Neuronal plasticity relies on tightly regulated control of protein levels at synapses. One mechanism to control protein abundance is the ubiquitin-proteasome degradation system. Recent studies have implicated ubiquitin-mediated protein degradation in synaptic development, function, and plasticity, but little is known about the regulatory mechanisms controlling ubiquitylation in neurons. In contrast, ubiquitylation has long been studied as a central regulator of the eukaryotic cell cycle. A critical mediator of cell-cycle transitions, the anaphase-promoting complex/cyclosome (APC/C), is an E3 ubiquitin ligase. Although the APC/C has been detected in several differentiated cell types, a functional role for the complex in postmitotic cells has been elusive. A novel postmitotic role for the APC/C at Drosophila neuromuscular synapses is described: independent regulation of synaptic growth and synaptic transmission. In neurons, the APC/C controls synaptic size via a downstream effector Liprin-α; in muscles, the APC/C regulates synaptic transmission, controlling the concentration of a postsynaptic glutamate receptor (van Roessel, 2004).
This study shows that the APC/C, a ubiquitin ligase known for its role in regulating cell cycle progression, functions in differentiated neurons to regulate synaptic growth, and in muscles to regulate synaptic transmission. The presence of the APC/C subunits Cdc27, Cdh1/Fizzy related, and APC2/Morula at synaptic structures, together with the accumulation of potential target proteins at synapses in APC2/mr mutants, suggest that the APC/C functions at synapses to regulate local ubiquitin-mediated protein degradation (van Roessel, 2004).
In neurons, the APC/C functions upstream of Liprin-α to constrain the number of synaptic boutons. Liprin-α interacts directly with Dlar, a protein tyrosine phosphatase, suggesting that the APC/C may modulate tyrosine phosphorylation at the synapse by negatively regulating Liprin-α levels. Genetic epistasis demonstrates that the APC/C requires Liprin-α to regulate synaptic bouton number. The regulation of Liprin-α by the APC/C may be indirect. However, the presence of three conserved destruction box motifs in Liprin-α, its accumulation following the loss of APC/C function in neurons, and the fact that Liprin-α is ubiquitylated in the nervous system in vivo, all suggest that it is a direct substrate of the APC/C. APC/C-dependent degradation of bacterially expressed Drosophila Liprin-α was sought in a Xenopus oocyte extract, but with little success. This could reflect the heterologous nature of the assay, or may indicate the requirement for a cofactor not present in Xenopus oocytes, as has been the case for other substrates of the APC/C (van Roessel, 2004).
The APC/C may regulate multiple proteins in differentiated neurons or muscle, just as it targets numerous substrates during the cell cycle. Indeed, other proteins degraded at the NMJ synapse, including Drosophila Unc-13, have putative destruction box sequence motifs. Interestingly, a role has previously been demonstrated for some cell cycle proteins at the Drosophila neuromuscular junction. For example a regulator of DNA replication, Latheo/ORC3, has been shown to affect synaptic function and behavior. Licensing of DNA replication is regulated during the cell cycle in part by the APC/C (van Roessel, 2004).
In muscles, the APC/C modulates muscle sensitivity to neurotransmitter and regulates the levels of a postsynaptic glutamate receptor. The upregulation of GluRIIa observed in APC2/mr mutants could result from: (1) an increase in local glutamate receptor synthesis, (2) inhibition of receptor turnover/degradation, or (3) an increase in glutamate receptor clustering at the synapse. In C. elegans, the glutamate receptor GLR-1 is ubiquitylated, and mutations that disrupt ubiquitylation led to increased accumulation of GLR-1 at central synapses. Although both GLR-1 and Drosophila GluRIIa each have a conserved destruction box motif, these lie in their extracellular domains, suggesting that glutamate receptors may not be direct targets of the APC/C. An alternative is that postsynaptic APC/C also functions through Liprin-α. In vertebrates, Liprin-α has a role in clustering glutamate receptors. Liprin-α mutations do not affect quantal size at the Drosophila NMJ, however, suggesting that Liprin-α is not necessary for regulating glutamate receptor levels in flies (van Roessel, 2004).
Recent evidence indicates that proteasome-mediated protein degradation regulates synaptic function in both vertebrates and invertebrates, although the molecular mechanisms for such regulation have been elusive. The putative ubiquitin ligase Highwire has been proposed to be a general regulator of the morphology and function of neuromuscular synapses in Drosophila. The physiological phenotypes of APC2/morula and highwire mutations, however, are dramatically different, indicating that ubiquitylation at the NMJ involves multiple ubiquitin ligase activities. Indeed there may be independent roles for regulatory monoubiquitylation and polyubiquitylation at Drosophila NMJs. A recent report has also described an acute requirement for ubiquitin-mediated proteolysis in regulating synaptic transmission at the Drosophila NMJ (Speese, 2003). Acute pharmacologic inhibition of the proteasome rapidly increases synaptic transmission, but does so via presynaptic mechanisms that increase transmitter release. Whether this role for ubiquitylation involves the APC/C or another ligase mechanism is unclear (van Roessel, 2004).
A role for protein degradation in regulating synaptic plasticity in Aplysia has been demonstrated. Longer term pharmacological inhibition of the proteasome facilitates serotonin-evoked synaptic strengthening at a central sensory-motor synapse. Presynaptic proteasome inhibition promotes growth of synaptic contacts, while postsynaptic inhibition increases the strength of glutamatergic synaptic inputs. Here, mutation of APC2/mr in Drosophila is sufficient to parallel both the pre- and post-synaptic effects of general proteasome inhibition at a central synapse in Aplysia. This leads to a suggestion that protein degradation via the APC/C may be an evolutionarily conserved mechanism for modulating synaptic strength. The APC/C may be a principal regulator of proteasome-dependent protein degradation at glutamatergic synapses, and thus a key effector of synaptic plasticity (van Roessel, 2004).
Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).
This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).
In brp5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRPD1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).
Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca2+ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).
The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the CacGFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca2+ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca2+ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca2+ channel protein it has been concluded that Ca2+ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca2+ channels and components of ribs/T-bars might well control the formation of Ca2+ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).
The mean Ca2+ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of CacGFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca2+ channels beneath the T-bar pedestal (Fouquet, 2009).
Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca2+ channel trafficking, clustering, and functional modulation (Fouquet, 2009).
The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca2+ channel levels at maturing AZs but not for initializing Ca2+ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca2+ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca2+ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).
In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca2+ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).
This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca2+ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).
Classical cadherin-mediated interactions between axons and dendrites are critical to target selection and synapse assembly. However, the molecular mechanisms by which these interactions are controlled are incompletely understood. In the Drosophila visual system, N-cadherin is required in both photoreceptor (R cell) axons and their targets to mediate stabilizing interactions required for R cell target selection. This study identifies the scaffolding protein Liprin-α as a critical component in this process. Mutations were isolated in Liprin-α in a genetic screen for mutations affecting the pattern of synaptic connections made by R1–R6 photoreceptors. Using eye-specific mosaics, a previously undescribed, axonal function for Liprin-α in target selection was demonstated: Liprin-α is required to be cell-autonomous in all subtypes of R1–R6 cells for their axons to reach their targets. Because Liprin-α, the receptor tyrosine phosphatase LAR, and N-cadherin share qualitatively similar mutant phenotypes in R1–R6 cells and are coexpressed in R cells and their synaptic targets, it is inferred that these three genes act at the same step in the targeting process. However, unlike N-cadherin, neither Liprin-α nor LAR is required postsynaptically for R cells to project to their correct targets. Thus, these two proteins, unlike N-cadherin, are functionally asymmetric between axons and dendrites. It is proposed that the adhesive mechanisms that link pre- and post-synaptic cells before synapse formation may be differentially regulated in these two compartments (Choe, 2006).
In the lamina, Liprin-α function is cell-autonomous to each R1–R6 cell subtype and is required before synapse formation. This phenotype is substantially identical to phenotypes described for N-cadherin and LAR in these cells. Expression studies reveal that these three genes are expressed in largely overlapping patterns. These extensive similarities suggest that these genes act at the same step in the target selection process in R1–R6 axons. However, further somatic mosaic analysis revealed a critical distinction amongst the functions of these genes. That is, whereas N-cadherin is required both pre- and postsynaptically, Liprin-α and LAR are required only in R1–R6 cell axons, not their targets. Because work in other systems has demonstrated that Liprin-α, LAR, and N-cadherin form a complex and that LAR can regulate the critical cadherin effector, β-catenin, it is speculated that homophillic, N-cadherin-mediated adhesive interactions might be differentially regulated between pre- and postsynaptic cells (Choe, 2006).
Previous studies of Liprin-α have demonstrated that it functions as a key regulator of active zone structure and synaptic function. Indeed, Liprin-α mutations cause significant defects in the size, structure, and physiology of synaptic boutons and defects in active zone size and the localization of synaptic vesicle components. Intriguingly, in these studies, axonal innervation of the postsynaptic target was completely normal. The current study has demonstrated that this observation is not true in the developing visual system: Photoreceptors lacking Liprin-α function frequently fail to reach their appropriate postsynaptic targets. Because ultrastructural analysis of the development of this system reveals that synapses do not form until well after photoreceptor axons have reached their terminal target, these studies have defined a previously undescribed function for Liprin-α in target selection (Choe, 2006).
What does Liprin-α do in this context? R1–R6 cells mutant for Liprin-α, LAR, or N-cadherin display identical axonal phenotypes, both in adult animals and during development, argue that these genes act in the same process during target selection. These genes also are required for the layer-specific targeting of R7 axons. In this context, the results are consistent with all three genes acting together during the second step of R7 layer-specific targeting; during the first step of the R7-targeting process, N-cadherin acts independently of LAR (and presumably of Liprin-α). Extensive evidence in other systems suggests biochemical and regulatory interactions between LAR and N-cadherin and between Liprin-α and LAR. Recent studies have proposed that LAR, N-cadherin, and Liprin-α are cotransported to the postsynaptic densities of excitatory synapses in adult brains and that LAR phosphatase activity regulates membrane insertion of this complex in dendrites. In addition, LAR associates directly with β-catenin and can influence its phosphorylation in vitro. Moreover, protein tyrosine phosphatase activity can modulate cadherin-dependent neurite outgrowth in culture. Taken together, it is speculated that Liprin-α and LAR act as regulators of N-cadherin-mediated adhesion in R1–R6 cell axons (Choe, 2006).
A critical, very early step in R1–R6 target selection is a homophillic, N-cadherin-mediated interaction between R cells and their presumptive targets that occurs before the ultimate choice of synaptic partner. One possibility is that Liprin-α acts before N-cadherin during the target selection process to control the trafficking of molecules necessary for R cell axons to stably contact their targets. Such a view would be conceptually consistent with the role for Liprin-α as a regulator of axonal trafficking. Indeed, N-cadherin itself or one of its effectors would be likely candidates. However, inconsistent with this notion, no gross changes in the levels or localization of N-cadherin, β-catenin, or LAR were detected in Liprin-α mutant R cell growth cones. The alternative model is that Liprin-α acts after N-cadherin, recruiting additional components to the presynaptic terminal that are involved in initiating active zone assembly and maintaining contact between pre- and postsynaptic cells. Here, the formation of N-cadherin-mediated adhesive interactions between R cell axons and their targets would alter the activity of Liprin-α at the future synapse, affecting the trafficking of synaptic vesicle components in the region. Such a notion also is consistent with the observed biochemical interactions in mammalian cells between Liprin-α and other presynaptic components, as well as genetic studies demonstrating that Liprin-α is required for active zone assembly and recruitment of synaptic vesicle components. Broadly speaking, a role for Liprin-α downstream of adhesion molecules involved in target selection raises the possibility that, in many contexts, Liprin-α may directly link the process of choosing a synaptic partner to synapse assembly (Choe, 2006).
Cadherin function has been studied extensively in the context of symmetric interactions between epithelial cells, and models derived from these studies have been applied to interactions between neurons. In this context, this work raises the possibility that cadherin function might be asymmetrically regulated between axons and dendrites. In particular, these experiments demonstrate that the mutant phenotypes associated with the loss of Liprin-α, LAR, or N-cadherin from R1–R6 cell axons are indistinguishable. However, although N-cadherin also is required postsynaptically, Liprin-α and LAR are not, demonstrating that the relative contributions of each component differ in R1–R6 cells and their targets. These results suggest that the molecular mechanisms that stabilize connections between R cell axons and their targets differ pre- and postsynaptically. Given that N-cadherin is a critical component on both sides of this interaction, and that LAR, in other contexts, has been shown to influence N-cadherin adhesivity, it was speculated that these differences may be reflected in how cadherin-mediated adhesion complexes are used or regulated in axons and dendrites (Choe, 2006).
An eyFLP-based mosaic screen in adult head sections was designed to identify genes required in photoreceptors for their normal axonal targeting. In addition to a new allele of LAR, a single allele of a second gene with a very similar phenotype was isolated, that was named out of step (oos). P element-induced male recombination was used to map the oos mutation to a small region that included the Liprin-α gene. oos mutants had a stop codon at amino acid 307 of Liprin-α, within the N-terminal coiled-coil domain, making oos likely to be a null allele of Liprin-α. The identity of oos was confirmed by demonstrating that pan-neuronal expression of Liprin-α cDNA could rescue the oos targeting defect. This allele is referred to as as Liprin-αoos (Hofmeyer, 2006).
In wild-type optic lobes, the R7 and R8 photoreceptors terminate in two distinct layers, with R7 projecting deeper into the medulla than R8. In Liprin-αoos mutants, as in LAR mutants, the R7 layer is largely absent; however, the Liprin-αoos phenotype is weaker than LAR, with 37% of R7 axons projecting beyond the R8 layer compared with 15% for LAR. Despite the lack of terminals in the R7 layer, the R7 cell body was present at the correct location in tangential sections of eyes with Liprin-αoos clones. Liprin-αoos mutant R7 cells also expressed the appropriate rhodopsin genes; in mutant clones, axons expressing a lacZ reporter specific for the Rh3 and Rh4 genes (PanR7-lacZ) projected to the medulla but terminated prematurely in the R8 layer. Using the Rh1-lacZ reporter, it was found that R1–R6 terminated in the appropriate target neuropil, the lamina, in the absence of Liprin-α (Hofmeyer, 2006).
Mutations in both Ncad and LAR show a similar R7 targeting defect in adult optic lobes. However, the R7 targeting defect in Ncad mutants is already apparent at 17 h after puparium formation, whereas LAR mutant R7 cells project normally at this stage but retract to the R8 layer later in pupal development. Liprin-α mutants had a normal R7 projection pattern at 24 h, a stage at which N-cad mutants show significant abnormalities but LAR mutants do not (Hofmeyer, 2006).
Liprin-α acts autonomously in the R7 cell to direct its axon to the appropriate target layer. Expression of Liprin-α in R7 (and R3 and R4, which terminate in the lamina) with the sevenless (sev)-GAL4 driver rescued the R7 targeting defect in Liprin-αoos mutants as effectively as pan-neuronal expression with elav-GAL4. Conversely, expression only in R8 cells by using the 109.68-GAL4 driver failed to rescue R7 targeting in Liprin-αoos mutants (Hofmeyer, 2006).
Both human and Drosophila Liprin-α have been shown to bind to the LAR intracellular domain (Kaufmann, 2002, Serra-Pages, 1995). Several lines of evidence argue that this interaction is important for R7 targeting: (1) both endogenous LAR and a tagged form of Liprin-α expressed in photoreceptors with GMR-GAL4 were transported to the growth cones of photoreceptors R1–R6; (2) the presence of LAR and Liprin-α in the same protein complex was confirmed by coimmunoprecipitation of epitope-tagged proteins from Drosophila embryos and S2R+ cells. Consistent with previous findings for human LAR (Serra-Pages, 1995), the distal D2 phosphatase domain of LAR was sufficient to coimmunoprecipitate Liprin-α. (3) The D2 domain is essential for LAR function in R7 targeting, because its absence in either the LARbypass truncation allele or a deleted LAR rescue construct resulted in a strong R7 projection defect. LARbypass generates a partially functional gene product, because its motor axon guidance phenotype is less penetrant than LAR null alleles and it supports normal egg elongation (Hofmeyer, 2006).
Liprin-α has 19 tyrosine residues, three of which are found at conserved positions in the LHD of mammalian and C. elegans Liprin-α homologues. In S2R+ cells, tagged Liprin-α immunoprecipitated by using two different tags was recognized by anti-phosphotyrosine antibody. Liprin-α is thus a potential substrate for LAR (Hofmeyer, 2006).
Previous studies have concluded that Liprin-α binds to LAR to control its subcellular localization. In C. elegans syd-2 mutants, the LAR homologue PTP-3 fails to cluster at synapses (Ackley, 2005), and in cultured mammalian cells, Liprin-α promotes the localization of LAR to focal adhesions (Serra-Pages, 1995, Serra-Pages, 1998). Therefore the intracellular distribution of LAR was analyzed in Liprin-αoos mutant photoreceptors. Despite its reported role in axonal transport of synaptic vesicle components (Miller, 2005), Liprin-α was not required for the transport of endogenous LAR protein to the growth cones of larval R1–R6 photoreceptors. The strong expression of LAR on medulla neurons prevented examination of endogenous LAR protein within the R7 growth cone. Instead, the distribution of epitope-tagged LAR expressed in photoreceptors was monitored. No difference was detected between WT and Liprin-αoos mutants in hemagglutinin (HA)-LAR localization within the R7 or R8 termini in adult head sections. Although the morphology of R7 growth cones that projected beyond the R8 layer was abnormal in Liprin-αoos mutants, HA-LAR was distributed into all regions of these growth cones that contained cytoplasmic β-gal (Hofmeyer, 2006).
To obtain higher resolution, the intracellular distribution of LAR was examined in adherent Drosophila S2R+ cells. In these cells, epitope-tagged Liprin-α colocalizes with the focal adhesion marker Talin, consistent with the localization of human LAR and Liprin-α1 to focal adhesions in MCF7 cells (Serra-Pages, 1995). It was found that even in the absence of cotransfected Liprin-α, HA-tagged LAR also localizes to focal adhesions marked by Talin. Endogenous expression of either Liprin-α or the related Liprin family member CG11206 was not detected in S2R+ cells by RT-PCR. In case Liprin-α was expressed below the level of detection of this assay, RNA interference was used to knock down any potential endogenous Liprin-α mRNA. Expression of a double-stranded RNA hairpin construct complementary to Liprin-α together with an HA-tagged form of Liprin-α strongly reduced HA immunoreactivity, demonstrating its effectiveness. However, LAR still colocalized with Talin in the presence of this RNA interference construct. Thus, LAR localization to focal adhesions in S2R+ cells requires neither endogenous nor ectopically expressed Liprin-α (Hofmeyer, 2006).
The strikingly similar effects of LAR and Liprin-α mutations on R7 targeting, as well as on neuromuscular synapse morphogenesis (Kaufmann, 2002), prompted an investigation of whether Liprin-α is required for other known functions of LAR. LAR has been shown to organize a network of actin filaments in the ovarian follicle cells that promotes egg elongation; females lacking LAR thus lay short, rounded eggs. In contrast, it was found that females lacking Liprin-α lay eggs with the WT shape. Egg elongation also does not require the D2 domain of LAR to which Liprin-α binds, because females carrying the LARbypass allele lay normally elongated eggs (Hofmeyer, 2006).
Zygotic LAR mutant embryos have a characteristic 'bypass' phenotype in the embryonic motor axon projection, resulting from a failure of the ISNb branch of the projection to defasciculate from the ISN branch. Embryos lacking zygotic Liprin-α do not show this bypass phenotype (Kaufmann, 2002). To test whether their normal development was due to perdurance of the maternal contribution of Liprin-α, embryos were generated lacking both maternal and zygotic Liprin-α; however, these embryos also did not display the bypass phenotype. Bypassing was not observed in 267 hemisegments examined in WT embryos or in 227 hemisegments examined in maternal/zygotic Liprin-αoos mutants. Liprin-α thus is not required for the function of LAR in this aspect of motor axon guidance (Hofmeyer, 2006).
If Liprin-α acted by controlling LAR localization, it should have no effect in the absence of LAR. However, it was found that overexpression of Liprin-α in photoreceptors in a LAR null mutant background could partially restore R7 targeting. The converse was not true, because overexpression of LAR had no effect on R7 targeting in a Liprin-α mutant background. In addition, clones lacking both Liprin-α and LAR had fewer correctly targeted R7 axons than clones homozygous for LAR. These data show that both endogenous and overexpressed Liprin-α can promote some normal R7 targeting even in the complete absence of LAR, suggesting that Liprin-α may act both downstream of and in parallel to LAR (Hofmeyer, 2006).
These functions of Liprin-α might require interactions with other proteins involved in R7 targeting. The majority of protein-binding sites in human Liprin-α have been mapped to the N-terminal coiled-coil domain, which also mediates homodimerization. This domain likewise is required for homodimer formation in Drosophila, because a truncated form of Liprin-α lacking the N terminus failed to coimmunoprecipitate with full-length Liprin-α protein in S2 cells. Although the remaining C-terminal domain is sufficient to mediate binding to LAR, expression of this truncation in vivo did not rescue the Liprin-α mutant phenotype. These data suggest that R7 targeting requires Liprin-α dimerization or the interaction of other factors with the N terminus of Liprin-α (Hofmeyer, 2006).
Reference names in red indicate recommended papers.
Ackley, B. D., et al. (2005). The two isoforms of the Caenorhabditis elegans leukocyte-common antigen related receptor tyrosine phosphatase PTP-3 function independently in axon guidance and synapse formation. J. Neurosci. 25(33): 7517-28. 16107639
Choe, K. M., Prakash, S., Bright, A. and Clandinin, T. R. (2006). Liprin-α is required for photoreceptor target selection in Drosophila. Proc. Natl. Acad. Sci. 103(31): 11601-6. 16864799
Dai, Y., et al. (2006). SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS. Nat. Neurosci. 9(12): 1479-1487. 17115037
Dunah, A. W., et al. (2005). LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses. Nat. Neurosci. 8(4): 458-67. 15750591
Fouquet, W., et al. (2009). Maturation of active zone assembly by Drosophila Bruchpilot. J. Cell Biol. 186(1): 129-45. PubMed Citation: 19596851
Hofmeyer, K., Maurel-Zaffran, C., Sink, H. and Treisman, J. E. (2006). Liprin-α has LAR-independent functions in R7 photoreceptor axon targeting. Proc. Natl. Acad. Sci. 103(31): 11595-11600. 16864797
Kaufmann, N., et al. (2002). Drosophila Liprin-α and the receptor phosphatase Dlar control synapse morphogenesis. Neuron 34: 27-38. 11931739
Ko, J., et al. (2003a). Interaction between Liprin-α and GIT1 is required for AMPA receptor targeting. J. Neurosci. 23: 1667-1677. 12629171
Ko, J., et al. (2003b). Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins. J. Biol. Chem. 278(43): 42377-85. 12923177
Kriajevska, M., et al. (2002). Liprin beta 1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, is a new target for the metastasis-associated protein S100A4 (Mts1). J. Biol. Chem. 277(7): 5229-35. 11836260
Miller, K. E., DeProto, J., Kaufmann, N., Patel, B. N., Duckworth, A. and Van Vactor, D. (2005). Direct observation demonstrates that Liprin-α is required for trafficking of synaptic vesicles. Curr. Biol. 15(7): 684-9. 15823543
Olsen, O., et al. (2005). Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex. J. Cell Biol. 170(7): 1127-34. 16186258
Patel, M. R., et al. (2006). Hierarchical assembly of presynaptic components in defined C. elegans synapses. Nat. Neurosci. 9(12): 1488-98. 17115039
Samuels, B. A., et al. (2007). Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK. Neuron 56(5): 823-37. PubMed citation: 18054859
Serra-Pages, C., et al. (1995). The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J. 14(12): 2827-38. Medline abstract: 7796809
Serra-Pages, C., et al. (1998). Liprins, a family of LAR transmembrane protein-tyrosine phosphatase-interacting proteins. J. Biol. Chem. 273(25): 15611-20. PubMed Citation: 9624153
Serra-Pages, C., Streuli, M., Medley Q. G. (2005). Liprin phosphorylation regulates binding to LAR: evidence for liprin autophosphorylation. Biochemistry 44(48): 15715-24. 16313174
Shin, H., et al. (2003). Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha. J. Biol. Chem. 278(13): 11393-401. 12522103
van Roessel, P., Elliott, D. A., Robinson, I. M., Prokop, A. and Brand, A. H. (2004). Independent regulation of synaptic size and activity by the anaphase-promoting complex. Cell 119(5): 707-18. 15550251
Weng, Y. L., Liu, N., Diantonio, A. and Broihier, H. T. (2011)
Wyszynski, M., et al. (2002). Interaction between GRIP and liprin-/SYD2 is required for AMPA receptor targeting. Neuron 34: 39-52. 11931740
Yeh, E., Kawano, T., Weimer, R. M., Bessereau, J. L. and Zhen, M. (2005). Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J. Neurosci. 25(15): 3833-41. 15829635
Zhen, M., and Jin. Y. (1999). The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature. 401: 371-375. 10517634
date revised: 15 October 2011
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