InteractiveFly: GeneBrief

Liprin-α: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Liprin-α

Synonyms -

Cytological map position- 27A1-27A1

Function - scaffolding protein

Keywords - synapse, axon guidance, oogenesis

Symbol - Liprin-α

FlyBase ID: FBgn0046704

Genetic map position - 2L

Classification - N-terminal coiled-coil domain, C-terminal LAR binding region

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Sugie, A., Hakeda-Suzuki, S., Suzuki, E., Silies, M., Shimozono, M., Mohl, C., Suzuki, T. and Tavosanis, G. (2015). Molecular remodeling of the presynaptic active zone of Drosophila photoreceptors via activity-dependent feedback. Neuron [Epub ahead of print]. PubMed ID: 25892303
Summary:
Neural activity contributes to the regulation of the properties of synapses in sensory systems, allowing for adjustment to a changing environment. Little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. This study found that after prolonged exposure to natural ambient light the presynaptic active zone in Drosophila photoreceptors undergoes reversible remodeling, including loss of Bruchpilot, DLiprin-alpha, and DRBP, but not of DSyd-1 or Cacophony. The level of depolarization of the postsynaptic neurons is critical for the light-induced changes in active zone composition in the photoreceptors, indicating the existence of a feedback signal. In search of this signal, this study has identified a crucial role of microtubule meshwork organization downstream of the divergent canonical Wnt pathway, potentially via Kinesin-3 Imac. These data reveal that active zone composition can be regulated in vivo and identify the underlying molecular machinery.
Bohme, M. A., Beis, C., Reddy-Alla, S., Reynolds, E., Mampell, M. M., Grasskamp, A. T., Lutzkendorf, J., Bergeron, D. D., Driller, J. H., Babikir, H., Gottfert, F., Robinson, I. M., O'Kane, C. J., Hell, S. W., Wahl, M. C., Stelzl, U., Loll, B., Walter, A. M. and Sigrist, S. J. (2016). Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat Neurosci [Epub ahead of print]. PubMed ID: 27526206
Summary:
Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-alpha, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission.
BIOLOGICAL OVERVIEW

In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. This study has identified a null mutation in the Liprin-α (for LAR-interacting protein) gene based on a similar R7 projection defect. Liprin-α physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-α nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-α proteins have been reported to control the localization of LAR, LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-α. In addition, Liprin-α overexpression or loss of function can affect R7 targeting in the complete absence of LAR. Despite its reported role in axonal transport of synaptic vesicle components (Miller, 2005), Liprin-α is not required for the transport of endogenous LAR protein to the growth cones of larval R1–R6 photoreceptors. It is concluded that Liprin-α does not simply act by regulating LAR localization but also has LAR-independent functions (Hofmeyer, 2006). A second publication (Choe, 2006) reports similar results.

The color-sensitive photoreceptors of the Drosophila visual system, R7 and R8, provide a simple system in which to study layer-specific axon targeting. Whereas the outer photoreceptors R1–R6 project their axons to the lamina, R7 and R8 project to the medulla, where R8 terminates in the more superficial M3 layer and R7 in the deeper M6 layer. This targeting occurs in two stages, with both R7 and R8 growth cones pausing in separate temporary layers before proceeding to their final positions. Several genes are known to contribute to the establishment of the R7 and R8 projection pattern. The transcription factor Runt is expressed in R7 and R8, and its misexpression is sufficient to target R2 and R5 to the medulla, suggesting that it controls the choice of optic neuropil. Endogenous expression of the homophilic cell adhesion molecule Capricious (Caps) in R8 or its ectopic expression in R7 directs these photoreceptors to terminate in the Caps-positive M3 layer. The transmembrane cadherin Flamingo (Fmi) is required for R8 targeting, whereas loss of either N-cadherin (Ncad) or one of the receptor protein tyrosine phosphatases (RPTPs), PTP69D or LAR, causes R7 to terminate inappropriately in the R8 layer (Hofmeyer, 2006).

Other functions of LAR include axonal patterning of photoreceptors R1–R6 and embryonic motor neurons, synapse morphogenesis at the larval neuromuscular junction (NMJ), and polarization of actin filaments in the follicle cells surrounding the oocyte, which promotes egg elongation along the anterior-posterior axis. It is unclear how LAR and other RPTPs signal within the cell to induce the cytoskeletal rearrangements that mediate these functions. Trio, a guanine nucleotide exchange factor for Rac, and Enabled (Ena), which regulates actin polymerization, show genetic interactions with LAR in both R7 targeting and motor axon guidance. LAR can dephosphorylate both Ena and its antagonist, the cytoplasmic kinase Abelson (Abl). Yeast two-hybrid screens for proteins that bind to the LAR intracellular domain identified both the human and Drosophila homologues of Liprin-α, a protein with an N-terminal coiled-coil domain and a C-terminal LAR-binding liprin homology domain (LHD) consisting of three sterile alpha motif domains (Kaufmann, 2002; Serra-Pages, 1995). Drosophila Liprin-α mutations have the same effects as LAR mutations on NMJ synapse morphology (Kaufmann, 2002), suggesting that the two proteins act together (Hofmeyer, 2006).

Several studies suggest a role for Liprin-α in protein localization. Synaptic vesicle proteins such as synaptotagmin and synaptobrevin are mislocalized in neurons mutant for either Drosophila Liprin-α or the Caenorhabditis elegans Liprin-α homologue syd-2 (Miller, 2005, Zhen, 1999). In Drosophila, this phenotype reflects a requirement for Liprin-α in axonal transport of synaptic vesicles through its binding to the Kinesin-1 (Khc) motor protein (Miller, 2005). In cultured hippocampal neurons, Liprin-α promotes glutamate receptor targeting to synapses (Ko, 2003a, Wyszynski, 2002). The observations that human Liprin-α2 promotes clustering of LAR within the plasma membrane in COS cells, and that SYD-2 and the C. elegans LAR homologue PTP-3 regulate each other’s localization along the nerve cord, suggest that localization also may be the mechanism by which Liprin-α influences LAR function (Hofmeyer, 2006).

This study identifies a likely null mutation in Drosophila Liprin-α that has the same effect on R7 photoreceptor targeting as LAR, yet does not affect two other LAR-dependent processes, egg elongation and motor axon guidance. Liprin-α physically interacts with LAR in embryos and cultured cells; however, the results do not support a primary role for Liprin-α in LAR localization. LAR localization to focal adhesions in cultured Drosophila cells and to photoreceptor growth cones in vivo is independent of Liprin-α. In addition, Liprin-α overexpression can partially restore R7 targeting, and removal of Liprin-α can further reduce R7 targeting in the complete absence of LAR, indicating that some functions of Liprin-α are independent of LAR (Hofmeyer, 2006).

Thus Liprin-α is specifically required in the R7 photoreceptor for its axonal targeting to the correct layer in the medulla. R7 terminates inappropriately in the R8 target layer in the absence of either Liprin-α, LAR, or the D2 domain of LAR to which Liprin-α binds. Liprin-α and LAR both localize to photoreceptor growth cones and are found in the same protein complex. These results imply that a protein–protein interaction between LAR and Liprin-α is important for R7 targeting. The data suggest that this interaction primarily contributes to signaling downstream of LAR, rather than mediating localization of the LAR protein. However, Liprin-α also appears to act in a parallel, LAR-independent pathway (Hofmeyer, 2006).

The C. elegans Liprin-α homologue, syd-2, was identified through its effects on the localization of presynaptic proteins (Zhen, 1999). Previous studies also have shown that in mammalian cells, LAR and Liprin-α colocalize at focal adhesion contacts (Serra-Pages, 1995), but in the absence of cotransfected Liprin-α, LAR is uniformly distributed throughout the plasma membrane (Serra-Pages, 1998). In contrast to these results, no effect of Liprin-α on LAR localization was detected. Despite the reported role for Liprin-α in axonal transport in motor neurons (Miller, 2005), endogenous LAR is correctly transported to the R1–R6 growth cones, and epitope-tagged LAR does not alter its distribution in R7 terminals in Liprin-α mutants. In S2R+ cells, LAR localizes to focal adhesions in the absence of cotransfected or detectable endogenous Liprin-α. It is unlikely that one of the other two Liprin family members encoded in the Drosophila genome can substitute for Liprin-α in LAR localization, because CG10743 is most homologous to human Liprin-β, which does not bind directly to LAR, and CG11206 is not expressed in S2R+ cells (Hofmeyer, 2006).

Because Liprin-α is not required for LAR localization but binds to LAR and has the same effect as LAR on R7 targeting, it may contribute to LAR signal transduction. Although the Liprin-α protein contains no predicted catalytic domains, human Liprin-α1 has been shown to undergo autophosphorylation (Serra-Pages, 2005), suggesting that it might act as a kinase. Alternatively, Liprin-α might promote the association of LAR with its substrates; mammalian Liprin-α family members have been shown to bind to a variety of synaptic molecules (Ko, 2003a; Wyszynski, 2002). Although the LAR substrate Ena binds directly to the intracellular domain of LAR, the Drosophila Trio homologue lacks the domains that mediate LAR binding by human Trio and might require an adaptor protein such as Liprin-α. Liprin-α also might promote interaction of LAR with a cadherin/catenin complex, because β-catenin is a substrate for human LAR in COS cells. Finally, Liprin-α might directly regulate the phosphatase activity of LAR. The D2 domain of LAR and other RPTPs can inhibit their phosphatase activity, in some cases by promoting dimerization. Although the LAR intracellular domain crystallizes as a monomer, homotypic interactions between the LAR transmembrane and intracellular domains have been observed in biochemical assays. Binding of Liprin-α to LAR-D2 might reduce its ability to dimerize (Hofmeyer, 2006).

LAR does not require Liprin-α for all of its functions. The R7 mistargeting phenotype is stronger in LAR mutants than in Liprin-αoos mutants, although the Liprinoos allele is likely to be a null. More dramatically, egg elongation and ISNb motor axon guidance are unaffected in Liprin-α mutants but strongly affected in LAR mutants. Conversely, removal or overexpression of Liprin-α can alter the extent of correct R7 targeting even in the complete absence of LAR. These observations indicate that Liprin-α does not function exclusively as an adaptor for LAR or a regulator of LAR activity but can also act in a parallel pathway, perhaps by localizing other proteins necessary for R7 targeting. Upstream input for this parallel function might come from Ncad or the RPTP PTP69D, because mutations in either show a LAR-like R7 targeting defect in the adult. However, Liprin-α could mediate only a subset of the functions of these molecules, because Ncad is required at an earlier stage and Ptp69D mutants cause an R1–R6 mistargeting phenotype. LAR and PTP69D might have substrates in common that control R7 targeting, because a chimeric protein with the extracellular domain of LAR and the intracellular domain of PTP69D can rescue R7 targeting in LAR mutants. Taken together, these results show that the simple model that Liprin-α acts by localizing LAR to the appropriate region of the plasma membrane (Serra-Pages, 1998) is unlikely to be correct. Understanding the mechanism of Liprin-α function will lead to new insights into RPTP signaling and layer-specific axonal targeting (Hofmeyer, 2006).


REGULATION

Protein Interactions

Drosophila Liprin-α and the receptor phosphatase Dlar control synapse morphogenesis

The synaptic function of the receptor protein tyrosine phosphatase (RPTP), Dlar, and an associated intracellular protein, Liprin-α, has been examined at the Drosophila larval neuromuscular junction. Liprin-α and Dlar are required for normal synaptic morphology. Synapse complexity is proportional to the amount of Dlar gene product, suggesting that Dlar activity determines synapse size. Ultrastructural analysis reveals that Dliprin-α and Dlar are required to define the size and shape of the presynaptic active zone. Accordingly, there is a concomitant decrease in synaptic transmission in both mutants. Finally, epistasis analysis indicates that Dliprin-α is required for Dlar's action at the synapse. These data suggest a model where Dliprin-α and Dlar cooperate to regulate the formation and/or maintenance of a network of presynaptic proteins (Kaufmann, 2002; full text of article).

The yeast two hybrid system was used to searched for Drosophila proteins that would bind directly to Dlar in the yeast interaction trap assay. Using the entire cytoplasmic domain of Dlar as bait, approximately ten million Drosophila cDNA clones were examined. Sequence of the first strong interacting clone revealed it to be the Drosophila homolog of Liprin-α. Mammalian Liprin-α belongs to a family of related mammalian proteins; however, scans of the Drosophila genome identify only one Liprin-α gene (Kaufmann, 2002).

The structural motifs in Liprin-α family members consist of an N-terminal coiled-coil domain, shown to multimerize the protein and a C-terminal LAR binding region. While Drosophila Liprin-α is 47% identical to mammalian Liprin-α overall, much higher levels of conservation are seen in individual domains. For example, a region of 70 amino acids near the N terminus of Drosophila Liprin-α is over 90% identical to mLiprin-α. In addition, the LAR binding domain of Drosophila Liprin-α recovered in the protein interaction screen is 75% identical to mLiprin-α and 68% identical to its C. elegans homolog (Kaufmann, 2002).

As a first step toward a functional analysis, the expression of Liprin-α was examined. Using in situ hybridization, Liprin-α mRNA was observed to be abundantly expressed in the embryo, from cellular blastoderm through hatching. A strong signal at early stages (stages 4 and 5) indicates that there is a substantial maternal contribution of Liprin-α. Liprin-α mRNA accumulates at highest levels in the developing central and peripheral nervous systems (CNS and PNS, respectively) and is not detected outside of the CNS and PNS late in embryonic development. In situs at the third instar stage also reveal persistent expression of liprin-α in the larval CNS and bodywall muscle (Kaufmann, 2002).

To determine Liprin-α protein localization, polyclonal antibodies were raised and affinity purified. In late stage embryos (stages 16 and 17), the highest levels of Liprin-α are restricted to the neuropil of the ventral nerve cord. This precisely matches the pattern of Dlar protein localization at these stages, consistent with direct biochemical association between Liprin and LAR family members. While Liprin-α protein localizes to both longitudinal and commissural neuropil in stage 16, it was noticed that the protein becomes restricted to longitudinal neuropil at stage 17 and first instar. Since developing synapses are found primarily in the longitudinal regions, this raised the question of whether Liprin-α is primarily synaptic in its localization. To address this, peripheral motor pathways were examined at larval stages when the NMJ is well formed and distinct from the axon. At this stage, Liprin-α accumulates specifically at the synapse, showing little or no staining along the axon. Higher resolution confocal images show that Liprin-α is both presynaptic and postsynaptic (compared to a neuronal membrane marker), often forming puncta at the membrane reminiscent of active zones. This pattern is nearly identical to the localization of the synaptic protein Discs large (Dlg) and strongly suggests a function for Liprin-α at the NMJ. Interestingly, Liprin-α localization appears grossly normal at Dlar null mutant NMJs. Although in situ hybridizations confirm that Dlar is expressed throughout the larval CNS, it is not detected in muscle during the same larval stages (Kaufmann, 2002).

The liprin-α gene maps to the cytological location 27B1. The closest P element was about 40 kb away from liprin-α at that time, though there is now an EP insertion (EP2141) approximately 600 base pairs upstream of liprin-α. To generate mutations in liprin-α, P elements from two separate lines, l(2)k00605 and l(2)k13315, were remobilized. Two independent strains were recovered from the l(2)k13315 remobilization with insertions in the liprin-α transcription unit. Both insertions fall in an intron in the 5'UTR of liprin-α within a few nucleotides of each other but are oriented in opposite directions. Staining for Liprin-α clearly shows lower levels of the protein in homozygous embryos from one of the insertion lines (J1), while protein levels seemed approximately normal in the other line (F3). Though dramatically lighter than wild-type controls, traces of residual Liprin-α are still seen in the CNS of the J1 line. This suggests that J1 is hypomorphic; however, the pattern is also consistent with the perdurance of maternally expressed Liprin-α; abundant staining in early embryos (stage 4) supports this theory (Kaufmann, 2002).

To generate stronger mutant alleles, the P element in the F3 line was remobilized. A number of liprin-α deficiency lines were generated by this method. One line, liprin-αF3ex15, was selected for detailed study. DNA sequence analysis shows that liprin-αF3ex15 retains some of the P element and leaves a small piece of the 5'UTR of liprin-α while the rest of the gene is deleted. Genomic mapping of this excision shows that the liprin-α coding region is completely removed in this line; however, in embryos, traces of protein staining are still observed in the embryonic neuropil, similar to that seen in the liprin-αJ1 insertion line; the localization of this antigen appeared normal. It is believed this is due to maternal contribution to the embryo. To generate additional alleles, a P element from the l(2)k00605 mobilization screen, which had inserted 600 base pairs upstream of liprin-α, was excised and excisions were analyzed. This latter screen generated deficiencies extending in both directions, some of which removed liprin-α, including liprin-α77ex17. While generating these alleles, two lines derived from excision of EP2141, liprin-αR60 and liprin-αR117 became available. Mapping of these excisions shows they are both small deficiencies, removing most of the upstream region between liprin-α and an adjacent transcription unit. Liprin-α protein levels are reduced in both lines. Dlar expression and localization is normal in all of these liprin-α mutant backgrounds (Kaufmann, 2002).

To determine the function of Liprin-α, a series of allelic combinations that disrupt liprin-α was created with removal of only one copy of flanking genes. Since a percentage of motor axons (~20%) from the intersegmental nerves b and d (ISNb and ISNd, respectively) require Dlar for guidance to their target muscles in embryonic stages, embryonic motor pathways were examined in liprin-α mutants. No defects in axon guidance were seen. Thus, no evidence is found that zygotic liprin-α is necessary for motor axons to reach their targets, although maternal expression may mask an early function. However, the absence of axon guidance defects in zygotic liprin-α mutants afforded the opportunity to examine the role of liprin-α in synapse development at the larval NMJ (Kaufmann, 2002).

The most thoroughly characterized NMJ in the Drosophila larva is the glutamatergic synapse at muscles 6 and 7. This synapse (6/7NMJ) is innervated by two neurons: RP3, which elaborates a terminal decorated with large boutons (type Ib), and MN6/7b, which generates smaller boutons (type Is). The number of boutons and the branch complexity in the 6/7NMJ at abdominal segments A2 and A3 is highly stereotyped during the wandering third larval instar stage. When liprin-α mutants are compared to the genetically matched parental control strains using immunohistochemistry, a 30%-50% reduction is found in the size of the 6/7NMJ, as assessed by the number of boutons per NMJ. Both type Ib and type Is boutons are reduced in number. Although more variable than bouton number, branch complexity of the terminal arbor is also decreased compared to wild-type. In addition, the same phenotype is seen in three liprin-α loss-of-function combinations derived from two independent parental strains, ruling out genetic background effects. Bouton number is normal in liprin-α/+ heterozygotes, demonstrating that liprin-α is not haploinsufficient (Kaufmann, 2002).

In order to prove that the reduction in synapse size was due solely to loss of liprin-α function, the J1 P element insertion was mobilized and revertants were sought that completely restored the liprin-α gene. Two precise excision lines were isolated, and anatomical analysis shows that they have normal bouton numbers and branching complexity at the 6/7NMJ. liprin-α is thus required for normal NMJ development (Kaufmann, 2002).

In order to better understand the defects in synaptogenesis, NMJ morphology was examined in liprin-α mutants using confocal microscopy. At the tips of wild-type synaptic arbors, a large end bouton is frequently surrounded by smaller boutons, presumably formed by budding of the parent bouton in the process of synapse growth. In contrast, liprin-α mutant terminals frequently lack these nascent bouton structures. Quantification of these structures in wild-type and heterozygous controls, compared to two different strong liprin-α mutant combinations, reveals a substantial reduction in the mutants. This suggests that Dliprin-α is important for the growth of the synapse through formation of new boutons at the ends of terminal branches. The reduction in branch complexity in liprin-α mutants is consistent with this hypothesis. Despite the reduction of nascent boutons, the expression and localization of several synaptic markers are unaffected by the liprin-α mutations; the markers include the scaffolding protein Discs large (Dlg), the cytoskeletal protein Futsch, and the glutamate receptor GluRII (Kaufmann, 2002).

The function of liprin-α at the synapse and the late larval expression of Dlar raised the question of whether Dlar might also be required for NMJ morphogenesis. In this regard, the low penetrance (~20%) of motor axon guidance defects in Dlar null alleles (e.g., Dlar13.2 and Dlar5.5) provided an opportunity to examine NMJ morphology at synapses innervated by the correct number of motor neurons. To identify the ~80% of larval segments where axon guidance was unaffected in Dlar mutants, the analysis was limited to 6/7NMJs that contained both type Ib and type Is boutons from axons that entered the target domain along the correct trajectory (indicating that both RP3 and MN6/7b were present). Antibodies that recognize the synaptic protein Dlg were used in order to distinguish between the two classes of boutons. Examination of bouton number and terminal branch complexity revealed that complete loss of Dlar results in a reduction in synapse size very similar to that observed in liprin-α loss-of-function mutants. Both type Ib and type Is boutons were reduced, suggesting that both neurons at the 6/7NMJ require Dlar activity, as observed in liprin-α mutants (Kaufmann, 2002).

The synapse is very sensitive to the amount of Dlar expressed. Although a null allele of Dlar (Dlar5.5) would be expected to show more penetrant phenotypes than a weaker hypomorph (Dlarbyp), reduction of Dlar gene dose by 50% (Dlar5.5/+) decreases NMJ size relative to wild-type. This is in contrast to all other known functions of Dlar and suggests that synapse growth is directly proportional to the amount of Dlar activity. If this model is correct, it is predicted that raising Dlar activity above wild-type levels should increase synapse size. Indeed, expression of a full-length Dlar transgene under control of two different postmitotic neural-specific GAL4 drivers increases bouton number in comparison to a wild-type strain. These experiments also suggest that Dlar acts presynaptically, as initially suggested by the neural-specific expression of endogenous Dlar (Kaufmann, 2002).

To determine if the effect of increased Dlar expression was manifest specifically during larval stages when the NMJ undergoes massive growth and to ask if the activity could be supplied by the catalytic domains alone, the cytoplasmic region of Dlar was expressed under a neural-specific GAL4 fused to a hormone receptor (GS-GAL4). This expression system provides both tissue and temporal specificity. Induction of Dlar expression during midlarval development with 50 µg/ml mifepristone increases bouton number (Kaufmann, 2002).

Previous studies have suggested a model where Liprin-α acts upstream of Dlar to localize the protein on the cell surface (Serra-Pagès, 1998). The Dlar gain-of-function NMJ phenotype presented an opportunity to perform an epistasis test by simultaneously removing liprin-α function while overexpressing Dlar in postmitotic neurons. While overexpression of Dlar does not rescue the liprin-α phenotype, the absence of Liprin-α protein prevents the increase in NMJ bouton number observed when Dlar is overexpressed in a wild-type background. This puts Liprin-α genetically downstream of Dlar, providing evidence that Liprin-α is required for the normal output of the LAR pathway (Kaufmann, 2002).

Genetic analysis in C. elegans shows that the Liprin-α homolog syd-2 is required to constrain the size of cholinergic active zones (AZs). To ask whether this Liprin-α function is conserved in Drosophila and whether it generalizes to a glutamatergic synapse, an analysis of liprin-α mutant NMJ ultrastructure was undertaken. liprin-α active zones (AZs) fall across a range of sizes from smaller than normal to far greater in size. Mutant AZs were always abnormal in either total size (area) or shape; the mean maximum dimension of mutant AZs (1319 nm) is nearly double that of wild-type (684 nm). Moreover, the mean total area of mutant AZs is 2.4-fold higher than wild-type (Kaufmann, 2002).

Since Liprin-α and Dlar associate directly and display nearly identical gross NMJ phenotypes, it was asked if Dlar is also required to define the dimensions of the presynaptic active zone. Like the liprin-α mutants, overall ultrastructure is normal in the Dlar mutants on both presynaptic and postsynaptic sides of the NMJ. However, 61% of individual AZs are larger than the largest AZ found in wild-type NMJs (Kaufmann, 2002).

To elucidate a potential synaptic function of Liprin-α and Dlar, the synaptic physiology of mutant larval NMJs were examined by whole-cell recordings of evoked excitatory junctional potentials (EJPs) and spontaneous excitatory junctional potentials (mEJPs). EJPs were reduced 36% in liprin-α and 34% in Dlar mutants, compared to precise excision controls; this was highly significant. The resting potential of the mutant muscle was normal (Kaufmann, 2002).

The decrease of synaptic transmission in liprin-α and Dlar mutants was not caused by a postsynaptic defect since quantal events (mEJPs) were normal. The mean amplitude and the frequency of unitary mEJPs in liprin-α and Dlar larvae also showed no significant differences from control. This indicates that the postsynaptic sensitivity to neurotransmitter and the neurotransmitter content of synaptic vesicles are not altered in mutants. However, the normal postsynaptic neurotransmitter reception pointed to a defect intrinsic to the evoked release process (Kaufmann, 2002).

To better quantify mutant synaptic transmission, the number of quanta released per stimulus was determined. Quantal content is defined by the ratio of EJP/mEJP amplitudes after correcting for nonlinear summation using a reversal potential of 0 mV. The quantal content of evoked release for both liprin-α and Dlar was reduced by over 50%. This highly significant reduction in presynaptic vesicle release is consistent with a fundamental defect in active zone structure and function (Kaufmann, 2002).

Analysis of the C. elegans Liprin-α gene syd-2 provided the first support for the model that Liprins are integral to the presynaptic protein complex that organizes neurotransmitter release (Zhen, 1999). This function is conserved. Data from C. elegans, Drosophila, and mammals indicate that the protein localization of Liprin-α family members to the synapse is also conserved (Zhen, 1999; Wyszynski, 2002). Interestingly, in all organisms, Liprin-α is found on both sides of the synapse. Analyses in C. elegans and mammalian neurons support both presynaptic and postsynaptic roles. In C. elegans, loss of syd-2 increases the dimensions of the AZ by approximately 2-fold, with a concomitant decrease in the efficiency of synaptic transmission (Zhen and Jin, 1999). Analysis in Drosophila not only confirms an increase in AZ size, but also reveals that liprin-α mutations have a dramatic effect on AZ shape. This latter phenotype is fully penetrant, suggesting that liprin-α is a requisite structural component of the AZ (Kaufmann, 2002).

Exactly what Liprin-α is doing on the postsynaptic side is unknown. Since Dlar expression is not detected in muscle, this also raises the question of what proteins act in partnership with Liprin-α in the subsynaptic reticulum. Studies in mammalian synapses suggest that Liprins interact with several pre- and post-synaptic proteins (Kaufmann, 2002).

But how do Liprins act to support LAR-family receptor function? Several models are possible. Liprin-α may act in the signaling mechanism downstream of the receptor. Although Liprins have not been shown to be phosphorylated PTP substrates, they could serve to recruit other necessary signaling proteins. The fact that mLiprin-α associates with synaptic scaffolding proteins is consistent with this hypothesis (e.g., GRIP; Wyszynski, 2002). Alternatively, Liprin-α may function to deliver LAR-family receptors to their sites of action in the cell, as suggested by experiments in nonneuronal cells. Without a functional delivery system, Dlar overexpression may fail to increase RPTP activity in the appropriate location (Kaufmann, 2002).

Direct observation demonstrates that Liprin-α is required for trafficking of synaptic vesicles

Axonal transport is required for the elaboration and maintenance of synaptic morphology and function; this study demonstrates that Liprin-α is required for trafficking of synaptic vesicles. Liprin-αs are scaffolding proteins important for synapse structure and electrophysiology. A reported interaction with Kinesin-3 (Kif1a) suggested Liprin-α may also be involved in axonal transport. Aberrant accumulations have been discovered, at the light and ultrastructural levels, of synaptic vesicle markers (Synaptotagmin and Synaptobrevin-GFP) and clear-core vesicles along Drosophila Liprin-α mutant axons. Analysis of presynaptic markers reveals reduced levels at Liprin-α synapses. Direct visualization of Synaptobrevin-GFP transport in living animals demonstrates a decrease in anterograde processivity in Liprin-α mutants but also an increase in retrograde transport initiation. Pull-down assays reveal that Liprin-α interacts with Drosophila Kinesin-1 (Khc) but not dynein. Together, these findings suggest that Liprin-α promotes the delivery of synaptic material by a direct increase in kinesin processivity and an indirect suppression of dynein activation. This work is the first to use live observation in Drosophila mutants to demonstrate the role of a scaffolding protein in the regulation of bidirectional transport. It suggests the synaptic strength and morphology defects linked to Liprin-α may in part be due to a failure in the delivery of synaptic-vesicle precursors (Miller, 2005; full text of article).

Three prominent models have been proposed to explain the regulation of bidirectional transport: (1) a substitution model in which only one set of motors is present on the cargo at a given time, (2) a tug-of-war model in which both anterograde and retrograde motors are bound and always active but differ in their number on the cargo, and (3) a coordinate-regulation model in which both sets of motors are bound but one group is inactive. The observations that dynein is associated with anterograde transported cargos and kinesin is associated with retrogradely transported vesicles containing synaptic components argue against the substitution model of transport for SVPs. If the tug-of-war model were correct, then the shift in flux that was observe in Liprin-α mutants would correspond to a change in the number of bound active motors and a skewing of the velocity profile. However, because no such shift is observed, the current results are most consistent with a model in which coordinate regulation mediated through Liprin-α modulates transport. Because Liprin-α interacts with kinesins but not dynein, the data suggest that Liprin-α directly promotes kinesin activity or cargo-association, which then leads to dynein inhibition through some additional component(s) (Miller, 2005).

In light of the observations that disruption of kinesin alters the morphology and electrophysiological properties of synapses, these observations suggest that the synaptic defects seen in mutants of LAR and the Anaphase Promoting Complex may be mediated in part by Liprin-α’s role in axonal transport. As a scaffolding protein with multiple known partners and motors, Liprin-α is in an ideal position for integrating and transducing information to regulate the delivery of cargoes to and from the synapse (Miller, 2005).

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, Drosophila Syd-1 (DSyd-1: RhoGAP100F), homolog of Syd-1 (synapse defective 1), a multidomain RhoGAP-like protein, that is required for C. elegans HSNL synapse assembly (Dai, 2006; Patel, 2006). was identified as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha (Owald, 2010).

Mechanisms which regulate assembly and maturation of presynaptic AZs are not well understood. This study identified the Drosophila Syd-1 homologue (DSyd-1) as a binding partner of BRP. DLiprin-α and DSyd-1 mark presynaptic sites where, subsequently, AZs (and adjunct PSDs) originate and mature, whereas BRP and Ca2+ channels accumulate at later time points than DLiprin-α and DSyd-1. DLiprin-α previously has been shown to be important for proper AZ formation. Thus, consistent with reduced numbers of AZs forming at NMJs of dsyd-1 and dliprin-α mutants and with both proteins being localized to AZs, the accumulation of DLiprin-α and DSyd-1 at nascent AZs may be instrumental for transforming selected sites into AZs, a process referred to as 'AZ nucleation activity.' However, as the morphological size of dsyd-1 NMJs is reduced, as is the AZ number, in principle, other growth processes might also become rate-limiting at dsyd-1 mutant NMJs. In other words, reduced AZ numbers could also be a consequence of a reduction in morphological NMJ growth. Studying the coupling between morphological growth and AZ formation will be important for determining the relevance of morphological size to total AZ number (Owald, 2010).

Work on en passant synapses of the C. elegans HSNL motor neuron implies that, in genetic terms, Syd-1 operates upstream of Syd-2/Liprin-α. This is based on the fact that a Syd-2/Liprin-α; dominant allele can bypass the requirement of syd-1, which indicates that the protein's essential role in AZ assembly at HSNL synapses is mediated via Syd-2/Liprin-α. This study provides evidence that DSyd-1 is required to properly target DLiprin-α to AZs. In the absence of DSyd-1, DLiprin-α distributes unevenly at NMJ terminals, sparing many AZs. Thus, direct evidence is provided that the RhoGAP DSyd-1 operates upstream in AZ assembly in vivo: DSyd-1 seemingly stalls DLiprin-α to developing AZs in order to allow for the AZ nucleation function of DLiprin-α to effectively operate (Owald, 2010).

DLiprin-α seems to be a direct substrate of DSyd-. The data imply that other presynaptic substrate proteins of DSyd-1 might exist at nascent synapses, a finding that is unexpected based on analysis of AZ formation in C. elegans. Therefore, it is deduced from these findings that presynaptic DSyd-1 (but apparently not DLiprin-α) plays an important role in shaping the PSD assembly. Embryos and larvae mutants for dsyd-1, and importantly, dliprin-α; dsyd-1 double mutant embryos (the double mutant is embryonic lethal), showed increased overall amounts of postsynaptic GluRs, whereas dliprin-α single mutant embryos and larvae did not. These increased amounts of GluRs in dsyd-1 mutants vanished after presynaptic reexpression of UAS–dsyd-1cDNA. It is tempting to speculate that the presynaptic DSyd-1 protein helps the AZ localization of an adhesion protein, which via trans-synaptic interaction might steer the incorporation of postsynaptic GluRs. A potential role of the Neurexin–Neuroligin axis should be evaluated in this context (Owald, 2010).

Drosophila NMJs express two functionally distinct GluR complexes, DGluRIIA and IIB, which influence the number of release sites formed. Individual PSDs form distinctly from preexisting ones, and mature over hours, switching from DGluRIIA to IIB incorporation throughout maturation in a manner dependant on presynaptic signaling. DSyd-1 might mediate such a maturation signal, as dsyd-1 mutants show excessive amounts of DGluRIIA incorporation at PSDs. This regulation is likely not (or only partially) due to compensation for reduced presynaptic glutamate release, as dliprin-α mutants (with similarly reduced transmission levels) do not show this dramatic increase in GluR levels (Owald, 2010).

Despite enlarged receptor fields and specifically elevated DGluRIIA levels, average miniature event amplitudes were comparable between dsyd-1 animals and controls, which currently cannot be accounted for. A possible explanation might comprise regulatory processes rendering populations of receptors non-/partially functional. Nonetheless, EJC decay time constants of dsyd-1 mutants resemble those found at dgluRIIB-deficient (and thus GluRIIA dominated) NMJs (Owald, 2010).

Which processes are downstream of the DSyd-1–mediated DLiprin-α activity at nascent AZs? Liprin family proteins steer transport in axons and dendrites (e.g., of AMPA receptors) to support synaptic specializations. Notably, in dsyd-1 mutants, although many AZs lacked proper amounts of DLiprin-α, large ectopic accumulations of DLiprin-α were observed. At the same time, ectopic accumulations of BRP/electron density were observed in the absence of DSyd-1. It is tempting to speculate that these ectopic pools of DLiprin-α provoke the aberrant accumulation of electron densities in dsyd-1 mutants, which is consistent with the transport function of DLiprin-α and the direct interaction of DLiprin-α/Syd-2 and ELKS/BRP. Consistently, large BRP accumulations observed in dsyd-1 embryos were no longer present in dsyd-1; dliprin-α double mutants, which indicates that the presence of DLiprin-α is needed to provoke these overaccumulations of BRP when DSyd-1 is missing (Owald, 2010).

In the absence of DSyd-1, BRP was inappropriately localized, even within the cytoplasm, forming ectopic electron-dense material (which is consistent with its role as building block for the electron-dense T bars). Such 'precipitates' also occurred at and close to non-AZ membranes. Moreover, at dsyd-1 AZs, large malformed T bars formed. Thus, it appears plausible that DSyd-1 keeps BRP 'in solution' to organize its proper consumption at AZs. An alternate and not mutually exclusive explanation may be that axonal BRP precipitates also reflect defects in axonal transport due to the absence of DSyd-1. The presence of several binding interfaces between BRP and DSyd-1 may be considered as a basis for regulating their interplay (Owald, 2010).

BRP accumulation in the center of the AZ is also in the center of the functional and structural AZ assembly process. It appears likely that BRP assembly is regulated on multiple levels. Notably, although BRP accumulation is severely compromised in mutants for the kinesin imac, it is not fully eliminated. Moreover, the serine/arginine protein kinase SRPK79D was recently shown to associate with BRP and to repress premature 'precipitation' of BRP in the axons. Furthermore, mutants for the serine/threonine kinase unc51 have recently been shown to suffer from BRP targeting defects. Phosphorylation of DSyd-1 (e.g., within serine-rich stretches toward the C terminus) might be involved in regulating proper longer-range transport ('blocking precipitation on the way') as well as proper delivery of BRP at nascent AZ sites (Owald, 2010).

Recently, the Rab3 GTPase has been shown to be crucial for effective nucleation of BRP at AZs (Graf, 2009). In an interesting parallel to dsyd-1 defects, rab3 mutant NMJs showed fewer BRP-positive AZs; however, if present, BRP levels were increased. Nonetheless, instead of overgrown T bars, as observed in dsyd-1 mutants, rab3 mutants rather showed multiple T bar AZs (Graf, 2009). It will be interesting to investigate whether these pathways act in parallel or converge, along with their relationships to other synaptogenic signals (Owald, 2010).

The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance

The multiprotein complexes that receive and transmit axon pathfinding cues during development are essential to circuit generation. Identified and characterized the Drosophila sterile α-motif (SAM) domain-containing protein Caskin, which shares homology with vertebrate Caskin, a CASK [calcium/calmodulin-(CaM)-activated serine-threonine kinase]-interacting protein. Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related (Lar) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of Ckn mediates its interaction with Lar. Like Caskin, Liprin-α is a neuronal adaptor protein that interacts with Lar via a SAM domain-mediated interaction. Evidence is presented that Lar does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on Lar. Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in Lar signal transduction is evolutionarily conserved. Last, several ckn mutants were characterized that retain Lar binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, the SH2/SH3 adaptor protein Dock was identified as a second Caskin-binding protein and it was found that Caskin binds Lar and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in Lar-dependent signaling during motor axon targeting, ckn and dock have overlapping roles in axon outgrowth in the CNS. Together, these studies identify caskin as a neuronal adaptor protein required for axon growth and guidance (Weng, 2011).

The Drosophila neuromuscular system is an excellent paradigm to decipher the molecular signals orchestrating the precise matching between individual motorneurons and their muscle partners. A number of guidance cues and receptors coordinately regulate motor axon pathfinding assuring the high fidelity of this process. The axon must integrate these disparate signals as it navigates through its environment. Multidomain adaptor proteins promote such integration since they serve as platforms to facilitate communication between signal transduction cascades (Weng, 2011).

The leukocyte common antigen-related (LAR)-related subfamily of receptor protein tyrosine phosphatases (RPTPs type IIA) are conserved regulators of axon pathfinding and synaptogenesis. This subfamily includes the Drosophila receptors Lar and PTP69D, and the vertebrate receptors LAR, protein tyrosine phosphatase ς (PTPς), and PTPδ. Family members contain a variable number of Ig and fibronectin (FN) III domains extracellularly, and two intracellular phosphatase domains. The membrane-proximal D1 phosphatase domain (D1) confers most if not all of the catalytic activity of the receptor, whereas the membrane-distal D2 domain is catalytically inactive and may contribute to LAR family function via interaction with downstream signaling components. These receptors exhibit neuronal expression patterns, and loss-of-function (LOF) mutants display defects in axon targeting and synapse formation. Heparan sulfate proteoglycans (HSPGs) are binding partners of LAR family members in axon pathfinding and synaptogenesis. In vertebrates, PTPδ is a neuronal receptor for chondroitin sulfate proteoglycan (CSPG) and inhibits axon regeneration after CNS injury. On the intracellular side, Lar activity in some contexts requires phosphatase activity, whereas in other contexts its function is independent of catalytic activity, suggesting a diversity of downstream signaling pathways. Indeed, a number of Lar-interacting proteins have been identified. Lar function in synaptic maturation requires Liprin-α, a sterile α-motif (SAM) domain-containing adaptor protein that interacts with Lar in vertebrates and invertebrates (Weng, 2011).

Drosophila Dock and vertebrate Nck are neuronal SH2/SH3-containing adaptor proteins that link guidance receptors to cytoskeletal remodeling. Given the widespread expression of Dock in the embryonic CNS and its central position linking guidance receptors to the actin cytoskeleton, it is notable that motor axons in dock LOF mutant embryos display only subtle defects, raising the possibility of compensation or redundancy. Dock/Nck interact directly with a number of receptors, including Robo, DSCAM (Down syndrome cell adhesion molecule), and the insulin receptor and bind directly to cytoskeletal effectors such as p21-activated protein kinase (Pak) and WASP (Wiskott-Aldrich syndrome protein), thereby presumably linking receptor activation to cytoskeletal rearrangement. Of particular relevance, vertebrate Caskin has been identified as a potential Nck interactor. Caskin was first identified as a novel protein binding the CaM kinase domain of CASK. Vertebrate Caskins are predicted scaffolding proteins with multiple ankyrin repeats, an SH3 domain, and two SAM domains, suggesting that Caskin is a component of a multiprotein complex. This study presents genetic, cell-biological, and biochemical evidence arguing that Drosophila Caskin is a Lar-binding partner and is required for Lar signal transduction in motor axon guidance (Weng, 2011).

This study has demonstrate that Caskin mediates a novel Lar RPTP signaling cascade during axonogenesis. Analysis of a panel of ckn LOF alleles indicates that ckn is necessary for motor axon pathfinding, since homozygous mutants display classic bypass defects in the ISNb motor nerve. This phenotype is identical with that displayed by Lar mutants, and genetic and biochemical interaction data demonstrate that Ckn is a Lar-interacting protein. These studies position Caskin to be a core member of a Lar-associated signaling complex that mediates its function during axonogenesis (Weng, 2011).

Vertebrate Caskin was identified as a binding partner of the synaptic adaptor protein CASK and competes for binding to the CaM kinase domain of CASK with the PDZ (postsynaptic density-95/Discs large/zona occludens-1) protein Mint1. The CASK-binding site on Caskin maps to an N-terminal region not conserved in Drosophila, suggesting that fly Caskin does not bind CASK. Consistent with this finding, Drosophila Caskin and CASK do not interact in a yeast interaction assay. However, both mouse and fly Caskin homologs bind LAR family members and Nck/Dock, in support of considerable shared functions. Furthermore, overexpression of the Lar-binding domain of mouse Caskin in Drosophila neurons yields a pathfinding phenotype like that of Lar and ckn LOF, suggesting that mouse Caskin competes with fly Ckn for binding to the Lar receptor to function as a dominant negative. These biochemical studies indicate that, whereas Caskins may have species-specific binding partners, Ckn function in Lar signal transduction is conserved. Drosophila Lar also physically interacts with the Abl tyrosine kinase and its substrate the cytoskeletal regulator Ena (Wills, 1999). This study was unable to detect physical interactions between Ckn and Abl or Ena, suggesting they bind Lar independently. This raises the possibility that Caskin and Abl/Ena constitute parallel pathways downstream of the Lar receptor (Weng, 2011).

The allelic series enabled analysis of the in vitro and in vivo activities of four Caskin mutant proteins: Ckn-A, Ckn-C, Ckn-K, and Ckn-Y. Ckn-A and Ckn-K contain alterations in the first SAM domain and block the interaction of Ckn with Lar, pointing to the importance of this domain for Lar/Ckn complex formation. The in vivo analysis of Ckn-A and Ckn-K is in strong agreement with the in vitro data as motor axon phenotypes are not associated with their overexpression, suggesting they do not interfere with Lar signaling in vivo. The behavior of Ckn-A and Ckn-K contrasts that of Ckn-C, which contains a C-terminal deletion. Ckn-C interacts with Lar, and its neuronal overexpression yields dominant-negative-like effects. In fact, the penetrance of ISNb bypass associated with Ckn-C overexpression is comparable with that observed in embryos lacking both maternal and zygotic Lar, suggesting that it effectively interferes with Lar activity. Although Ckn-C binds Lar, cknC homozygous LOF mutants display a 'Lar-like' ISNb phenotype, indicating that Lar signaling is blocked downstream of receptor binding. Ckn-C does not interact with Dock, but this interaction is insufficient to explain the cknC mutant phenotype since ISNb bypass is not associated with dock LOF. The pathfinding phenotype observed in cknC embryos argues that the allele also disrupts the interaction between Caskin and another downstream protein(s) essential for Lar signaling (Weng, 2011).

Dock/Nck are SH2/SH3-containing adaptor proteins that couple phosphotyrosines on activated receptors to downstream signaling molecules via SH2 and SH3 domain interactions, respectively. Dock also engages in a ligand-regulated SH3 domain interaction with the Robo receptor, demonstrating that it is involved in diverse interactions downstream of guidance receptors. This work has demonstrate that Caskin interacts with the second SH3 domain of Dock (SH3-2). This domain has also been shown to interact with the cytoskeletal effector Pak, raising the issue of the relationship between Caskin and Pak. It will be informative to determine whether Dock forms alternative complexes with Caskin and Pak, or whether Dock binds Caskin and Pak simultaneously (Weng, 2011).

The contrast between the ckn and dock single- and double-mutant phenotypes demonstrates that the adaptors have mostly redundant functions. Single-mutant analyses indicate that ckn plays a nonredundant role in Lar signaling, whereas dock has a unique role in synaptogenesis of the RP3 motorneuron. However, the outgrowth defects observed in dock ckn double mutants argue that these adaptors have overlapping roles in a number of signaling events. These data caution against drawing conclusions of cellular function based solely on single mutant analysis, as this obviously uncovers only the nonredundant functions of a protein. The issue of genetic redundancy may be particularly acute in signaling systems involving multi-subunit complexes with many opportunities for parallel functions. It will be important to identify additional binding partners of dock and ckn to determine whether they have a common set of interactors, or whether they impinge on the cytoskeleton via distinct, yet redundant, paths (Weng, 2011).

The Lar receptor is a member of the type IIA subfamily of RPTPs, comprising Lar and PTP69D in flies. The single-mutant phenotypes of Lar and PTP69D indicate they have nonredundant functions in motor axon guidance, NMJ growth, and photoreceptor axon targeting. Several observations hint that the unique functions implied by the divergent phenotypes of Lar and PTP69D stem in part from distinct ligand-binding activities. Lar and PTP69D alkaline phosphatase fusion proteins have be shown to possess distinct embryonic staining patterns suggesting the presence of unique ligands. Furthermore, overexpression of a chimeric receptor composed of the Lar extracellular domain fused to the PTP69D intracellular domain rescues the LOF photoreceptor defect of Lar, whereas a PTP69D extracellular domain fusion to the Lar intracellular domain does not, arguing that Lar and PTP69D have overlapping intracellular partners and (at least partially) nonoverlapping extracellular ones. However, more recent data open the door for functional differences between the intracellular pathways activated by Lar and PTP69D. R7 photoreceptor axon targeting is independent of Lar phosphatase activity, but dependent on PTP69D phosphatase activity, suggesting that the receptors have distinct binding partners. These findings are consistent with the work presented in this study. Both fly and vertebrate Caskins interact with subsets of LAR family receptors, raising the possibility that the intracellular signaling cascade(s) organized by Ckn contributes to the functional differences between Lar and PTP69D (Weng, 2011).

the physical relationship between Lar, Ckn, and Liprin-α was investigated, and no ternary complex was detected. These binding data support mapping studies indicating that Ckn and Liprin-α both interact with the D2 phosphatase domain of Lar via SAM domain-mediated interactions. They further suggest sequential/competitive binding of Ckn and Liprin-α to the Lar receptor and raise the possibility of distinct neuronal functions. It is conceivable that Ckn and Liprin-α both act downstream of Lar to mediate its activity during axon outgrowth/pathfinding and synaptogenesis, respectively. To determine whether Ckn function is specific for Lar signaling during axonogenesis, it will be informative to test whether ckn LOF mutants exhibit defects in the assembly/localization of presynaptic components similar to that observed in Lar mutants. Alternatively, the function of Liprin-α in Lar signaling may be primarily to localize or maintain Lar at the presynaptic terminal, whereas Ckn functions in downstream signal transduction. This hypothesis is supported by evidence for a conserved function for Liprin-α in synaptic protein targeting or anchoring. A role for Liprin-α in trafficking is further bolstered by conserved physical interactions between Liprin-α and Kinesin, suggesting it is an adaptor protein for anterograde transport of synaptic proteins. In this scenario, it is notable that Liprin-α function is not required for pathfinding, arguing either that another protein serves to localize Lar during guidance or that Lar activity in this process does not require its tight localization to the axon terminal. This model is consistent with the broad axonal localization of Lar during embryogenesis (Weng, 2011).

Extracellularly, LAR family members interact with HSPGs and CSPGs. In Drosophila, mutations in the HSPG syndecan (sdc) interact with Lar in motor axon guidance, but homozygous LOF sdc embryos do not display appreciable bypass phenotypes, arguing that other ligands are involved. Once these ligands are identified, it will be critical to determine whether ligand binding influences the association of intracellular adaptors such as Liprin-α and Caskin with Lar. Recently, vertebrate LAR family members have moved into the spotlight in the field of axon regeneration, as PTPsigma has been shown to be a receptor for CSPGs, which are dramatically upregulated at the lesion site and are strongly inhibitory to axon growth. Strikingly, axons in PTPsigma mutant mice have a greatly enhanced ability for long-distance regeneration relative to wild-type mice. These studies suggest that blocking PTPsigma signaling in injured axons might enhance recovery after spinal cord injury. Hence, the truncated forms of fly and vertebrate Caskins that interfere with Lar signaling are particularly interesting. The identification of such dominant-negative reagents allowing the blockade of Lar signal transduction in vivo may have clinical implications in neuronal regeneration (Weng, 2011).

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).

During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal. Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation. Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation. Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly. Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ. However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side. The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants. Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).

This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cell. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).

This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).

Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).

Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).

Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca2+ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).

Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).

What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104, a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants. However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).

Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca2+ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).

The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).

A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2. Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins RhoGAP100F/Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission (Bohme, 2016).

All presynaptic AZs accumulate scaffold proteins from a canonical set of few protein families, which are characterized by extended coiled-coil stretches, intrinsically unstructured regions and a few classical interaction domains, particularly PDZ and SH3 domains. These multidomain proteins collectively form a compact 'cytomatrix' often observable by electron-dense structures covering the AZ membrane, which have been found to physically contact SVs, and thus have been suggested to promote SV docking and priming as well as to recruit Ca2+ channels. Still, how the structural scaffold components (ELKS, RBP, RIM and Liprin-α) tune the functionality of the SV-release machinery has remained largely enigmatic. Liprin-α is crucial for the AZ assembly process and at Drosophila NMJ AZs, Liprin-α-Syd-1 cluster formation initializes the assembly of an 'early' scaffold complex, which subsequently guides the accumulation of a 'late' RBP-BRP scaffold complex. This study provides evidence that these scaffold complexes together operated as 'molecular rulers' that confer a remarkable degree of order, patterning AZ composition and function in space and time: the 'early' Liprin-α-Syd-1 clusters recruited Unc13B, and this scaffold served as a template to accumulate the 'late' BRP-RBP scaffold, which recruited Unc13A. Unc13 isoforms were precisely organized in the tens of nanometers range, which the data suggest to be instrumental to control SV release probability and SV-Ca2+ channel coupling. As a molecular basis of this patterning and recruitment, a multitude of molecular contacts was identified between the Unc13 N termini and the respective scaffold components using systematic Y2H analysis. As one out of several interactions, this study identified a cognate PxxP motif in the N terminus of Unc13A to interact with the second and third SH3 domains of RBP. Point mutants within the PxxP motif interfered with the binding of the RBP-SH3 domains II and III on the Y2H level but did not have a major impact on Unc13A localization and function when introduced into an Unc13 genomic transgene. Nonetheless, elimination of the scaffold components BRP and RBP on the one hand or Liprin-α on the other hand drastically impaired the accumulation of Unc13A or Unc13B. It is suggested that these results are explained by a multitude of parallel interactions that provide the avidity needed to enrich the respective Unc13 isoforms in their specific 'niches' and may cause a functional redundancy among interaction motifs, as was likely observed in the case of the Unc13A PxxP motif. Future analysis will be needed to investigate these interaction surfaces in greater detail, and address how exactly 'early' and 'late' scaffolds coordinate AZ assembly (Bohme, 2016).

Unc13 proteins have well-established functions in SV docking and priming. Accordingly, it was observed that loss of Unc13A resulted in overall reduced SV docking without affecting T-bar-tethered SVs, which is qualitatively opposite to a function of BRP in SV localization, whose C-terminal amino acids function in T-bar-tethering, but not docking. Variants lacking these residues suffer from increased synaptic depression, suggesting a role in SV replenishment. Therefore, in addition to its role in localizing Unc13A to the AZ reported here, BRP may also cooperate functionally with Unc13A by facilitating SV delivery to docking sites (Bohme, 2016).

Synapses are highly adapted to their specific features, varying widely concerning their release efficacy and short-term plasticity. These features impact information transfer and may provide neurons with the ability to detect input coherence, maintain stability and promote synchronization. Differences in the biochemical milieu of SVs can tune priming efficacy and release probability, which largely affects short-term plasticity. In the current experiments, it was found that loss of Unc13A resulted in dramatically (~90%) reduced synaptic transmission, which exceeded the (~50%) reduction in SV docking, pointing to an additional function in enhancing release efficacy. These changes were paralleled by drastically increased short-term facilitation as well as EGTA hypersensitivity and could be due to decreased Ca2+ sensitivity of the molecular release machinery, for example, mediated by different Synaptotagmin-type Ca2+ sensors, or different numbers of SNARE complexes. However, although a rightward shift was observed of the dependence of normalized release amplitudes on extracellular Ca2+ concentration at Unc13A-deficient synapses, its slope and thus Ca2+ cooperativity was unaltered, arguing against fundamentally different Ca2+-sensing mechanisms. Instead a scenario is favored in which SV Ca2+ sensing is conserved, but local Ca2+ signals at SV positions are attenuated because of their larger distances to Ca2+ channels upon loss of Unc13A. Both Unc13 isoforms were clearly segregated physically with different distances to the Ca2+ channel cluster, and loss of Unc13A selectively reduced the number of docked SVs in the AZ center. These findings are best explained by Unc13A promoting the docking and priming of SVs closer to Ca2+ channels than Unc13B. In fact, mathematical modeling reproduced the data by merely assuming release from two independent pathways with identical Ca2+ sensing and fusion mechanisms that only differed in their physical distance to the Ca2+ source in the AZ center. The distances estimated by the model were in very good agreement with the positions of the two Unc13 isoforms defined by STED microscopy. Thus, the data suggest that differences in the distance of SVs in the tens of nanometer range to the Ca2+ channels mediated by the two Unc13 isoforms likely contributed profoundly to the observed phenotypes. It is proposed that the role of the N terminus is to differentially target the isoforms into specific zones of the AZ, while the conserved C terminus confers identical docking and priming functions at both locations. Notably, recent work in Caenorhabditis elegans also characterized two Unc13 isoforms, with fast release being mediated by UNC-13L, whereas slow release required both UNC-13L and UNC-13S44. The proximity of the UNC-13L isoform to Ca2+ entry sites was mediated by the protein's N-terminal C2A-domain (not present in Drosophila) and was critical for accelerating neurotransmitter release, and for increasing/maintaining the probability of evoked release assayed by the fraction of AP- to sucrose-induced release. In contrast, the slow SV release form dominantly localized outside AZ regions. Thus it would be interesting to investigate the sub-AZ distribution of C. elegans Unc-13 isoforms and test whether the same scaffold complexes as in Drosophila mediate the localization of the different Unc-13 isoforms (Bohme, 2016).

Notable differences in short-term plasticity have been reported for mammalian Unc13 isoforms. The mammalian genome harbors five Munc13 genes. Of those, Munc13-1, -2 and -3 are expressed in the brain, and function in SV release; differential expression of Munc13 isoforms at individual synapses may represent a mechanism to control short-term plasticity. Thus, it might be warranted to analyze whether differences in the sub-active zone distribution of Munc13 isoforms contribute to these aspects of synapse diversity in the rodent brain (Bohme, 2016).

Fast and slow phases of release have recently been attributed to parallel release pathways operating in the calyx of Held of young rodents (56 nm and 135 nm) qualitatively matching the coexistence of two differentially positioned release pathways described in this study. The finding of discretely localized release pathways with distances larger than 60 nm is further in line with the recent suggestion that, at some synapses, SVs need to be positioned outside an 'exclusion zone' from the Ca2+ source (~50 nm distance to the center of the SV for the calyx of Held). At mammalian synapses, developmental changes in the coupling of SVs and Ca2+ channels have been described, which qualitatively matches the sequential arrival of loosely and tightly coupled Unc13B and Unc13A isoforms during synaptogenesis described here. Thus, this work suggests that differential positioning of Unc13 isoforms couples functional and structural maturation of AZs. To what degree modulation of this process contributes to the functional diversification of synapses is an interesting subject of future analysis (Bohme, 2016).


DEVELOPMENTAL BIOLOGY

Embryonic

Independent regulation of synaptic size and activity by the anaphase-promoting complex requires Liprin-α

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).

Maturation of active zone assembly by Drosophila Bruchpilot

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).


EFFECTS OF MUTATION

Liprin-α is required for photoreceptor target selection in Drosophila

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).

Liprin-α has LAR-independent functions in R7 photoreceptor axon targeting

Liprin-α is required for retinal axon targeting: 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 Liprinoos (Hofmeyer, 2006).

Liprin-α is autonomously required for R7 axons to terminate in the correct target layer: 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 Liprinoos mutants, as in LAR mutants, the R7 layer is largely absent; however, the Liprinoos 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 Liprinoos clones. Liprinoos 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 Liprinoos 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 Liprinoos mutants (Hofmeyer, 2006).

A direct interaction between LAR and Liprin-α is required for R7 targeting: 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).

Liprin-α is not required for LAR localization in photoreceptors: 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 Liprinoos 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 Liprinoos mutants, HA-LAR was distributed into all regions of these growth cones that contained cytoplasmic β-gal (Hofmeyer, 2006).

Liprin-α does not influence LAR localization in S2R+ cells: 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).

Liprin-α is not required for all of the functions of LAR: 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 Liprinoos mutants. Liprin-α thus is not required for the function of LAR in this aspect of motor axon guidance (Hofmeyer, 2006).

Liprin-α can function independently of LAR: 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).

Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 (RhoGAP100F) as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. This study shows that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. The earlier defect coincides with a failure to localize synaptic vesicles (SVs), suggesting that it reflects a failure in presynaptic assembly. The relationship between syd-1 and Liprin-α in R7s was analyzed. It was found that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: Liprin-α was shown to promote Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. It was shown that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. It is therefore concluded that the two defects are caused by distinct molecular mechanisms. Trio overexpression was found to rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity (Holbrook, 2012).

GFP-fused SV proteins, such as Syt-GFP, are classic tools for studying presynaptic development but have not been used previously to analyze R7s. This study found that, as expected, Syt-GFP within R7s is enriched at sites known by electron microscopy to contain active zones. Loss of LAR, Liprin-α, or syd-1 causes R7 terminals to fail to contact their normal, M6, target layer. This study demonstrated that this morphological defect correlates temporally with a failure to localize SVs to presynaptic sites and is therefore likely to reflect a defect in R7 presynaptic development rather than simply in target layer selection (Holbrook, 2012).

Liprin-α is not only a scaffold for the assembly and retention of presynaptic components, including SVs, at presynaptic sites but also a positive regulator of Kinesin-3/Unc-104/Imac-dependent axon transport of those components. This study shows that, unlike Liprin-α, Syd-1 is not required for normal Kinesin-3/Unc-104/Imac-mediated transport. However, SVs are similarly mislocalized in Liprin-α and syd-1 mutant R7 axons that contact M6. A simple interpretation is that this mislocalization reflects a requirement for Liprin-α and syd-1 in retaining SVs within R7 terminals; in support of this, it was found that SVs are localized normally to syd-1 mutant R7 axon terminals at 24 h APF, before synaptogenesis. It was hypothesized that the additional disruption of axon transport in Liprin-α mutant R7s is reflected in their greater inability to maintain contact with M6; in support of this, it was found that imac mutant R7 axons also lose contact with M6 (Holbrook, 2012).

Although both Liprin-α and syd-1 are required for the clustering of SVs at en passant synapses in worm, syd-1 is not required for the localization of SVs to NMJ terminals in fly. The molecular mechanisms underlying presynaptic development at NMJ and in R7s have been shown previously to differ in several respects. The current finding further highlights the importance of analyzing synapse development using multiple neuron types (Holbrook, 2012).

Although mitochondria are often enriched at synapses, it remains unclear what proportion of them might be stably associated with presynaptic sites rather than transported there in response to acute energy needs. Within at least some axons, most clusters of stationary mitochondria reside at nonsynaptic sites. In R7s, Mito-GFP was found to be enriched at presynaptic sites. Because arthropod photoreceptor neurons continuously release neurotransmitter in response to light, this enrichment might simply be caused by continuous energy needs. However, this study found that mitochondria remained enriched at R7 terminals even in the absence of light-evoked activity, indicating that either spontaneous release is sufficient for their recruitment or an activity-independent mechanism is responsible. It is speculated that the permanently high energy demands at photoreceptor synapses may have selected for the activity-independent association of mitochondria with R7 synapses and that this localization requires syd-1 and Liprin-α. Mito-GFP is mislocalized in imac mutant R7s, despite previous work indicating that Kinesin-3/Unc-104/Imac is not required for transport of mitochondria. It is therefore thought that mitochondria are normally tethered at R7 presynaptic sites and that loss of imac indirectly causes their mislocalization by disrupting transport of the components required for tethering to occur (Holbrook, 2012).

Previous work identified two different phenotypes associated with loss of the LAR/Liprin/trio pathway: loss of LAR or Liprin-α caused R7 axons to terminate before their M6 target layer, whereas loss of Liprin-β or trio caused R7 axons to project extensions beyond M6. One possibility is that these two defects are simply different manifestations of the same cellular defect: a decrease in the stability of the synaptic contact between R7s and their targets. However, this study has shown that loss of a single gene, syd-1, causes both defects and that the defects occur at distinct developmental time points, suggesting that they occur by distinct mechanisms. In support of this, Liprin-α overexpression can rescue the early but not the late syd-1 defect (Holbrook, 2012).

The earlier defect, failure to contact M6, correlates with the failure to localize SVs, suggesting, as mentioned above, that this represents a failure to assemble synapses. However, the cause of the later morphological defect and the precise nature of the extensions remain unclear. It is noted that the extensions often terminate in small varicosities that can contain Syt-GFP, and Mito-GFP, indicating that they are not simply filopodia but may instead represent sites of ectopic presynaptic assembly. One possibility is that, as at NMJ, loss of syd-1 causes ectopic accumulations of Liprin-α, Brp, Nrx-1, or other presynaptic proteins and that these might then promote ectopic, abnormal presynaptic assembly. A second possibility is that the extensions may instead be an indirect consequence of the role of syd-1 in postsynaptic development: perhaps the extensions are the response of the syd-1 mutant R7 terminal to defects in its postsynaptic target. Loss of Liprin-α causes no such postsynaptic effect, providing an explanation for why Liprin-α mutant R7s do not form extensions. A third possibility is that R7s form distinct types of synapses at different time points. Failure to assemble one type of synapse, which R7s assemble first, causes decreased contact with M6, whereas failure to assemble a second type, which occur later, results in extensions. Consistent with this model, R7s form synapses with more than one neuron type (Holbrook, 2012).

Loss of syd-1 has a significantly weaker effect on fly NMJ development than does loss of Liprin-&alpha. Likewise, this study shows that the early phase of R7 terminal development, during which presynaptic components are localized, is less affected by loss of syd-1 than by loss of Liprin-α. A possible explanation for this difference is identified: loss of Liprin-α, but not of syd-1, significantly decreases Kinesin-3/Unc-104/Imac-mediated axon transport, and Kinesin-3/Unc-104/Imac is required for R7s to form boutons in M6 (Holbrook, 2012).

In both worm and fly, Syd-1 is required for the normal localization of Liprin-α and Brp/ELKS to presynaptic sites. In worm, loss of syd-1 can be rescued either by overexpressing full-length wild-type Liprin-α, or by overexpressing a domain of Liprin-α that promotes oligomerization of Liprin-α proteins, or by a mutation that enhances the ability of Liprin-α to bind Brp/ELKS. These results suggest that the primary function of Syd-1 is to potentiate Liprin-α activities. However, this sutyd found that Liprin-α overexpression only partially rescues the early defect that syd-1 mutant R7s have in assembling synapses. This suggests that, as in worm, Liprin-α can act partly independently of Syd-1 during presynaptic assembly but that, unlike in worm, Syd-1 also has some Liprin-α-independent function. In contrast, Liprin-α overexpression does not at all rescue the late extensions caused by loss of syd-1. As it speculated above, one possibility is that these extensions might be caused by mislocalized Liprin-α, Brp, or Nrx-1 (Holbrook, 2012).

Unlike Liprin-α, Trio overexpression fully rescues the early and partly rescues the late defect caused by loss of syd-1, suggesting that Syd-1 promotes R7 synaptic terminal development primarily by potentiating Trio activity. Consistent with this model, loss of trio phenocopies loss of syd-1 from R7s, and overexpressing Syd-1 or Trio bypasses the need for LAR to similar degrees. At fly NMJ, Trio promotes presynaptic development by acting as a GEF for Rac1. Syd-1 has a RhoGAP domain, albeit one that has not been shown to interact with GTPases. Syd-1 may act distantly upstream of Trio. However, it is also possible that Syd-1 might instead regulate one or more small GTPases in parallel with Trio. GAPs and GEFs have opposite effects on GTPases, but loss of trio or syd-1 causes similar defects at both NMJ and in R7s. One possibility, therefore, is that Syd-1 acts as a GAP not for Rac1 but for Rho, which often functions in opposition to Rac. Alternatively, Syd-1 might act as an atypical GAP for Rac1 -- perhaps lacking GAP activity but able to bind and protect Rac1-GTP from conventional GAPs -- or Syd-1 might yet act as a conventional GAP for Rac1 if it is the rate of cycling between GDP- and GTP-bound states of Rac1 (rather than simply the amount of the GTPase that is in the 'active,' GTP-bound, state) that promotes presynaptic development (Holbrook, 2012).


EVOLUTIONARY HOMOLOGS

Liprin-α in C. elegans

At synaptic junctions, specialized subcellular structures occur in both pre- and postsynaptic cells. Most presynaptic termini contain electron-dense membrane structures, often referred to as active zones, which function in vesicle docking and release. The components of those active zones and how they are formed are largely unknown. This study reports that a mutation in the C. elegans syd-2 (for synapse-defective) gene causes a diffused localization of several presynaptic proteins and of a synaptic-vesicle membrane associated green fluorescent protein (GFP) marker. Ultrastructural analysis revealed that the active zones of syd-2 mutants are significantly lengthened, whereas the total number of vesicles per synapse and the number of vesicles at the prominent active zones are comparable to those in wild-type animals. Synaptic transmission is partially impaired in syd-2 mutants. syd-2 encodes a member of the liprin (for LAR-interacting protein) family of proteins which interact with LAR-type (for leukocyte common antigen related) receptor proteins with tyrosine phosphatase activity (RPTPs). SYD-2 protein is localized at presynaptic termini independently of the presence of vesicles, and functions cell autonomously. It is proposed that SYD-2 regulates the differentiation of presynaptic termini in particular the formation of the active zone, by acting as an intracellular anchor for RPTP signalling at synaptic junctions (Zhen, 1999).

Active zones are presynaptic regions where synaptic vesicles fuse with plasma membrane to release neurotransmitters. Active zones are highly organized structurally and are functionally conserved among different species. Synapse defective-2 (SYD-2) family proteins regulate active zone morphology in C. elegans and Drosophila. This study demonstrates by immunoelectron microscopy that at C. elegans synapses, SYD-2 localizes strictly at active zones and can be used as an active zone marker when fused to green fluorescent protein (GFP). By driving expression of SYD-2::GFP fusion protein in GABAergic neurons, it is possible to visualize discrete fluorescent puncta corresponding to active zones in living C. elegans. During development, the number of GABAergic synapses made by specific motoneurons increases only slightly from larvae to adult stages. In contrast, the number of SYD-2::GFP puncta doubles, suggesting that individual synapses accommodate the increasing size of their synaptic targets mainly by incorporating more active zone materials. Furthermore, this marker was used to perform a genetic screen to identify genes involved in the development of active zones. 16 mutants were recovered with altered SYD-2::GFP expression, including alleles of five genes that have been implicated previously in synapse formation or nervous-system development. Mapping of 11 additional mutants suggests that they may represent novel genes involved in active zone formation (Yeh, 2005).

Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. The long isoform, PTP-3A, localizes specifically at synapses and the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1, and the intracellular adaptor α-liprin, syd-2. nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. It is proposed that PTP-3A acts as a molecular link between the extracellular matrix and α-liprin during synaptogenesis (Ackley, 2005).

The presynaptic regions of axons accumulate synaptic vesicles, active zone proteins and periactive zone proteins. However, the rules for orderly recruitment of presynaptic components are not well understood. This study systematically examined molecular mechanisms of presynaptic development in egg-laying synapses of C. elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, have key roles in presynaptic assembly. SYD-2 (liprin-alpha) regulate the size and the shape of active zones. In syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell-autonomously at presynaptic terminals, downstream of synaptic specificity molecule SYG-1. SYD-1 is likely to act upstream of SYD-2 to positively regulate its synaptic assembly activity. These data imply a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components (Patel, 2006).

A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-alpha, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in C. elegans syd-2 Liprin-alpha alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. A missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2gf mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2gf also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2gf requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role (Dai, 2006).

Mammalian Liprin-α

LAR family transmembrane protein-tyrosine phosphatases function in axon guidance and mammary gland development. In cultured cells, LAR binds to the intracellular, coiled coil LAR-interacting protein at discrete ends of focal adhesions, implicating these proteins in the regulation of cell-matrix interactions. Seven LAR-interacting protein-like genes are described in humans and Caenorhabditis elegans that form the liprin gene family. Based on sequence similarities and binding characteristics, liprins may be subdivided into either α- or beta-type. The C-terminal, non-coiled coil regions of α-liprins bind to the membrane-distal phosphatase domains of LAR family members, as well as to the C-terminal, non-coiled coil region of beta-liprins. Both α- and beta-liprins homodimerize via their N-terminal, coiled coil regions. Liprins are thus multivalent proteins that potentially form complex structures. Some liprins have broad mRNA tissue distributions, whereas others are predominately expressed in the brain. Co-expression studies indicate that liprin-α2 alters LAR cellular localization and induces LAR clustering. It is proposed that liprins function to localize LAR family tyrosine phosphatases at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates (Serra-Pages, 1998).

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors. GRIP binds to the liprin-α/SYD2 family of proteins that interacts with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) that are implicated in presynaptic development. In neurons, liprin-α and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-α is important for postsynaptic as well as presynaptic maturation (Wyszynski, 2002).

Metastasis-associated protein S100A4 (Mts1) induces invasiveness of primary tumors and promotes metastasis. S100A4 belongs to the family of small calcium-binding S100 proteins that are involved in different cellular processes as transducers of calcium signal. S100A4 modulates properties of tumor cells via interaction with its intracellular targets, heavy chain of non-muscle myosin and p53. A new molecular target of the S100A4 protein has been identified, liprin beta1. Liprin beta1 belongs to the family of leukocyte common antigen-related (LAR) transmembrane tyrosine phosphatase-interacting proteins that may regulate LAR protein properties via interaction with another member of the family, liprin alpha1. This study shows by the immunoprecipitation analysis that S100A4 interacts specifically with liprin beta1 in vivo. Immunofluorescence staining demonstrated the co-localization of S100A4 and liprin beta1 in the cytoplasm and particularly at the protrusion sites of the plasma membrane. The S100A4 binding site maps at the C terminus of the liprin beta1 molecule between amino acid residues 938 and 1005. The S100A4-binding region contains two putative phosphorylation sites by protein kinase C and protein kinase CK2. S100A4-liprin beta1 interaction results in the inhibition of liprin beta1 phosphorylation by both kinases in vitro (Kriajevska, 2002).

Liprin-α is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-α regulates the development of presynaptic active zones and that the association of liprin-α with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Liprin-α directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-α, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-α interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-α interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes (Ko, 2003a).

Liprin-alpha/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-alpha is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. This study reports that liprin-alpha directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-alpha and that liprin-alpha1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-alpha1 in cultured neurons and forms a complex with liprin-alpha1 in brain. Liprin-alpha1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-alpha1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-alpha-binding region, is sufficient for the synaptic localization of liprin-alpha1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-alpha may be involved in the presynaptic localization of liprin-alpha and the molecular organization of presynaptic active zones (Ko, 2003b).

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of AMPA glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. This study reports that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including AMPA receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons (Shin, 2003).

The LAR transmembrane tyrosine phosphatase associates with liprin-α proteins and colocalizes with liprin-α1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extent of in vitro and in vivo phosphorylation of these mutants was reduced relative to that of wild-type liprin-α1. Treatment of liprin-α1 with calf intestinal phosphatase weakens its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibits liprin phosphorylation does not bind to LAR as assessed by coprecipitation studies. Endogenous LAR binds phosphorylated liprin-α1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, immunoprecipitates of liprin-α1 expressed in COS-7 cells were found to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-α1 derived from Sf-9 insect cells retains the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-α1 truncation mutant incorporates phosphate after denaturation and/or renaturation in SDS gels. Finally, binding assays show that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LH region mutations that inhibit liprin phosphorylation stabilize the association of liprin with ATP-agarose. Taken together, these results suggest that liprin autophosphorylation regulates its association with LAR (Serra-Pages, 2005).

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. It is proposed that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP (Dunah, 2005).

Neurotransmitter release regulated by a MALS-liprin-α presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. The MALS protein complex was purified from brain and it was found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone (Olsen, 2005).

The ternary scaffolding protein complex of MALS/Veli (mammalian LIN-7/vertebrate homologue of LIN-7), CASK (peripheral plasma membrane protein), and Mint-1 (munc-18 interacting protein 1), consist of vertebrate homologues of a complex first identified in Caenorhabditis elegans that mediates vulval development. In mammalian brain, the MALS–CASK–Mint-1 complex occurs on both sides of synaptic junctions and is thought to serve distinct roles in these two locations. Presynaptically, this complex links to neurexin, an adhesion molecule that binds across the synapse to postsynaptic neuroligin. Furthermore, Mint-1 associates with Munc18-1, an essential component of the synaptic vesicle fusion machinery. Postsynaptically, MALS binds to the N-methyl-D-aspartate (NMDA)–type of glutamate receptors and is reported to transport NMDA receptor vesicles along microtubules (Olsen, 2005 and references therein).

Genetic studies have failed to establish the essential roles of the MALS–CASK–Mint-1 complex in brain. Three MALS genes exist in mammals, and targeted disruption of MALS-1 and MALS-2 leads to compensatory up-regulation of MALS-3 in the CNS. Mint-1 mutant mice show no defects in excitatory synaptic transmission and only a subtle defect in inhibitory synaptic transmission . Also, no synaptic analysis has been reported for CASK knockouts that die at birth due to midline defects (Olsen, 2005 and references therein).

Several molecules that mediate synapse development have been identified through invertebrate genetic studies. For example, mutation of C. elegans syd-2 disperses presynaptic active zones (Zhen, 1999). A similar structural defect occurs in flies lacking the Drosophila melanogaster syd orthologue liprin-alpha, which exhibits a concomitant decrease in synaptic transmission (Kaufmann, 2002). Liprin-α binds to a receptor protein tyrosine phosphatase, Dlar (Serra-Pages, 1998), suggesting a model whereby liprin-α and Dlar cooperate to organize presynaptic active zones. How liprin-α links to the synaptic vesicle machinery remains uncertain (Olsen, 2005 and references therein).

To define the essential roles for the MALS complex in mammals, the MALS complex was purified from brain. Isolation of the MALS complex revealed an association with a family of cytoskeletal and presynaptic adhesion molecules. Importantly, liprin-α1, -α2, -α3, and -α4 in the MALS complex. Association with this complex is mediated through the SAM domains in liprin-α and an NH2-terminal region in CASK. Using the sterile α motif (SAM) domains of liprin-α as a dominant negative, the MALS–liprin complex was disrupted in dissociated neurons. To understand the function of the MALS complex, mutant mice were generated lacking all three MALS genes. Mice lacking any single gene were viable and fertile. However, mice lacking all three MALS genes died within one hour of birth. This perinatal lethality is associated with impaired presynaptic function, reflecting the presynaptic deficits of invertebrates lacking liprin-α orthologues. These studies establish a crucial role for the MALS complex in synaptic vesicle exocytosis and implicate liprin-α in this process (Olsen, 2005).

Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK: Cdk5 regulates the interaction between CASK and liprin-α

Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins was investigated in a mammalian system. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. It was found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis (Samuels, 2007).

Homologs of liprin-α proteins are essential for presynaptic terminal formation in C. elegans and Drosophila . Mutations in C. elegans syd-2 result in a diffuse localization of several presynaptic proteins and abnormally sized active zones, and loss- and gain-of-function experiments demonstrate that presynaptic organization is dependent on syd-2. Likewise, Dliprin-α is required for normal synaptic morphology including the size and shape of the presynaptic active zone in Drosophila . Cdk5-dependent phosphorylation of CASK occurs in both the CaMK and L27 domains, and only mutation of both sites yields a localization phenotype. Since liprin-α proteins require the presence of both domains to interact with CASK, the phosphorylation sites are in a prime spot to mediate the interaction. According to the model described in this study, liprin-α is required for initial CASK localization to presynaptic terminals. Since, liprin-α binds directly to the kinesin motor KIF1A and in Drosophila liprin-α mutant axons there is decreased anterograde processivity resulting in reduced levels of presynaptic markers at terminals, it is feasible that liprin-α acts as a cargo receptor that delivers CASK, as well as other components, to and within the developing synapse. Cdk5-dependent phosphorylation could then act to coordinate distinct pools of CASK that are bound to liprin-α or are bound to other components of the presynaptic machinery. Importantly, it is not believed that Cdk5 loss of function generally affects liprin-α-mediated transport since synaptophysin, a marker of synaptic vesicles, is still properly localized within synaptosomes. In this model, there would be advantages of having locally enhanced Cdk5 activity within the presynaptic terminal relative to some other cellular compartments. Supporting this idea, phospho-CASK is particularly enriched at synaptic membranes, and Cdk5 has been shown to phosphorylate and regulate several proteins, including Munc-18, Dynamin-1, Amphiphysin-1, and Synaptojanin-1, that function to control multiple rounds of the synaptic vesicle cycle. Synapsin-1 is also a Cdk5 substrate. With regard to the role of liprin-α, it will ultimately be essential to assay synapse formation and CASK localization in mammalian liprin-α loss-of-function models (Samuels, 2007).


REFERENCES

Search PubMed for articles about Drosophila Liprin-α

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

Bohme, M. A., Beis, C., Reddy-Alla, S., Reynolds, E., Mampell, M. M., Grasskamp, A. T., Lutzkendorf, J., Bergeron, D. D., Driller, J. H., Babikir, H., Gottfert, F., Robinson, I. M., O'Kane, C. J., Hell, S. W., Wahl, M. C., Stelzl, U., Loll, B., Walter, A. M. and Sigrist, S. J. (2016). Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat Neurosci [Epub ahead of print]. PubMed ID: 27526206

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

Graf, E. R., et al. (2009). Rab3 dynamically controls protein composition at active zones. Neuron 64: 663-677. PubMed Citation: 20005823

Holbrook, S., Finley, J. K., Lyons, E. L. and Herman, T. G. (2012). Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development. J Neurosci 32: 18101-18111. PubMed ID: 23238725

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

Hu, Z., Tong, X. J. and Kaplan, J. M. (2013). UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans. Elife 2: e00967. PubMed ID: 23951547

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

Li, L., Tian, X., Zhu, M., Bulgari, D., Bohme, M. A., Goettfert, F., Wichmann, C., Sigrist, S. J., Levitan, E. S. and Wu, C. (2014). Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons. J Neurosci 34: 8474-8487. PubMed ID: 24948803

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

Owald, D., et al. (2010). A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila. J. Cell Biol. 188(4): 565-79. PubMed Citation: 20176924

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). The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance. J. Neurosci. 31(12): 4421-33. PubMed Citation: 21430143

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


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