads to an activity-dependent increase in synaptic size and transmitter release. It is believed that this model will prove highly beneficial to the large community of researchers who investigate synaptic plasticity in Drosophila. The utility of more recent techniques (such as the ChannelRhodopsin or the newly reported temperature sensitive TrpA1 channel transgenes) to induce neural activity-dependent synaptic plasticity at Drosophila motor synapses has not been tested yet and it will be interesting to see if these afford greater experimental flexibility in the future (Freeman, 2010).

Signal transduction through the Ras cascade has been shown to affect both dendritic and pre-synaptic plasticity in invertebrate and vertebrate model systems. In mammalian neurons, Ras signaling has been linked to hippocampal slice LTP, changes in dendritic spine architecture and plasticity of cultured neurons. In this context, Ras signaling has been shown to impinge on downstream MAP kinase signaling, thus implicating a canonical signaling module already established as a mediator of long-term plasticity in vertebrates. In Drosophila, expression of a mutant constitutively active Ras that is predicted to selectively target ERK leads to synapse expansion and increased localized phosphorylation of ERK at pre-synaptic terminals. In light of these observations, tests were performed to see if Ras signaling os necessary and sufficient for synaptic plasticity in CK. The results suggest that synaptic changes in CK are driven by stimulated Ras/ERK signaling in Drosophila motor neurons, and these can be replicated by directly enhancing Ras signaling in these cells. Furthermore Ras activation was found to be sufficient to cause stable elevation in pre-synaptic transmitter release. Finally, evidence is provided to show that synaptic effects of Ras activation require the function of both Fos and CREB in motor neurons. The consistency of signaling events in CK with those observed in mammalian preparations makes this a more useful and generally applicable genetic model of synaptic plasticity (Freeman, 2010).

In vivo reporters of neural activity have been difficult to design but offer better experimental resolution and flexibility over standard immuno-histochemical or RNA in situ methods to detect changes in gene expression in the brain. Thus, a good reporter permits increased temporal and spatial resolution, the option of live imaging (for fluorescent reporters) and in the case of transcriptional reporters, better understanding of cis-regulatory elements that control activity-dependent gene expression. This paper describes two genetically encoded reporters with utility clearly beyond the current study; a Raf based reporter to detect Ras activation in neurons and an enhancer based reporter to detect transcription of Fos (Freeman, 2010).

The Ras binding domain of Raf has been used previously to detect Ras expression in yeast, mammalian cell lines, and recently in hippocampal neuron dendrites. This study used a similar strategy to model the reporter using the conserved Ras binding domain and the cysteine-rich domain (RBD + CRD) from Drosophila Raf, under the reasonable assumption that this would provide sensitive reporter activity in neurons. This is the first time that a Ras reporter has been utilized in an intact metazoan organism to measure changes in endogenous Ras activity. In addition to confirming Ras activation in CK brains, it is expected that this reporter will find widespread use in tracing Ras activation in multiple tissues through development and in response to signaling changes in the entire organism. Since the reporter is based on the GAL4-UAS system, it can be expressed in tissues of choice, limiting reporter activity to regions of interest. Indeed, the experiments with the eye-antennal imaginal disc illustrate the utility of this reporter in identifying regions of activated Ras signaling during eye development (Freeman, 2010).

The Fos transcriptional reporter is one of the very few activity-regulated reporters in existence in Drosophila and it should find broad acceptance as a tool to map neural circuits in the fly brain that show activity-dependent plasticity. The reporter believed to be reasonably accurate since it is expressed in expected tissue domains (embryonic leading edge cells, for instance), and also co-localizes extensively with anti-Fos staining in the larval brain. There are several recognizable transcription factor binding motifs that can be detected in this 5 kb region of DNA (including binding sites for CREB, Fos, Mef2 and c/EBP). Which of these transcription factors regulate activity-dependent Fos expression from this enhancer is currently unknown. However, future experiments that dissect functional elements in this large enhancer region are expected to refine and identify these regulatory elements. Such studies are likely to lead the way in the development of a new generation of neural activity reporters in the brain (Freeman, 2010).

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, this study used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. Sixteen candidates were identified that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release, was identified. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, Comt/NSF was validated to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. The possible functional relevance of this modification was tested using Ari-1 loss of function mutants that displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca(2+) dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. These results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination (Ramirez, 2021).

Neurotransmitter release is mediated by a set of protein-protein interactions that include the N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). These proteins assemble into a tripartite complex in order to elicit synaptic vesicle fusion, which is formed by one synaptic vesicle membrane SNARE protein (v-SNARE), Synaptobrevin, and two plasma membrane SNARE proteins (t-SNAREs), Syntaxin and the 25-kDa synaptosome-associated protein. Following vesicle fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs. Free t-SNAREs from the plasma membrane can then participate in new priming reactions, while the v-SNAREs can be incorporated into recycled synaptic vesicles. These interactions, also routinely used for intracellular vesicle trafficking in all cell types, are conserved across species, including Drosophila (Ramirez, 2021).

Deviations on the rate of neurotransmitter release are at the origin of multiple neural diseases, including Parkinson's disease. Under physiological conditions, the Leucine-rich repeat Serine/Threonine-protein kinase 2 (LRRK2) phosphorylates NSF to enhance its ATPase activity, which facilitates the disassembly of the SNARE complex. However, the most common Parkinson's disease mutation in LRRK2 causes an excess of kinase activity that interferes with the vesicle recycling. Similarly, α-Synuclein, another Parkinson's disease protein, alters neurotransmitter release by preventing the v-SNARE vesicle-associated membrane protein (VAMP)-2, also known as Synaptobrevin-2, from joining the SNARE complex cycle. Correct neural functioning, therefore, requires delicate regulation in vesicle trafficking. This regulation can be achieved by posttranslational modifications, such as ubiquitination. In fact, ubiquitination of certain proteins can affect their activity or life span. At the presynaptic side, for example, increased neurotransmitter release correlates with decreased protein ubiquitination. Similarly, acute pharmacological proteasomal inhibition causes rapid strengthening of neurotransmission (Ramirez, 2021).

Ariadne 1 is an E3 ubiquitin-ligase, first identified in Drosophila, from a conserved gene family defined by two C3HC4 Ring fingers separated by a C6HC in-Between-Rings domain (the RBR motif). Ari-1 had been described to be essential for neuronal development, and its mutants reported to exhibit reduced eye rhabdomere surface and endoplasmic reticulum, as well as aberrant axonal pathfinding. However, despite its importance, no neuronal substrates have been reported so far. Only three Ari-1 substrates have been postulated, either in cultured cells or in vitro, while three Parkin substrates were reported to interact with Ari-1 in COS-1 cells. For this reason, with the aim to identify neuronal Ari-1 substrates, advantage was taken of two methodologies. The first one, the bioUb strategy, allows the identification of hundreds of ubiquitinated proteins from neuronal tissues. The system relies on the overexpression of a tagged ubiquitin that bears a 16 amino acid long biotinylatable peptide, which can be biotinylated by the Escherichia coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo. Remarkably, this approach can be efficiently applied to identify neuronal E3 ligase substrates. In contrast, the second methodology favors the isolation of GFP-tagged proteins under denaturing conditions to further characterize their ubiquitination pattern under the presence or absence of an E3 ligase (Ramirez, 2021).

This study has combined the bioUb strategy with the overexpression of Ari-1 and identified 16 putative neuronal substrates of Ari-1. Among those, focus was placed on Comatose (Comt), the fly NSF orthologue, due to its relevance in normal and pathological function at the synapse. By the isolation of GFP-tagged Comt from Drosophila photoreceptor neurons overexpressing Ari-1, this study confirmed Comt/NSF as an Ari-1 ubiquitin substrate and showed that it is mostly monoubiquitinated. Furthermore, Ari-1 loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side. These defects in the mutants are compatible with a deregulation of Com/NSF activity. Altogether, these data show that Ari-1 regulates neurotransmitter release by controlling Comt/NSF activity through ubiquitination (Ramirez, 2021).

This study identified sixteen novel putative substrates of Ari-1 in Drosophila photoreceptor neurons in vivo by means of an unbiased proteomic approach. Remarkably, despite Ari-1 being recently shown to regulate the positioning of the cell nucleus in muscles via a direct interaction with Parkin (Tan, 2018), as well as to interact with some Parkin substrates, there is no overlap between the substrates identified for Ari-1 and those previously identified for Parkin in flies. Taken together, the available data suggest that the 16 targets identified in this study are specifically regulated by Ari-1 in Drosophila photoreceptor neurons and that this E3 ligase has a wide functional repertoire (Ramirez, 2021).

This study focused on Comt, an ATPase required for the maintenance of the neurotransmitter release. Ubiquitination of proteins involved in vesicle trafficking and neurotransmitter release had been previously reported. Similarly, the importance of the ubiquitination machinery for the proper neuronal function has also been demonstrated. The alterations produced on synaptic transmission by ubiquitination are typically attributed to an acute control of synaptic protein turnover. However, many of these presynaptic proteins have been reported to be mainly mono- or di-ubiquitinated, a type of ubiquitin modification that is not usually associated with protein degradation. In line with this, the results showed that Comt/NSF is preferentially monoubiquitinated by Ari-1/ARIH1, suggesting that Ari-1/ARIH1 could be regulating Comt/NSF activity, rather than its life span or expression levels (Ramirez, 2021).

Ari-1 mutations result in abnormal synaptic function at the larval stage, a result consistent with a regulatory function of NSF. All mutant alleles examined exhibit a reduced frequency of spontaneous synaptic release. In addition, ari-12 mutants exhibit a large calcium-dependent evoked release. Analysis of the mechanism for enhanced evoked release in ari-12 suggests that the primary defect consists in an increased probability of vesicle fusion in response to calcium entry in the presynaptic side. First, by comparing the amplitude and time course of spontaneously occurring postsynaptic events in mutant and control animals, the possibility of a postsynaptic modification was ruled out. Since no significant differences were found, it is concluded that the receptor field size and kinetic properties of postsynaptic receptors are normal in the mutant (Ramirez, 2021).

The ari-1 functional defects could result from alterations of synaptic transmission during development; therefore, the number of synaptic contacts was quantified, assuming that most release sites occur within varicosities. No significant difference was observed between mutant and control. Although this study does not include electron microscopy quantification of synaptic vesicles, th confocal microscopy and electrophysiology data point toward an upregulation of release from single synapses (Ramirez, 2021).

The failure analysis from single varicosities represents direct evidence that, at relatively low calcium concentrations, mutant terminals release more quanta than controls in response to an action potential. Whether increased quantal release could be explained on the basis of more release sites being concentrated on mutant terminals was examined. The focal recordings using saturating calcium concentrations argue against this possibility. When mutant terminals are exposed to high calcium, in order to increase the likelihood that all active zones within the bouton will release a quantum, EJP amplitudes in the mutant are indistinguishable from that of controls. These data suggest that the number of release sites in mutant and control terminals is similar and favor the hypothesis that, at physiological calcium concentrations, the probability of vesicle fusion upon calcium entry is increased in the mutant (Ramirez, 2021).

It was found that ari-1 mutants have opposite effects on spontaneous and evoked release. Classically, the two modes of vesicular release have been considered to represent a single exocytotic process that functions at different rates depending on the Ca2+ concentration. However, recent work challenges this idea and supports the alternative model where spontaneous and evoked response might come from different vesicles pools. Several experimental evidences indicate that both forms of release may represent separate fusion pathways. Employing a state-of-the-art optical imaging in larval NMJ, it has been shown that evoked and spontaneous release can be segregated across active zones. Thus, three types of active zones could be defined: those that only release vesicles in response to a rise of intracellular calcium (evoked release), a second population that only participates in spontaneous release, and a third small proportion (around 4%) that participates in both evoked and spontaneous release. This result advocates for a different molecular and spatial segregation of both modes of release (Ramirez, 2021).

Differential content or activity of regulatory SNARE binding proteins could discriminate between spontaneous and evoked release. It has been shown that the presence of the Vamp-7 isoform could participate in this differential release. Vamp-7 preferentially labels vesicles unresponsive to stimulation, and it colocalizes only partially with the endogenous synaptic vesicle glycoprotein Sv2 and the vesicular glutamate transporter Vglut1, suggesting that this vesicle pool does not support evoked transmitter release. Recently, it was shown that the double knockout mouse for Synaptobrevin genes, syb1 and syb2, results in a total block of evoked release, while spontaneous release was increased in both frequency and quantal size without changes in the number of docked vesicles at the active zone, confirming the idea that evoked and spontaneous releases are differentially regulated. Interestingly, Vamp-7 was found by MS to be less ubiquitinated when Ari-1 is overexpressed, suggesting that Ari-1 mutants could be favoring evoked release through NSF and reducing spontaneous release through Vamp7. Thus, Ari-1 could be acting as a repressor and activator of evoked and spontaneous release, respectively. All together, these results evidence a new layer of complexity over the actual fine-tuning of synaptic transmission. A physiological regulatory mechanism for both types of release has been recently demonstrated for inhibitory synapses at the trapezoid body, an important brain area in auditory integration. In this nucleus, activation of metabotropic glutamate receptor mGluR1 differentially modulates both spontaneous and evoked release in both GABAergic and Glycynergic synapses (Ramirez, 2021).

Early functional studies of NSF employing the fly thermo-sensitive mutant allele comtts17, have reported a reversible reduction of synaptic transmission. Consistent with a role of NSF on SNARE dissociation, this inhibition parallels an increase in the number of synaptic vesicles at the presynaptic terminal. At this point, it can only be speculated how specifically Ari-1/ARIH1 regulates Comt/NSF activity within the presynaptic terminal. Opposite to the role of NSF mutant comtts17, which impairs SNARE complex disassembly, a change that enhances NSF functionality due to the lack of its ubiquitination would favor the dissociation of the so-called trans-SNARE complex. Further, this would build up the number of SNARE complexes assembled per vesicle, thus increasing the efficiency of fusion machinery in a Ca2+-dependent manner. Consistent with this interpretation, it has been shown that fast release of a synaptic vesicle requires at least three SNARE complexes, whereas slower release may occur with fewer complexes (Ramirez, 2021).

Interestingly, some of the additional putative substrates identified are also related to synapse physiology and neurotransmitter release. PPO1 is an enzyme with L-DOPA monooxygenase activity, hence, may be involved in the metabolism of dopamine neurotransmitter. Similarly, GstO3 is involved in glutathione metabolism, another type of neurotransmitter. Vha44 and Vha68-1 are components of the vacuolar proton-pump ATPase, whose mutations have been reported to impair neurotransmitter release. Vha44 has also been described as an enhancer of Tau-induced neurotoxicity, and CG15117, orthologue of human GUSB, has been associated with neuropathological abnormalities. The long recovery time from paralysis observed in comt6 ari2/comt6 females could result from the role of Ari-1 in the ubiquitination of these additional substrates, in addition to the role of Comt in tissues other than the nervous system (Ramirez, 2021).

The data reported in this study may be relevant in the context of Parkinson's disease. It should be noted that most Parkinson's-related genes encode proteins involved in vesicle recycling and neurotransmitter release at the synapse. Thus, the kinase LRRK2 phosphorylates NSF to enhance its ATPase activity upon the SNARE complex and facilitate its disassembly. Pathological mutations in this protein, such as G2019S, cause an excess of kinase activity that interferes with vesicle recycling. Deregulated synaptic aggregates of α-Synuclein may target VAMP-2 hampering the formation of the SNARE complex. Parkin is a structural relative of Ari-1, based on their common Cysteine rich C3HC4 motif, which is also at the origin of some forms of Parkinson's disease. All these genes and their corresponding mechanisms of activity sustain the scenario in which several types of Parkinson's disease seem to result from a defective activity of the synapse. In this context, the role of Ari-1/ARIH1 emerges as a mechanism to regulate a key component of the SNARE complex, Comt/NSF. Conceivably, Ari-1/ARIH1 may become a suitable target for either diagnosis or pharmacological treatment of Parkinson's and related diseases (Ramirez, 2021).

A neuroprotective function of NSF1 sustains autophagy and lysosomal trafficking in Drosophila

A common feature of many neurodegenerative diseases is the accumulation of toxic proteins that disrupt vital cellular functions. Degradative pathways such as autophagy play an important protective role in breaking down misfolded and long-lived proteins. Neurons are particularly vulnerable to defects in these pathways, but many of the details regarding the link between autophagy and neurodegeneration remain unclear. Previous studies found that temperature-sensitive (ts) paralytic mutants in Drosophila are enriched for those exhibiting age-dependent neurodegeneration. This study shows that one of these mutants, comatose (comt), in addition to locomotor defects, displays shortened lifespan and progressive neurodegeneration, including loss of dopaminerigic (DA) neurons. comt encodes N-ethyl-maleimide sensitive fusion protein (NSF1), which has a well-documented role in synaptic transmission. However, the neurodegenerative phenotypes observed in comt mutants do not appear to depend on defects in synaptic transmission, but rather from their inability to sustain autophagy under stress, due at least in part to a defect in trafficking of lysosomal proteases such as Cathepsin-L. Conversely, over-expression of NSF1 rescues alpha-synuclein-induced toxicity of DA neurons in a model of Parkinson's Disease. These results demonstrate a neuroprotective role for NSF1 that involves mediation of fusion events crucial for degradative pathways such as autophagy, providing greater understanding of cellular dysfunctions common to several neurodegenerative diseases (Babcock, 2004; PubMed).


EVOLUTIONARY HOMOLOGS

NSF function in disassembling SNARE complexes

Homotypic vacuole fusion in yeast requires Sec18p (NSF), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). Vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, since the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (1) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release; (2) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (3) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes (Ungermann, 1998).

SNARE proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Several lines of evidence demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption (Weber, 2000).

Homotypic vacuole fusion occurs in ordered stages of priming, docking, and fusion. Priming, which prepares vacuoles for productive association, requires Sec17p (the yeast homolog of alpha-SNAP), Sec18p (the yeast NSF, an ATP-driven chaperone), and ATP. Sec17p is initially an integral part of the cis-SNARE complex together with vacuolar SNARE proteins and Sec18p (NSF). Previous studies have shown that Sec17p is rapidly released from the vacuole membrane during priming as the cis-SNARE complex is disassembled, but the order and causal relationship of these subreactions has not been known. Addition of excess recombinant his(6)-Sec17p to primed vacuoles can block subsequent docking. This inhibition is reversible by Sec18p, but the reaction cannot proceed to the tethering and trans-SNARE pairing steps of docking while the Sec17p block is in place. Once docking has occurred, excess Sec17p does not inhibit membrane fusion per se. Incubation of cells with thermosensitive Sec17-1p at nonpermissive temperature causes SNARE complex disassembly. These data suggest that Sec17p can stabilize vacuolar cis-SNARE complexes and that the release of Sec17p by Sec18p and ATP allows disassembly of this complex and activates its components for docking (Wang, 2000).

In yeast, assembly of exocytic SNARE complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs (Ssop and Sec9p) occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane (Grote, 2000).

NSF interaction with alpha-SNAP

N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. NSF is able to associate with Golgi membranes in an ATP-dependent fashion. This association is dependent on three peripheral membrane proteins, termed soluble NSF attachment proteins (SNAPs). alpha-SNAP and NSF are associated in a 20S complex with three membrane proteins: syntaxin, SNAP-25 (synaptosomal associated protein of 25 kD) and vesicle-associate membrane protein (VAMP), collectively termed SNAP receptors (SNAREs). In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for the ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids has little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids results in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP are able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs reveals only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine [alpha-SNAP(L294A)] results in a decrease in the ability to stimulate NSF ATPase activity but has no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285) and alpha-SNAP (L294A) are able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) are all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to fully disassemble the 20S complex and do not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis (Barnard, 1997).

Regulation of NSF function

Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble NSF attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. The depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes is reported. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF (Matveeva, 2001).

Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. A yeast two-hybrid screen was performed to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. The D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca(2+)-dependent exocytosis, the effect was examined of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K(+)-stimulated growth hormone release. Taken together, these findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system (Liu, 2006).

Mutational alteration of NSF

An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts (Steel, 2000).

NSF interaction with receptors

Specific interaction has been demonstrated between the GluR2 (AMPA) receptor subunit C-terminal peptide (see Drosophila Glutamate receptor IIA and Glutamate receptor IIB), an ATPase N-ethylmaleimide-sensitive fusion protein (NSF), and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as the dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. This paper provides evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. A model is proposed in which NSF functions as a chaperone in the molecular processing of the AMPA receptor (Osten, 1998).

The findings that NSF and alpha- and beta-SNAPs interact with GluR2 in a complex, which in several respects resembles the interaction of NSF and SNAP at the SNARE, can be interpreted to support a functional model of the GluR2-NSF-SNAP binding. In this model, the NSF-SNAP complex is required in chaperone-like priming of the AMPA receptors during a continuous process required for receptor function. This process could involve receptor recycling between the postsynaptic membrane and a cytoplasmic pool. As has been proposed for NSF function at the SNARE complexes, the interaction of NSF and SNAP with the AMPA receptor could involve the disruption of multiprotein complexes, such as those formed between the membrane-inserted receptor and the proteins of the postsynaptic density (such as GRIP). NSF-driven disassembly of these complexes could be required for the proper sorting of these proteins at specific times during development, as for example, prior to a new cycle of insertion and anchoring, or in the processing of newly synthesized receptors (Osten, 1998 and references).

NSF interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission is also observed when an anti-NSF monoclonal antibody (mAb) is loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function (Nishimune, 1998).

NSF specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function (Song, 1998).

Disruption of NSF-GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) causes a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). NMDA receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduces the surface expression of AMPA receptors, whereas NMDA receptor surface expression in the same living cells is unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity is unchanged, and a punctate distribution of GluR2 is observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses. Based on these findings and the known properties of NSF, a model is favored in which the interaction between NSF and GluR2 is involved in the part of the cycling process that is necessary for the insertion and/or stabilization of AMPA receptors at the postsynaptic membrane. By analogy with its known presynaptic functions, NSF could act at the AMPA receptor complex by stripping the receptors of associated proteins. Candidate proteins interacting with GluR2 include the PDZ-containing proteins GRIP, ABP, and PICK1. Removal of associated proteins could prime or "reset" the AMPA receptor complex to a naive state, thereby allowing insertion into the postsynaptic membrane. If the action of NSF is prevented, for example, by peptide block, the receptors cannot be appropriately processed, and insertion/reinsertion of the reconfigured receptors into the postsynaptic membrane cannot occur (Noel, 1999).

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF causes a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis causes a gradual increase in the AMPAR EPSC. These manipulations have no effect on the NMDAR EPSC but prevent the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process (Luscher, 1999).

An investigation was carried out to see whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevents homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevents the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD are due to changes in quantal size and quantal content but are not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses (Luthi, 1999).

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity, which may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions (Hanley, 2002).

AMPAR trafficking is thought to involve constitutive cycling of receptors by endocytosis/exocytosis, as well as regulated events as part of LTD (endocytosis) and LTP (exocytosis). AMPAR endocytosis during some forms of LTD is dependent upon GluR2 phosphorylation and regulation of accessory protein binding. The NSF-mediated disassembly of the GluR2-PICK1 complex described in this study is therefore likely to be crucial in limiting endocytosis of AMPARs to maintain constitutive cycling at a constant rate and hence maintain a constant level of receptors at the synaptic membrane. From this baseline, LTD could be induced (in conjunction with phosphorylation events) by reducing the activity of NSF, possibly by modulation of SNAP-PICK1 binding, to stabilize GluR2-PICK1 interactions, and consequently enhance receptor endocytosis. This study has identified the molecular mechanism for the activity of NSF in AMPA receptor trafficking, and has demonstrated that NSF can function as a disassembling molecular chaperone in a protein complex other than the 20S SNARE complex. As additional NSF binding partners are identified, it is possible that this ATPase, previously thought to be faithful to the SNARE complex, will show more promiscuous chaperone behavior (Hanley, 2002).

NSF and Golgi membrane fusion

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, a mutation was introduced into mammalian NSF that, in Drosophila NSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs (Muller, 1999).

Results obtained with the comatose-like mutant were substantiated by using a distinct mutant, NSF(E329Q), that is defective in ATPase activity and SNARE disassembly. This mutation results in a ~75% reduction in ATPase activity, which completely abolishes its ability to stimulate fusion in an intra-Golgi transport assay. These results indicate a positive correlation between the membrane-fusion-promoting function of NSF and its ATPase activity. In contrast, the ATPase-defective NSF (E329Q), like NSF(G274E), is capable of promoting cisternal regrowth to ~80% of the level of wild-type NSF. Together, data obtained using the mutant NSF proteins indicate that NSF's ATPase activity may not be directly linked to postmitotic Golgi membrane fusion (Muller, 1999).

Since NSF(G274E) and NSF(E329Q) both lack the ability to break up SNARE complexes, NSF-dependent SNARE disassembly seems to be uncoupled from membrane fusion of postmitotic Golgi fragments. The break-up of SNARE complexes is thought to be essential for the recycling of these proteins for further rounds of fusion; thus, these data indicate that recycling may not be needed for Golgi reassembly in the cell-free assay. On the basis of current models, it is therefore predicted that there is an abundant source of disassembled SNAREs on mitotic Golgi fragments before reassembly. If true, this could help to explain the discrepancy in the requirement for NSF's ATPase activity in other published membrane-fusion assays but not during mitotic Golgi reassembly (Muller, 1999).

However, this leaves open the nature of the distinct role for NSF during the membrane-fusion process. Interestingly, assembly of synaptic 20S complexes is temperature sensitive in the presence of the NSF mutant, which indicates that the presence of NSF in a SNARE complex might be critical. One possibility is that NSF is needed to prime (for example, by folding or assisting in accessory-factor recruitment) the SNAREs on MGFs in preparation for fusion. Although priming of vacuole and, perhaps, Golgi SNAREs correlates with the presence of Mg-ATP, it is not clear that ATP hydrolysis is needed for this process. Another possibility is that a checkpoint exists to ensure that NSF is recruited to the fusion site in preparation for its later function in breaking up SNARE complexes. This would certainly explain why NSF has been found on synaptic and clathrin-coated vesicles that still have to dock and fuse. Such recruitment would target NSF to the site at which its action will be needed. A final possibility is that NSF takes part in the actual fusion process itself. It is, therefore, interesting that NSF-SNAPs can directly fuse liposomes together in an ATP-dependent manner. Furthermore, this happens most efficiently when the NSF-SNAP complex has the lowest ATPase activity, the key feature of the NSF mutant (Muller, 1999).

In conclusion, these studies of the Drosophila comatose analog in mammalian NSF provide clear evidence that NSF has a role in membrane fusion that is divorced from its ability to break up SNARE complexes. The likelihood is that NSF has multiple roles and further structure/function studies should provide the means for their dissection (Muller, 1999).

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants reveals an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that is essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments reveals that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyzes the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. It is suggested that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function (Muller, 2002).

nsf is essential for organization of myelinated axons in zebrafish

Myelinated axons are essential for rapid conduction of action potentials in the vertebrate nervous system. Of particular importance are the nodes of Ranvier, sites of voltage-gated sodium channel clustering that allow action potentials to be propagated along myelinated axons by saltatory conduction. Despite their critical role in the function of myelinated axons, little is known about the mechanisms that organize the nodes of Ranvier. Starting with a forward genetic screen in zebrafish, an essential requirement for nsf (N-ethylmaleimide sensitive factor) was identified in the organization of myelinated axons. Previous work has shown that NSF is essential for membrane fusion in eukaryotes and has a critical role in vesicle fusion at chemical synapses. Zebrafish nsf mutants are paralyzed and have impaired response to light, reflecting disrupted nsf function in synaptic transmission and neural activity. In addition, nsf mutants exhibit defects in Myelin basic protein expression and in localization of sodium channel proteins at nodes of Ranvier. Analysis of chimeric larvae indicates that nsf functions autonomously in neurons, such that sodium channel clusters are evident in wild-type neurons transplanted into the nsf mutant hosts. Through pharmacological analyses, it has been shown that neural activity and function of chemical synapses are not required for sodium channel clustering and myelination in the larval nervous system. It is concluded that zebrafish nsf mutants provide a novel vertebrate system to investigate Nsf function in vivo. These results reveal a previously unknown role for nsf, independent of its function in synaptic vesicle fusion, in the formation of the nodes of Ranvier in the vertebrate nervous system (Woods, 2006).

Neuronal Ndrg4 is essential for nodes of Ranvier organization in zebrafish

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. This study identified N-myc downstream-regulated gene 4, ndrg4 (see Drosophila MESK2), as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 (see Drosophila Synapse protein 25) and nsf (see Drosophila Nsf2) are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery (Fontenas, 2016).

NSF and schizophrenia

Microarray expression profiling of prefrontal cortex from matched pairs of schizophrenic and control subjects and hierarchical data analysis reveals that transcripts encoding proteins involved in the regulation of presynaptic function (PSYN) were decreased in all subjects with schizophrenia. Genes of the PSYN group showed a different combination of decreased expression across subjects. Over 250 other gene groups did not show altered expression. Selected PSYN microarray observations were verified by in situ hybridization. Two of the most consistently changed transcripts in the PSYN functional gene group, N-ethylmaleimide sensitive factor and synapsin II, were decreased in ten of ten and nine of ten subjects with schizophrenia, respectively. The combined data suggest that subjects with schizophrenia share a common abnormality in presynaptic function (Mirnics, 2000).


REFERENCES

Search PubMed for articles about Drosophila comatose and N-ethylmaleimide-sensitive factor 2

Aoto J., Martinelli D. C., Malenka R. C., Tabuchi K., Südhof T. C. (2013) Presynaptic neurexin-3 alternative splicing trans-synaptically controls postsynaptic AMPA receptor trafficking. Cell 154: 75-88. PubMed ID: 23827676

Babcock, D. T., Shen, W. and Ganetzky, B. (2014). A neuroprotective function of NSF1 sustains autophagy and lysosomal trafficking in Drosophila. Genetics 199(2):511-22. PubMed ID: 25519897

Barnard, R. J., Morgan. A. and Burgoyne, R. D. (1997). Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis. J. Cell Biol. 139(4): 875-883. PubMed Citation: 9362506

Boulianne, G. L. and Trimble, W. S. (1995). Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila. Proc. Natl. Acad. Sci. 92: 7095-7099. 7624376

Dellinger, B., Felling, R. and Ordway, R. W. (2000). Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony. Genetics 155: 203-211. 10790395

Dunne, J. C., Kondylis, V. and Rabouille, C. (2002). Ecdysone triggers the expression of Golgi genes in Drosophila imaginal discs via Broad-complex. Dev. Biol. 245(1): 172-86. 11969264

Fontenas, L., De Santis, F., Di Donato, V., Degerny, C., Chambraud, B., Del Bene, F. and Tawk, M. (2016) . Neuronal Ndrg4 is essential for nodes of Ranvier organization in zebrafish. PLoS Genet 12: e1006459. PubMed ID: 27902705

Freeman, A., et al. (2010). A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila. Brain Res. 1326: 15-29. PubMed Citation: 20193670

Godenschwege, T. A., et al. (2004). Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour. Eur. J. Neurosci. 20: 611-622. PubMed Citation: 15255973

Golby, J. A., Tolar, L. A. and Pallanck, L. (2001). Partitioning of N-ethylmaleimide-sensitive fusion (NSF) protein function in Drosophila melanogaster: dNSF1 is required in the nervous system, and dNSF2 is required in mesoderm. Genetics 158(1): 265-78. 11333235

Grote, E., Carr, C. M. and Novick, P. J. (2000). Ordering the final events in yeast exocytosis. J. Cell Biol. 151(2): 439-52. 11038189

Hanley, J. G., Khatri, L., Hanson, P. I. and Ziff, E. B. (2002). NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex. Neuron 34: 53-67. 11931741

Ho, A., Morishita, W., Atasoy, D., Liu, X., Tabuchi, K., Hammer, R. E., Malenka, R. C., Südhof, T. C. (2006) Genetic analysis of Mint/X11 proteins: essential presynaptic functions of a neuronal adaptor protein family. J. Neurosci. 26: 13089-13101. PubMed ID: 17167098

Jahn, R. and Sudhof, T. C. (1999). Membrane fusion and exocytosis. Annu. Rev. Biochem. 68: 863-911. 10872468

Kawasaki, F., Mattiuz, A. M. and Ordway, R. W. (1998). Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release. J. Neurosci. 18(24): 10241-9. 9852561

Kawasaki, F. and Ordway, R. W. (1999). The Drosophila NSF Protein, dNSF1, plays a similar role at neuromuscular and some central synapses. J. Neurophysiol. 82(1): 123-130. PubMed Citation: 10400941

Kawasaki, F., Collins, S. C. and Ordway, R. W. (2002). Synaptic calcium-channel function in Drosophila: Analysis and transformation rescue of temperature-sensitive paralytic and lethal mutations of Cacophony. J. Neurosci. 22(14): 5856-5864. 12122048

Kawasaki, F. and Ordway, R. W. (2009). Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants. Proc. Natl. Acad. Sci. 106(34): 14658-63. PubMed Citation: 19706552

Klenchin, V. A. and Martin, T. F. (2000). Priming in exocytosis: attaining fusion-competence after vesicle docking. Biochimie 82: 399-407. 10865127

Laviolette, M. J., Nunes, P., Peyre, J. B., Aigaki, T. and Stewart, B. A. (2005). A genetic screen for suppressors of Drosophila NSF2 neuromuscular junction overgrowth. Genetics 170(2): 779-92. 15834148

Li, T., Tan, Y., Li, Q., Chen, H., Lv, H., Xie, W. and Han, J. (2015). The Neurexin-NSF interaction regulates short-term synaptic depression. J Biol Chem. 290(29): 17656-17667. PubMed ID: 25953899

Li, J., Ashley, J., Budnik, V., Bhat, M. A. (2007) Crucial role of Drosophila neurexin in proper active zone apposition to postsynaptic densities, synaptic growth, and synaptic transmission. Neuron 55: 741-755. PubMed ID: 17785181

Lin, R. C. and Scheller, R. H. (2000) Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16: 19-49. 11031229

Littleton, J. T., et al. (1998). Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly. Neuron 21(2): 401-13. PubMed Citation: 9728921

Littleton, J. T., et al. (2001). SNARE-complex disassembly by NSF follows synaptic-vesicle fusion. Proc. Natl. Acad. Sci. 98: 12233-12238. 11593041

Liu, Y., et al. (2006). Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis. J. Biol. Chem. 281(15): 9852-8. PubMed Citation: 16461345

Luscher, C., et al. (1999). Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron 24: 649-658. 10595516

Luthi, A., et al. (1999). Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction. Neuron 24: 389-399. 10571232

Matveeva, E. A., et al. (2001). Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes. J. Biol. Chem. 276(15): 12174-81. 11278345

Mirnics, K., et al. (2000). Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron. 28(1): 53-67. 11086983

Mohtashami, M., et al. (2001). Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis. J. Neurochem. 77(5): 1407-17. 11389191

Moressis, A. et al. (2009). A dual role for the adaptor protein DRK in Drosophila olfactory learning and memory. J. Neurosci. 29: 2611-2625. PubMed Citation: 19244537

Muller, J. M., et al. (1999). An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion. Nat. Cell Biol. 1: 335-340. 10559959

Muller, J. M., et al. (2002). Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. J. Cell Biol. 157(7): 1161-73. 12070132

Nishimune, A., et al. (1998). NSF binding to GluR2 regulates synaptic transmission. Neuron 21: 87-97. 9697854

Noel, J., et al. (1999). Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism. Neuron 23: 365-376. PubMed Citation: 10399941

Nunes, P., Haines, N., Kuppuswamy, V., Fleet, D. J. and Stewart, B. A. (2006). Synaptic vesicle mobility and presynaptic F-actin are disrupted in a N-ethylmaleimide-sensitive factor allele of Drosophila. Mol. Biol. Cell 17(11): 4709-19. Medline abstract: 16914524

Osten, P., et al. (1998). The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs. Neuron 21(1): 99-110. PubMed Citation: 9697855

Pallanck, L., et al. (1995). Distinct roles for N-ethylmaleimide-sensitive fusion protein (NSF) suggested by the identification of a second Drosophila NSF homolog. J. Biol. Chem. 270: 18742-18744. 7642522

Ramirez, J., Morales, M., Osinalde, N., Martinez-Padron, I., Mayor, U. and Ferrus, A. (2021). The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF. J Biol Chem: 100408. PubMed ID: 33581113

Sanyal, S., Basole, A. and Krishnan, K. S (1999). Phenotypic interaction between temperature-sensitive paralytic mutants comatose and paralytic suggests a role for N-ethylmaleimide-sensitive fusion factor in synaptic vesicle cycling in Drosophila. J. Neurosci. 19(24): RC47. 10594091

Sanyal, S. and Krishnan, K. S. (2001). Lethal comatose mutation in Drosophila reveals possible role for NSF in neurogenesis. Neuroreport. 12(7): 1363-6. 11388412

Siddiqi, O. and Benzer, S. (1976). Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. 73: 3253-3257. 184469

Song, I., et al. (1998). Interaction of the N-ethylmaleimide-sensitive factor with AMPA receptors. Neuron 21: 393-400. 9728920

Steel, G. J., et al. (2000). A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis. Mol. Biol. Cell 11(4): 1345-56. 10749934

Stewart, B.A., Mohtashami, M., Zhou, L., Trimble, W. S. and Boulianne, G. L. (2001). SNARE-dependent signaling at the Drosophila wing margin. Dev. Biol. 234(1): 13-23. 11356016

Tolar, L. A. and Pallanck, L. (1998). NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking. J. Neurosci. 18(24): 10250-6. PubMed Citation: 9852562

Ungermann, C., et al. (1998). A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. J. Cell. Biol. 140(1): 61-9. 9425154

Wang, L., Ungermann, C. and Wickner, W. (2000). The docking of primed vacuoles can be reversibly arrested by excess Sec17p (alpha-SNAP). J. Biol. Chem. 275(30): 22862-7. 10816559

Weber, T., et al. (2000). SNAREpins are functionally resistant to disruption by NSF and alphaSNAP. J. Cell. Biol. 149(5): 1063-72. 10831610

Wickner, W. and Haas, A. (2000). Yeast homotypic vacuole fusion: a window on organelle trafficking mechanisms. Annu. Rev. Biochem. 69: 247-275. 10966459

Woods, I. G., Lyons, D. A., Voas, M. G., Pogoda, H. M. and Talbot, W. S. (2006). nsf is essential for organization of myelinated axons in zebrafish. Curr. Biol. 16(7): 636-48. 16581508

Xu, H., et al. (2002). Syntaxin 5 is required for cytokinesis and spermatid differentiation in Drosophila. Dev. Bio. 251: 294-306. 12435359


Biological Overview

date revised: 17 December 2021

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