In a first attempt to understand Highwire function, both its mRNA and protein distribution were examined. For protein localization, a monoclonal antibody was generated against Hiw. Analysis by in situ hybridization shows that HIW mRNA is largely, if not exclusively, neural specific. Antisense probes label the ventral neurogenic region as early as stage 5. The staining persists in the neurogenic region at later stages and reaches its peak around stage 13, when both CNS and PNS neurons clearly express this transcript. After embryonic stage 14, the strength of CNS and PNS cell body expression starts to decrease. By stages 16 and 17, weak staining of neuronal cell bodies is observed throughout the CNS. However, by this stage, darker staining was also observed in a pattern within the CNS that coincides with the longitudinal axon tracts found where the synaptic neuropil forms; the same pattern was observed with four independent RNA in situ probes). This expression of mRNA is not associated with specific cell bodies, and thus it is concluded that it represents mRNA in axonal processes or synaptic regions (Wan, 2000).
Hiw protein is detectable at early embryonic stages in the CNS. In contrast to the mRNA expression, a peak of protein expression around stage 13 is not seen. Rather, antibody staining reveals that Hiw protein expression in the CNS is, by and large, constant from stage 13 through the end of embryogenesis. Within the CNS, most of the protein is expressed in the region of the longitudinal axon tracts around which the synaptic neuropil forms. No protein is seen in the CNS nerve roots, none in the peripheral motor and sensory nerves, and very little in the commissural axon pathways. Thus, Hiw expression correlates with where the synaptic neuropil forms in the CNS. Outside the CNS, the predominant location of Hiw protein expression is at NMJ synapses in the embyro. Protein expression is first observed during stage 16, just as these synapses are forming (Wan, 2000).
In third instar larvae, protein expression is observed at the NMJ and at the CNS neuropil. Type I synaptic boutons are clearly stained at all NMJs on all muscles. Weaker staining of type II boutons is detected. The synaptic staining is largely absent at the NMJ in hiw mutant alleles ND8, -9, and -69, and is greatly reduced in hiw mutant alleles hiwEMS, ND16, and ND51. In addition, a transgenic line of flies (pUAS-HIWC1-LD) was generated that ectopically expresses a fragment of Hiw protein containing the epitope used for generating the antibody. The Hiw mAb detects this Hiw protein fragment ectopically expressed in muscles or in tracheal branches, further confirming its specificity. When the horseradish peroxidase (HRP) product is overdeveloped, the antibody also faintly stains the surface of muscles and each of their attachment sites. However, it is believed that this staining is due to the recognition of an unrelated protein(s) since this staining is still present at about the same level in all of the hiw mutants (Wan, 2000).
To study the localization of Hiw at individual boutons, confocal microscopy was used. The staining is not uniform in boutons but rather is confined to patches. Double labeling experiments were performed with Hiw and other synaptic markers. Antibodies against Syt and Dlg label synaptic terminals in a rather uniform fashion. Double labeling with anti-Hiw and anti-Syt, as well as anti-Dlg, confirms that Hiw is not uniform within synaptic boutons (Wan, 2000).
To further identify the position and significance of these Hiw patches, antibodies that label active zones versus the areas around active zones, called periactive zones, were sought. The postsynaptic membrane just opposite the presynaptic active zones can be labeled with antibodies against an epitope tag on transgenic glutamate receptor subunits (MHC-myc-DGluRIIB). For double labeling with anti-Hiw, however, another active zone marker that was not a mouse monoclonal antibody was needed. The Drosophila homolog of the Pak cytoplasmic kinase is such a probe. The glutamate receptor subunit and Pak are precisely colocalized. By double labeling Hiw and Pak, it was found that most Hiw patches are nonoverlapping with Pak staining spots and form a pattern complementary to that of Pak. These data indicate that Hiw is localized to the periactive zones which surround the active zones (Wan, 2000).
A similar pattern of expression is seen for the CAM Fas II. Fas II staining is seen in nonoverlapping areas around Pak staining. The staining with both Fas II and Hiw fills the space outside the Pak staining areas, suggesting that both are localized around but not in active zones (Wan, 2000).
One additional antibody was used to confirm the distribution of Hiw protein outside active zones. Dap160 is a presynaptic membrane-associated protein that physically interacts with dynamin and is organized into an 'endocytic honeycomb' that surrounds active zones. Double labeling between Hiw and Dap160 reveals that the two proteins have largely overlapping distributions, further supporting the idea that Hiw protein is localized to subboutonic regions outside the active zones (Wan, 2000).
To examine the periactive zone localization of Hiw protein in greater detail, immuno-EM analysis was performed. Hiw protein is found to be associated with both the plasma membrane of boutons in all periactive zone areas and with clusters of vesicles outside active zones. However, active zones, T bars, and vesicles clustered at active zones are devoid of staining. Some Hiw staining is also observed associated with postsynaptic membranes opposite periactive zones, even though in situ hybridization analysis does not reveal muscle expression of hiw mRNA. In immuno-EM serial section reconstruction of boutons containing 20 active zones, the nonstaining active zones (which contained T bars and clusters of clear synaptic vesicles) measured 570 ± 50 nm, while the areas between them (the periactive zones) were all stained by the anti-HIW antibody and measured 1389 ± 144 nm. Given the variability of these measurements, the Hiw exclusion zone width of 570 ± 50 nm in the immuno-EM is in the same range as, but is a bit wider than, an independent assessment of active zone width in conventional EM of 469 ± 24 nm. It is not known whether this ~50 nm (~10%) difference on each side represents a real difference or whether the width of the active zone was simply underestimated by classic ultrastructural criteria (Wan, 2000).
Highwire is a large, evolutionarily conserved protein that is required to restrain synaptic growth and promote synaptic transmission at the Drosophila neuromuscular junction. Current models of highwire function suggest that it may act as a ubiquitin ligase to regulate synaptic development. However, it is not known in which cells highwire functions, whether its putative ligase domain is required for function, or whether highwire regulates the synapse during development or alternatively sets cell fate in the embryo. A series of transgenic rescue experiments were performed to test the spatial, structural, and temporal requirements for highwire function. Presynaptic activity of highwire is both necessary and sufficient to regulate both synapse morphology and physiology. The Highwire RING domain, which is postulated to function as an E3 ubiquitin ligase, is required for highwire function. In addition, highwire acts throughout larval development to regulate synaptic morphology and function. Finally, it is shown that the morphological and physiological phenotypes of highwire mutants have different dosage and temporal requirements for highwire, demonstrating that highwire may independently regulate the molecular pathways controlling synaptic growth and function (Wu, 2005).
Hiw is reported to localize to periactive zones, the regions surrounding but not including the active zone, in presynaptic terminals. It has been proposed that the periactive zone is a specialized domain that regulates synaptic growth because at least three proteins important for regulating synaptic growth localize there: Hiw, FasII, and Still life. However, the evidence that Hiw localizes to periactive zones comes from staining with the monoclonal antibody 6H4. 6H4 does not recognize Hiw. Synaptic staining with 6H4 persists in all hiw alleles, including excision mutants that delete the region of the protein against which the antibody was raised. In addition, 6H4 is unable to recognize misexpressed transgenic Hiw. To determine where Hiw is localized, sera were generated to 10 independent peptide and fusion protein antigens. Although a single serum was found that recognizes Hiw on an immunoblot, sera that work for immunocytochemistry were not generated. As such, the endogenous localization pattern of Hiw cannot be determined. GFP-tagged UAS-GFP-Hiw transgenes were generated that are fully capable of rescuing hiw mutants. The GFP-tagged Hiw does localize to synapses, although it does not extensively colocalize with synaptic vesicles, periactive zones, or active zones. The transgenic protein does appear to be enriched in synapses, because it is barely detectable in the preterminal axon. However, this overexpressed protein is visible in cell bodies and axon bundles. Similar results are found with GFP-tagged RPM-1, which is detected in synapses, axons, and cell bodies of C. elegans. It is concluded that Hiw is likely present at synapses (Wu, 2005).
A large number of hiw alleles have been generated, including many with stop codons. All of these nonsense alleles express truncated protein and indeed also express some full-length protein attributable to readthrough of the stop codon. This finding raises the possibility that no hiw alleles are true nulls. In fact, both screens that identified hiw alleles selected for viable mutants, which may have biased the screens against true null alleles. Two new excision mutants of hiw have been generated: one that deletes at least the N-terminal 2784 amino acids (hiwDeltaN), and another that appears to delete the C-terminal 2449 amino acids (hiwDeltaC). Although either allele could potentially express a truncated but partially functional protein, these are the first alleles that do not express any full-length protein. Like the previously identified alleles, these mutants are viable and have synaptic phenotypes that are essentially identical to a previously characterized genetic null allele, hiwND8. Therefore, the previously described hiw phenotype is likely the null phenotype (Wu, 2005).
RNA in situ analysis has demonstrated that hiw is abundantly expressed in the CNS, but that cannot exclude the possibility of lower-level expression in other tissues, such as muscle. In fact, in vertebrates, PAM has been detected in skeletal muscles. This study has addressed the problem of where hiw functions. Presynaptic hiw was found to be both necessary and sufficient for generating a morphologically and physiologically normal synapse. Postsynaptic expression has no detectable effect on synaptic development. These results are consistent with findings in worms and zebrafish. The conserved site of action for hiw supports the view that the mechanism of action of hiw may also be conserved (Wu, 2005).
It is likely that Hiw and its homologs function as E3 ubiquitin ligases. It contains a RING motif that can function as E3 ubiquitin-protein ligase in vitro, and this domain of the Hiw protein is the most highly conserved across species. In flies, hiw genetically interacts with the deubiquitinating protease fat facets, and, in worms, RPM-1 physically interacts with proteins that form an E3 ubiquitin ligase complex. In addition, mutations have been identified in flies and worms that are predicted to disrupt the RING domain; however, these mutant proteins may not be expressed. Therefore, it is unknown whether Hiw ligase activity is necessary for none, some, or all of the functions of hiw at the synapse. An hiw transgene encoding a mutant RING domain has been expressed. The mutations disrupt two conserved cysteine residues that are required for activity of RING domain ubiquitin ligases. This mutant protein is abundantly expressed, but cannot rescue any of the hiw mutant phenotype. Moreover, this RING mutant acts as a potent dominant negative. This suggests that the mutant protein is not grossly misfolded but instead likely interacts with Hiw-binding proteins. This supports the assertion that the RING domain, and ubiquitin ligase activity, is essential for hiw-dependent regulation of both synaptic morphology and transmission (Wu, 2005).
Because Hiw restrains synaptic growth, it was predicted that overexpression of Hiw could reduce synaptic complexity, generating small NMJs with few boutons. No such gain-of-function was observed, indicating that endogenous Hiw levels are not limiting for synaptic growth control. No gain-of-function phenotypes have been reported for rpm-1 either. These results are consistent with the finding that RPM-1 binds to a multiprotein ubiquitin ligase complex. Overexpressing a single member of that complex may not lead to increased function. In fact, the very weak dominant-negative phenotype caused by overexpression of wild-type Hiw suggests that excess Hiw may disrupt the stoichiometry of such a functional complex (Wu, 2005).
Attempts were made to distinguish between two models for the temporal requirement for hiw function. In hiw mutants, synaptic growth is excessive from the time of initial synapse formation. Hence, hiw may act exclusively during embryogenesis to set cell fate, programming the motoneuron for normal or excessive growth. Alternatively, hiw may function throughout development to regulate the synaptic growth rate of the motoneuron. This second model would allow for modification of synaptic growth during development and would be consistent with a function for hiw in activity-dependent synaptic plasticity. To investigate the temporal requirements for hiw, advantage was taken of the GeneSwitch Gal4 system for tissue-specific and temporally specific control of gene expression. It was found that expression of hiw after embryogenesis leads to a decrease in synaptic bouton number and a complete rescue of synaptic function. Hence, hiw not only acts in the embryo but can function throughout the larval growth period to control synaptic morphogenesis and transmission. It is noted that addition of hiw during larval development does not reverse the excess growth that occurred in the embryo and early larval stage. This may explain why similar studies in C. elegans found a requirement for rpm-1 early in synaptic development. hiw is required early in development, but the quantitative analysis of synaptic growth and function that is feasible at the Drosophila NMJ reveals that hiw also functions later in development (Wu, 2005).
Through manipulation of the timing of hiw expression, differential rescue of the morphological and physiological phenotypes of hiw was found. When hiw is expressed late, synaptic transmission is fully rescued, although there is still a twofold to threefold increase in bouton number. Hence, the physiological phenotype is not secondary to developmental defects at the synapse. The morphological and physiological phenotypes of hiw are genetically separable, because an hiw, fat facets double mutant shows a partial rescue of physiology with no rescue of morphology. However, the complete rescue of physiology by the late expression of hiw does more than show that morphology and physiology are independent: it raises the intriguing possibility that hiw functions as a short-term regulator of synaptic function. If hiw has an ongoing role in setting synaptic strength in addition to a developmental role controlling synaptic growth, it is in an ideal position to integrate and coordinate signals that regulate morphological and physiological synaptic plasticity (Wu, 2005).
Each abdominal hemisegment in the Drosophila embryo and larva has a stereotyped pattern of 30 muscles, each identifiable by its size, shape, body wall insertion position, and expression of molecular markers. About 45 motor neurons extend axons to innervate specific muscles in each hemisegment (Wan, 2000).
At the larval NMJ, there are two major types of synaptic terminals. Type I synapses have larger boutons and use glutamate as their neurotransmitter, while type II synapses have smaller boutons containing a variety of vesicles, including dense core vesicles filled with other transmitters, such as neuropeptides and biogenic amines. Type I boutons are surrounded postsynaptically by a conspicuous subsynaptic reticulum (SSR) consisting of multiple folds and invaginations of the muscle cell membrane. Type I synapses can be further divided into type Ib (big) and type Is (small) based on the sizes of their boutons. These synapses undergo dynamic growth during larval development. As the muscle increases its volume, there is a parallel increase in the number of synaptic boutons and the number and complexity of branches (Wan, 2000).
In a pilot effort to look for synaptic structural mutants, a collection of viable mutants on the X chromosome was screened, looking for structural defects at the NMJ of third instar larvae. These 230 mutant lines have various degrees of walking defects in adults. Antibodies against synaptotagmin (Syt) and Fas II were used as markers to label NMJ synaptic terminals of third instar larvae. One mutant line, hiw, has a dramatic synaptic structure phenotype. This initial hiwEMS mutant allele and all 12 subsequent hiwND mutant alleles, as well as 3 P element alleles, behave as viable recessive alleles. They are all viable as transheterozygotes with each other or with deficiencies that remove the gene (Wan, 2000).
In hiw mutants, the presynaptic boutons visualized by Syt staining are more numerous at all NMJs. Compared with wild-type synapses, hiw synapses have a greatly expanded branching pattern. The presynaptic terminal arbors contain many more Fas II-positive branches, the branches are longer, and the branch orders are increased. The synaptic area, the muscle surface area occupied by presynaptic structures, is also increase in hiw mutants. This synaptic overgrowth phenotype is 100% penetrant because expanded presynaptic arborizations and an increased number of boutons are observed at every NMJ in every segment of every mutant third instar animal (Wan, 2000).
Several aspects of the mutant phenotype were quantified. The number of type I boutons on muscles 6/7 and on muscle 4 were counted. hiw mutants have a 2-fold or greater increase in the number of boutons. After correcting for muscle area, hiw synapses also have a 2-fold increase in synaptic size compared with wild type. The combination of hiwEMS chromosome in trans over deficiencies show essentially identical phenotypes. Developmental curves of both hiw and wild-type larvae can be fitted with similar types of power functions, but hiw synapses have a larger synaptic size at first instar and grow at a higher rate during further larval stages. On average, there is a decrease of bouton size in hiw mutants. Given the dramatic increase in the number of boutons, this decrease in bouton size might reflect some limitation of one or more structural components as a result of oversprouting. Type Ib and type Is boutons still can be distinguished from one another in hiw mutants, although both are smaller than their wild-type counterparts. Type II boutons and branches seem more numerous in hiw mutants. However, this phenotype is less dramatic and more difficult to score, and thus was not quantitated (Wan, 2000).
To compare branching, three aspects of the branching pattern on muscle 12 were measured. Muscle 12 was chosen for this analysis because its innervation is relatively planar and accessible, and the branching pattern of that innervation is relatively simple in wild-type larvae, making it easier to quantitate branch number and complexity. First, the total length of this synapse (of only those branches containing type I boutons) was examined as a function of the length of the muscle. From the first larval instar stage onward, the synaptic length in hiw mutants is greater than in wild type. This relationship is maintained throughout larval development (Wan, 2000).
Second, the length of the two longest branches was measured. These branches are more than 2-fold longer in hiw mutants compared with wild type. Third, the branching pattern of this synapse was examined. The branches containing only type I boutons were divided into different groups based on their branch order and the number of branches were counted. There is a dramatic increase in the complexity of branches at hiw synapses; mutant synapses have many more branches and higher order branches. The average number of branches is 16.5 at hiw synapses on muscle 12, compared with 7.7 at the same synapse in third instar wild-type larvae. hiw synapses on muscle 12 often have several fifth order branches, something that is rarely observed in wild type (Wan, 2000).
Although the morphology of NMJ synapses is dramatically expanded in hiw mutants, the specificity of connections is not altered. The initial motor axon pathfinding and target selection appear normal. Antibodies that label both CNS and peripheral axon pathways (anti–Fas II and BP102 mAbs) reveal a normal pattern of axon pathways and a normal pattern of muscle innervation. Longer and larger presynaptic terminal branches were observed on the surfaces of muscles in hiw mutants toward the end of embryogenesis, but the specificity of contacts was normal. It appears that hiw mutants display the synaptic overgrowth phenotype from the onset of synaptogenesis during embryogenesis. This is consistent with the observation that hiw first instar larvae, even 30 min after hatching, already display a dramatic increase in bouton number (34 versus 17 in wild type at muscles 6/7) and increase in synaptic span (56% of muscle length in hiw versus 27% in wild type at muscle 12) (Wan, 2000).
To what extent are synaptic components localized normally in hiw mutant synapses? Postsynaptic glutamate receptors appear to be correctly localized just opposite active zones as determined by expression of the MHC-myc-DGluRIIA and MHC-myc-DGluRIIB transgenes in hiw mutant larvae. The presynaptic protein Syt and the synaptic CAM Fas II are expressed normally at hiw mutant synapses. Several other synaptic proteins (Discs-large [DLG], Pak, and Dap160) are also localized correctly in hiw mutants. Thus, many of the molecular events associated with synapse formation, including clustering of postsynaptic receptors and organization of presynaptic vesicles, appear normal in hiw mutants. This is consistent with the observation that hiw mutants are viable and are not severely impaired in motor behaviors (Wan, 2000).
To further examine the relationship of FasII and hiw, a test was performed to see whether overexpression of Fas II either pre- or post-synaptically can rescue the hiw mutant phenotype. Fas II overexpression cannot rescue the hiw mutant phenotype. This suggests that FasII is not downstream of hiw in the genetic control of synaptic growth (Wan, 2000).
Given the dramatic structural phenotype observed at hiw NMJ synapses, it was of interest to see whether the ultrastructure of these mutant synapses is affected. Serial sections of both hiw and wild-type boutons at muscle 6 (segment A3) of third instar larvae were examined in the electron microscope (EM). It was found that the hiw mutant synapses have active zones with T bars, clusters of synaptic vesicles, and other features that appear wild-type. The postsynaptic SSR also appears normal in size and shape. Some of these characteristics were quantitated from serial sections of 9 hiw boutons and they were compared with similar data from 18 wild-type boutons. Although hiw boutons are, on average, smaller than wild-type boutons, they nevertheless have the same relative bouton surface area per active zone. In addition, the average maximum width of individual active zones (as measured by membrane apposition, location of T bars, and clusters of synaptic vesicles) was 441 in hiw compared with 469 in wild type. Finally, the number of T bars per active zone is statistically similar. It is concluded from these data that the fundamental organization, size, and spacing of active zones in hiw mutants is relatively normal (Wan, 2000).
Given that hiw synapses, although greatly expanded, appear normal ultrastructurally, it was of interest to see whether synaptic transmission is altered at these mutant synapses. One might imagine that the expanded structure would lead to a stronger synapse. Surprisingly, it was found that these expanded synapses are actually weaker physiologically. Intracellular recordings were performed from muscle 6 (segment A3) of third instar larvae. This muscle is innervated by type I glutamatergic boutons from motor neurons RP3 and 6/7b. The postsynaptic responses to both spontaneous and evoked transmitter release were measured. The mean amplitude of spontaneous miniature junctional potentials (mEJPs), also called quantal size, is typically considered a measure of postsynaptic sensitivity to transmitter (in principle, it might also reflect the amount of neurotransmitter packed into each synaptic vesicle, although this is less likely). In contrast, the mean amplitude of the excitatory junctional potential (EJP) depends on both the postsynaptic receptor function and the amount of transmitter released from the presynaptic terminal (Wan, 2000).
The average mEJP amplitude is only slightly reduced in hiw mutants (0.65 ± 0.03 mV versus 0.91 ± 0.04 mV in wild type). However, the evoked release is greatly reduced; the mutant shows a 66% decrease compared with wild type (7.75 ± 0.86 mV versus 22.58 ± 1.73 mV, respectively). Thus, the quantal content (number of vesicles released) of the mutant synapses (13.18 ± 1.41, which is estimated by dividing the mean EJP amplitude by the mean mEJP amplitude and adjusting for nonlinear summation), is about 34% that of wild type (39.22 ± 4.64). Quantal content is an indication of presynaptic function. Since an obvious reduction in synaptic vesicle distribution was not observed in hiw mutants, the reduction in quantal content suggests more subtle defects in the release mechanism (Wan, 2000).
As a first step to gaining insight into the mechanisms of Hiw function, transgenic flies carrying different Hiw fragments were generated. One such transgene (pUAS-HIWC1-LD) contains a Hiw fragment between amino acids 2418 and 3461, fused to enhanced green fluorescent protein (EGFP). When this transgene was expressed panneurally using elav-Gal4 in an otherwise wild-type background, expanded synaptic structures were observed at the NMJ of third instar larvae. This phenotype is qualitatively similar to that of hiw loss-of-function mutants, although quantitatively it is not as severe. When the same transgene was expressed in muscles using the panmuscle 24B-Gal4 enhancer trap line, no phenotype at the NMJ was observed. This result further suggests that Hiw functions on the presynaptic side to control synaptic growth (Wan, 2000).
The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease Fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination (Diantonio, 2001).
Synaptic morphology is dynamic; once formed, synapses expand, retract, and remodel throughout life. This plasticity underlies the refinement of neuronal circuits during development and may be critical for plasticity in the adult brain. To identify molecular mechanisms regulating the morphological growth of synapses, a genetic screen was performed for molecules whose neuronal overexpression disrupts synaptic growth control at the Drosophila NMJ. A collection of flies capable of the targeted overexpression of endogenous Drosophila genes was screened and two lines, EP(3)381 and EP(3)3520, were identified whose overexpression in the nervous system leads to synaptic overgrowth. Both EP(3)381 and EP(3)3520 overexpress fat facets (faf), a deubiquitinating protease. Endogenous faf transcript is strongly and widely expressed in the developing central nervous system (CNS), demonstrating that neuronal expression of faf from the EP lines produces overexpression, not misexpression, of the transcript (Diantonio, 2001).
Anatomical analysis at the NMJ reveals that neuronal expression from both EP(3)381 and EP(3)3520 leads to an increase both in the number of synaptic boutons and in the synaptic span (the extent of the muscle covered by the synapse). This increase is not seen in flies that do not overexpress faf v or that overexpress a non-functional faf gene (elav-Gal4 crossed to EP(3)381faf-). Neuronal overexpression of faf also causes an increase in the number of synaptic branches as quantified by the number of branch points. Postsynaptic expression of faf does not affect synaptic morphology (Diantonio, 2001).
To assess the physiological consequence of neuronal faf overexpression, both spontaneous and evoked neurotransmitter release were analysed at muscle 6 of third instar larvae. Despite the greatly expanded size of the NMJ with faf overexpression, the amplitude of evoked excitatory junctional potentials (EJPs) is markedly reduced. Given that the amplitude of miniature EJPs (mEJPs) shows only a small, albeit significant, reduction, a large decrease was measured in quantal content (the number of vesicles released by the nerve) as measured by dividing the EJP amplitude by the mEJP amplitude. Neuronal overexpression of faf also leads to a reduction in the frequency of spontaneous mEJPs. The reduction in both quantal content and mEJP frequency indicates a presynaptic defect in neurotransmitter release. Other presynaptic mutants with even greater reductions in quantal content do not show a structural overgrowth, thus the anatomical phenotype described above is probably a direct consequence of faf overexpression and not a secondary consequence of this physiological phenotype (Diantonio, 2001).
faf antagonizes ubiquitin-dependent mechanisms by deubiquitinating target proteins. Alterations in synaptic structure and function owing to overexpression of faf suggest that ubiquitin-dependent mechanisms normally act to regulate the developing synapse. However, faf, a characterized deubiquitinating protease, might have other functions. To investigate the role of deubiquitination in the regulation of synaptic development, transgenic flies were generated capable of the targeted overexpression of the yeast deubiquitinating protease UBP2. This enzyme antagonizes ubiquitin-dependent mechanisms in yeast and has overlapping substrate specificity with FAF+9. Overexpression of yeast UBP2 in the nervous system of Drosophila leads to marked synaptic overgrowth and a severe reduction in presynaptic transmitter release. This phenotype is very similar to that seen with faf overexpression. Hence, antagonizing ubiquitin-dependent mechanisms by overexpression of deubiquitinating proteases markedly affects synaptic development (Diantonio, 2001).
To identify molecular pathways regulated by faf, a genetic interaction screen was performed to identify genes that enhance the faf overexpression phenotype. The X chromosome was screened for viable mutations that are lethal in combination with neuronal overexpession of faf. 7,000 chromosomes were screened and 15 lethal enhancers, 12 of which form one complementation group, were identifed. These 12 mutants are alleles of the highwire (hiw) gene and share the synaptic overgrowth phenotype described for loss-of-function hiw mutants. The hiw loss-of-function phenotypes are very similar to the faf gain-of-function phenotypes described here, with a large increase in the number of synaptic boutons, branches, and synaptic span, a small decrease in quantal size, and a large decrease in quantal content. The hiw transcript encodes a greater than 5,000 amino-acid protein that is localized to synapses and that contains a RING-H2 finger, a domain recently identified in a large family of E3 ubiquitin ligases. Hence, a potential synaptic E3 ligase was identified as a lethal enhancer of neuronal overexpression of faf. This genetic interaction provides further evidence that ubiquitination may have a central role in regulating synaptic growth and function (Diantonio, 2001).
To further investigate the genetic relationship between hiw and faf, double mutants were generated between loss-of-function alleles of hiw and faf. faf loss-of-function mutants have phenotypes in the developing eye and female germ line. In faf mutants, no defects were found in either synaptic morphology or function, possibly due to genetic redundancy between faf and one of the 17 other putative deubiquitinating proteases in the Drosophila genome. Although no phenotype was found for faf mutants in otherwise wild-type flies, in the sensitized background of a hiw mutant a requirement for faf is found. Two different loss-of-function alleles of faf both suppress the physiological phenotype of hiw, leading to a more than doubling of both quantal content and mEJP frequency. Hence endogenous faf activity acts to inhibit neurotransmitter release in a hiw background, much as increased faf activity inhibits neurotransmitter release in an otherwise wild-type background. Mutants of faf do not suppress the synaptic overgrowth seen in hiw, indicating that the physiological and morphological phenotypes in hiw are genetically separable. This suggests that either these phenotypes are mediated by different hiw substrates, or the anatomical phenotype is more sensitive to disruption of ubiquitin-dependent mechanisms. Finally, the inability to suppress the small quantal size defect suggests that this phenotype, seen in both hiw mutants and with overexpression of faf, may be a secondary consequence of synaptic overgrowth (Diantonio, 2001).
Decreases in postsynaptic activity induce a compensatory increase in presynaptic transmitter release, demonstrating that a homeostatic mechanism regulates synaptic strength during development. To assess the relationship between homeostatic and ubiquitin-dependent regulation, glutamate receptor function was disrupted in a hiw mutant. Postsynaptic expression of a dominant negative glutamate receptor (DgluRIIA-M/R) in a hiw mutant leads to an 18% decrease in quantal size. Since homeostatic compensation still occurs in a hiw mutant, it is suggested that homeostatic and ubiquitin-dependent regulation are mechanistically distinct (Diantonio, 2001).
The data presented here indicate that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila NMJ and suggest that a balance between positive and negative regulators of ubiquitination controls the structure and function of the synapse. Antagonizing ubiquitination by the neuronal overexpression of the deubiquitinating proteases faf or yeast UBP2 leads to synaptic overgrowth and defects in neurotransmitter release. This phenotype is very similar to the loss-of-function phenotype of hiw, a putative synaptic E3 ubiquitin ligase. Gain-of-function mutants of faf enhance hiw and loss-of-function alleles of faf suppress hiw. It is proposed that hiw-dependent ubiquitination controls the level or activity of critical regulatory molecules at the synapse, and that these molecules can be deubiquitinated by faf and other deubiquitinating proteases. Ubiquitinated proteins have been identified at mammalian synapses, and ubiquitin-processing enzymes can regulate long-term potentiation and facilitation, therefore control of ubiquitination by molecules such as HIW and FAF could be a widely used mechanism for regulating synaptic growth and function (Diantonio, 2001).
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date revised: 20 May 2007
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