RIM-binding protein: Biological Overview | References
Gene name - RIM-binding protein
Cytological map position - 88F3-88F3
Function - scaffolding protein
Keywords - neuromuscular junction, essential for the integrity of the synaptic active zone scaffold and for exocytotic neurotransmitter release
Symbol - Rbp
FlyBase ID: FBgn0262483
Genetic map position - chr3R:15,374,635-15,400,011
Cellular location - intracellular presynapse
This study defines activities of RIM-binding protein (RBP) that are essential for baseline neurotransmission and presynaptic homeostatic plasticity. At baseline, rbp mutants have a approximately 10-fold decrease in the apparent Ca2+ sensitivity of release that this study attributes to (1) impaired presynaptic Ca2+ influx, (2) looser coupling of vesicles to Ca2+ influx, and (3) limited access to the readily releasable vesicle pool (RRP). During homeostatic plasticity, RBP is necessary for the potentiation of Ca2+ influx and the expansion of the RRP. Remarkably, rbp mutants also reveal a rate-limiting stage required for the replenishment of high release probability (p) vesicles following vesicle depletion. This rate slows approximately 4-fold at baseline and nearly 7-fold during homeostatic signaling in rbp. These effects are independent of altered Ca2+ influx and RRP size. It is proposed that RBP stabilizes synaptic efficacy and homeostatic plasticity through coordinated control of presynaptic Ca2+ influx and the dynamics of a high-p vesicle pool (Müller, 2015).
Homeostatic signaling systems stabilize the active properties of nerve and muscle cells. The homeostatic modulation of presynaptic neurotransmitter release, referred to here as 'presynaptic homeostasis', is an evolutionarily conserved form of homeostatic plasticity that has been documented at neuromuscular junctions (NMJ) in systems ranging from Drosophila to human. There is also evidence for presynaptic homeostasis at synapses throughout the mammalian central nervous system. At the NMJ, inhibition of postsynaptic neurotransmitter receptors elicits an increase in presynaptic release that precisely offsets the magnitude of receptor perturbation and, thereby, restores postsynaptic muscle depolarization to baseline. Remarkably, different magnitudes of postsynaptic receptor perturbation induce different levels of presynaptic homeostatic compensation. Therefore, presynaptic homeostasis requires mechanisms that can accurately tune neurotransmitter release over a wide range and, then, stably maintain the newly established levels of presynaptic release (Müller, 2015).
Two presynaptic processes have been shown to be essential for presynaptic homeostasis: 1) modulation of presynaptic Ca2+ influx through the CaV2.1 Ca2+ channel (Frank, 2006; Müller, 2012) and 2) modulation of the readily-releasable pool of synaptic vesicles (RRP; Müller, 2012; Weyhersmüller, 2011). Recent evidence suggests that these processes are molecularly separable. The homeostatic modulation of presynaptic Ca2+ influx requires the expression of a presynaptic DEG/ENaC sodium leak channel (Younger, 2013), whereas homeostatic RRP modulation requires the presynaptic adaptor protein RIM (Rab3-Interacting Molecule; Müller, 2012). It remains unknown how the homeostatic modulation of Ca2+ influx and RRP size is coordinated, because molecules that participate in both processes have not been identified. Furthermore, it seems likely that additional presynaptic processes will be engaged during homeostatic plasticity in order to sustain potentiated release for prolonged periods of time. Yet, these additional processes and their mechanistic control remain unknown (Müller, 2015).
This study makes several advances, placing RIM-Binding Protein (RBP) at the forefront of presynaptic homeostatic plasticity. RBPs biochemically interact with two proteins that are central to presynaptic Ca2+ signaling and RRP modulation: Cav2.1 Ca2+ channels and RIM (Hibino, 2002; Liu, 2011). It was recently shown that RBP resides at the center of presynaptic active zones, where it surrounds presynaptic CaV2.1 Ca2+ channels (Liu, 2011). This previous study analyzed RBP's role in baseline synaptic transmission, finding that loss of RBP causes impaired Ca2+ channel clustering and impaired presynaptic Ca2+ influx with an associated deficit in neurotransmitter release (Liu, 2011; Müller, 2015 and references therein).
This study now demonstrates that RBP is essential for the homeostatic modulation of neurotransmitter release, being required for both the modulation of calcium influx and the RRP. As such, RBP is the first presynaptic protein that could reasonably participate in coordinating these two homeostatic changes. Then, another novel activity of RBP is defined, demonstrating that RBP is essential for the normal resupply of high-release probability vesicles. Loss of rbp was shown to slow the resupply of high-release probability vesicles by ~400%. This change is an order of magnitude more pronounced than previously described for Ca2+-dependent effects on the rate of vesicle resupply. Finally, this new activity of RBP during vesicle resupply was connected to the mechanisms of presynaptic homeostatic plasticity. Thus evidence is provided that presynaptic homeostasis acts primarily upon a pool of high-release probability vesicles that are thought to reside at the active zone in close proximity to presynaptic Ca2+ channels and RBP. It is proposed that RBP is a molecular keystone with actions that are uniquely important for the stabilization of baseline neurotransmission, coupling of vesicles to sites of Ca2+ influx, and the stable, persistent homeostatic modulation of presynaptic neurotransmitter release (Müller, 2015).
RBP specifically localizes to a domain that surrounds the presynaptic CaV2.1 Ca2+ channel (Liu, 2011). The data are consistent with previous analysis of baseline transmission in rbp mutants where it was shown that loss of RBP impairs the abundance and organization of Ca2+ channels at the active zone, causes a 30% drop in the amplitude of spatially averaged presynaptic Ca2+ transients and causes a striking >90% decrease in neurotransmitter release at physiological Ca2+ (Liu, 2011). Even though there is a supralinear relationship between Ca2+ influx and release, it is striking that release is nearly abolished in rbp while Ca2+ influx is decreased by only one third (Müller, 2015).
Several lines of evidence are presented that the defect in release observed in rbp mutants is due to the combined influence of 1) looser coupling of vesicles to the sites of presynaptic Ca2+ entry, and 2) impaired access to the RRP of synaptic vesicles. First, it was confirmed that there is only a modest, 28% decrease in presynaptic Ca2+ influx in rbp compared to wild type. Remarkably, this difference is diminished or eliminated as external Ca2+ is elevated beyond physiological levels, whereas release remains strongly impaired. The absence of an apparent difference in Ca2+ influx between wild type and rbp at elevated external Ca2+ levels could be due to effects of elevated external Ca2+ acting on presynaptic AP repolarization or channel properties that eliminate the difference observed at lower Ca2+ concentrations. Regardless, when Ca2+ influx is analyzed there is no difference comparing wild type and rbp at 3mM external Ca2+ and yet, under these precise recording conditions, there is a pronounced defect in presynaptic release. Thus, RBP must have an additional role in vesicle release. Second, it was demonstrated that loss of rbp confers a very high EGTA sensitivity to synaptic transmission. This effect cannot be attributed to decreased presynaptic Ca2+ influx in the rbp mutant, because (1) wild-type synapses display a very modest EGTA sensitivity when normalizing for presynaptic Ca2+ influx, and (2) a mutation that causes a similar defect in presynaptic Ca2+ influx (rim) induces a less pronounced EGTA sensitivity as compared to rbp (Müller, 2011). Since EGTA application to rbp mutant synapses reduces EPSC amplitudes by greater than 50%, the release-ready vesicles that support release in rbp mutants either have an 'intrinsically' low Ca2+ affinity that is independent of vesicle localization, or they are localized more distant from the Ca2+ channels compared to wild type. The latter possibility agrees with previous electron microscopy data showing a decrease in the number of vesicles located within 5nm of the presynaptic T-bar, which identifies the location of Ca2+ channels at this presynaptic active zone (Liu, 2011). These data are consistent with the conclusion that loss of RBP impairs either the formation or maintenance of a high-release probability vesicle pool that resides in close proximity to presynaptic Ca2+ channels where RBP has been shown to localize (Liu, 2011) (Müller, 2015).
Analysis of recovery from synaptic depression reveals another activity of RBP that is essential for the formation and/or maintenance of a high-release probability pool of synaptic vesicles. The slow kinetics of synaptic vesicle recovery following a depleting stimulus train was found to be dramatically impaired (by ~400%). This effect far exceeds previously published studies characterizing a role for Munc-13, bassoon, or calmodulin in vesicle recovery after repetitive stimulation. The loss of any of these genes altered the slow phase of vesicle recovery by a maximum of 40%. Truncation of the C-terminal end of the presynaptic protein Bruchpilot specifically impairs the fast recovery phase , while further truncation of the C-terminus of Bruchpilot affects both the slow and the fast recovery phase. By comparison, the absence of RBP predominantly decelerates the slow recovery phase, and therefore likely plays a major role in stabilizing a high release-probability vesicle pool, an idea that is consistent with the other rbp phenotypes. By controlling the size and stability of the high-release probability vesicle pool, RBP would also effectively couple a change in Ca2+ influx to modulation of the RRP, both of which are required for the expression of homeostatic synaptic plasticity (Müller, 2015).
The homeostatic potentiation of presynaptic neurotransmitter release requires an increase in the number of release-ready vesicles based upon either cumulative EPSC amplitude analysis during brief, high-frequency stimulus trains, or variance-mean EPSC-amplitude analysis following single AP stimulation at low frequency (Müller, 2012; Weyhersmüller, 2011). At mammalian central synapses, RRP size correlates with extracellular Ca2+ concentration. This raises the possibility that the homeostatic modulation of the RRP is simply a consequence of enhanced presynaptic Ca2+ influx. The current data demonstrate that this cannot be the case. First, the relationship between extracellular Ca2+ and RRP size is sublinear and quite shallow at high extracellular Ca2+ concentrations. During synaptic homeostasis, a ~20-30% increase in presynaptic Ca2+ influx was observed, which would not seem to be sufficient to achieve a doubling of the RRP based on the relationship between extracellular Ca2+ and RRP size. Furthermore, it was previously demonstrated that RIM is necessary for the homeostatic potentiation of RRP size, while not perturbing the homeostatic modulation of presynaptic Ca2+ influx (Müller, 2012), thereby providing a molecular separation between these two processes (Müller, 2015).
It is worth noting that baseline RRP size, estimated at physiological Ca2+, is smaller in both rbp and Rab3 interacting molecule (rim) mutants. As such, it is possible that loss of RIM or RBP could simply lead to a decrease in RRP size and thereby prevent homeostatic plasticity. However, data are presented that argue against this possibility for rbp mutants. The absolute RRP size is equivalent when comparing wild type and rbp mutants at super-physiological extracellular Ca2+ (15mM), while the homeostatic modulation of RRP size is completely blocked. It appears, therefore, that the total number of release-ready vesicles is not affected. Rather, access to the pool is impaired in rbp. Remarkably, RRP size is not nearing saturation when recording at 15mM extracellular Ca2+ because it was possible to observe a homeostatic doubling of RRP size under these condition at wild-type terminals. Based on these data, it is hypothesized that RBP recruits a biochemical activity during synaptic homeostasis through one of its protein interaction domains to expand the size of the RRP (Müller, 2015).
In wild type, when recordings are made at super-physiological extracellular Ca2+ (3mM), the induction of presynaptic homeostasis has no effect on the kinetics of synaptic vesicle replenishment following a stimulus train. Thus, recovery rates are insensitive to the absolute number of vesicles that are released during a depleting stimulus train, since release is doubled following the induction of synaptic homeostasis at all Ca2+ concentrations tested. However, in rbp mutants, the induction of homeostatic plasticity decelerates the slow phase of vesicle recovery by ~300%. This is remarkable because the slow rate of recovery is already decelerated at baseline in rbp mutants by ~400% compared to wild type. The combined effect is that vesicles that take ~6s to recover in wild type now take nearly 60s to recover in rbp following the induction of homeostatic plasticity. The additional deceleration observed in rbp following the induction of homeostatic plasticity is not a secondary consequence of releasing additional vesicles during the depleting stimulus train, because this does not happen in the rbp mutant. It is concluded that the absence of RBP has revealed a step in the process of vesicle replenishment that becomes rate limiting following the induction of presynaptic homeostasis. It is possible that this step could be normally acted upon by the homeostatic signaling system to ensure that the homeostatically enhanced state of release can be sustained during the repetitive stimulation that is characteristic of normal neuromuscular activity. Indeed, this RBP-dependent step could be related to the observation that the size of the RRP actually decreases instead of increasing following the induction of presynaptic homeostasis (Müller, 2015).
Several additional observations argue that the homeostasis-dependent deficit in vesicle recovery is not simply a secondary consequence of an impaired active zone. First, the homeostasis-dependent deceleration of replenishment kinetics is largely confined to the high-release probability population of vesicles, because the fast component of recovery is essentially unaltered. Second, the slow rate of recovery is unaltered if one probes recovery of the vesicle pool using stimulus trains. During stimulus trains, as opposed to single AP stimulation, Ca2+ levels rise substantially. It is concluded that the rate limiting stage of vesicle recovery is sensitive to the concentration of intracellular Ca2+, consistent with the known role of Ca2+ in accelerating vesicle replenishment. This argues that the process of vesicle recovery is not simply broken following the loss of RBP. Finally, previous ultrastructural examination of the active zone in rbp showed no evidence of accumulated membrane material that might indicate a 'traffic jam' at the active zone (Müller, 2015).
It is straightforward to suggest that the loss of rbp prevents the capture or stabilization of vesicles at the active zone, thereby dramatically decelerating the rate of vesicle replenishment to this pool under baseline conditions. If the number of vesicles being replenished remains unaltered in rbp mutants following the induction of synaptic homeostasis, then what accounts for the additional deceleration? It seems that the induction of homeostatic plasticity induces an additional presynaptic process that becomes rate limiting in the absence of RBP. This study demonstrated that the RRP can be doubled, even in the presence of 15mM extracellular Ca2+, suggesting a rather remarkable change in vesicle usage. It seems likely that this is achieved through the conversion of vesicles from the reserve pool to the RRP. One possibility is that this conversion process is still induced, but it becomes rate limiting in the absence of RBP. This would position RBP downstream in the cascade of events that lead to a functional doubling of the RRP, which is consistent with the localization of RBP at the Ca2+ channel. Finally, since loss of RIM has no effect on the rate of recovery yet also blocks the expansion of the RRP, the mechanism by which RIM acts on the RRP must be distinct. This possibility remains consistent with the genetic interaction experiments (Müller, 2015).
In addition to being crucial for the homeostatic modulation of RRP size, evidence is also provided that RBP is required for the homeostatic enhancement of presynaptic Ca2+ influx. A RBP-mediated modulation of presynaptic Ca2+ influx is conceivable because RBP biochemically interacts with the C- terminus of the presynaptic voltage-gated Ca2+ channel (Hibino, 2002; Liu, 2011). Moreover, RBP has been recently shown to be required for normal Ca2+ channel density at active zones and normal Ca2+ influx levels (Liu, 2011). Recent data indicate that low-voltage modulation of the presynaptic membrane via insertion of DEG/ENaC sodium leak channels may be a mechanism for altering presynaptic Ca2+ influx during presynaptic homeostasis (Younger, 2012). In this context, RBP may be required for the responsivity of the presynaptic CaV2.1 Ca2+ channels to membrane voltage changes (Müller, 2015).
Biochemical data provide insight into the unique activities of RIM and RBP. Drosophila RBP specifically interacts with the PxxP motifs of both RIM and the presynaptic voltage-gated Ca2+ channel through its third SH3 domain (Liu, 2011). It is therefore likely that RBP is either bound to RIM or to the Ca2+ channel, and that RBP's role in RRP size regulation and Ca2+ influx modulation might be biochemically separable. RIM interacts with a specific sequence at the very end of the C-terminus of the Ca2+ channel through its PDZ domain. Thus, RIM and RBP bind to different domains of the Ca2+ channel's C-terminus, which might explain the different roles of RBP and RIM in the homeostatic regulation of Ca2+ influx (Müller, 2015).
The molecular machinery mediating the fusion of synaptic vesicles (SVs) at presynaptic active zone (AZ) membranes has been studied in detail, and several essential components have been identified. AZ-associated protein scaffolds are viewed as only modulatory for transmission. This study discovered that Drosophila Rab3-interacting molecule (RIM)-binding protein (DRBP) is essential not only for the integrity of the AZ scaffold but also for exocytotic neurotransmitter release. Two-color stimulated emission depletion microscopy showed that DRBP surrounds the central Ca2+ channel field. In drbp mutants, Ca2+ channel clustering and Ca2+ influx were impaired, and synaptic release probability was drastically reduced. These data identify RBP family proteins as prime effectors of the AZ scaffold that are essential for the coupling of SVs, Ca2+ channels, and the SV fusion machinery (Liu, 2011).
In response to Ca2+ influx, synaptic vesicles (SVs) fuse with the active zone (AZ) membrane. An elaborate protein-based cytomatrix covering the AZ membrane is meant to facilitate the release process, but its components and operational principles are poorly understood. Previous studies have found that Bruchpilot is an essential building block of the Drosophila AZ cytomatrix (the T bar). This study used antibodies to screen for additional cytomatrix components (Liu, 2011).
Rab3-interacting molecule (RIM)-binding proteins (RBPs) are enriched at presynaptic terminals (Hibino, 2002; Spangler, 2007) and interact with voltage-gated Ca2+ channels (VGCCs). Mammals harbor three rbp family loci; Drosophila has a single rbp gene (drbp) (Mittelstaedt, 2007). This study generated antibodies against Drosophila RBP (DRBP), which stained presynapses at neuromuscular junctions (NMJs). The size of AZ cytomatrices is below the diffraction limit of light microscopy. To overcome this, two-color stimulated emission depletion (STED) microscope providing 50-nm resolution in the focal plane was used. Relative to the Bruchpilot C-terminal label, DRBP C-terminal immunoreactivity localized toward the AZ center. Vertically, the DRBP label localized ~100 nm closer to the AZ membrane than the Bruchpilot C-terminal label. Postembedding immunogold labeling against DRBP C terminus yielded similar results. DRBP C- and N-terminal labels are similarly distributed in planar views, and in vertical views the centers of the DRBP C- and N-terminal signals are on average ~30 nm apart. The Cacophony (Cac) VGCC localizes beneath the scaffold formed by Bruchpilot in the AZ center. DRBP C terminus encircled a small (50 to 100 nm) AZ-central field of CacGFP (Liu, 2011).
The drbp locus was subjected to genetic analysis. A strain with a transposon inserted between exons 6 and 7 (MB02027, drbpMinos) was positioned in trans over a deficiency including drbp, as well as a neighboring locus [Df(3R)S201, short Df]. In these larvae, DRBP levels at NMJs were reduced to one-third of control levels. These drbp hypomorphic flies hatched below expected Mendelian ratios, and mutant larvae showed reduced locomotion. Chemical mutagenesis screening provided alleles with premature stop codons (drbpSTOP1, drbpSTOP2, and drbpSTOP3). Animals carrying these alleles over Df only rarely reached adulthood, and mutant larvae barely moved. DRBP immunoreactivity at mutant larval NMJs was completely absent when either N- or C-terminal antibodies were used. Thus, these alleles are considered to be null. Using pacman technology, a genomic transgene encompassing the entire drbp locus (Rescue) was produced. One copy of this construct partially restored NMJ staining and partially rescued drbpSTOP1-3 adult vitality. Mutant larval NMJ terminals reached normal morphological size, and had normal synapse numbers, as also seen in transmission electron microscopy (TEM). Postsynaptic glutamate receptor (GluR) fields appeared enlarged (Liu, 2011).
Bruchpilot spots in drbp mutants appeared largely unaltered at confocal resolution. However, with STED, Bruchpilot signals emerged as atypically organized, no longer forming regular donuts but adopting irregular shapes. Bruchpilot signals extended into the cytoplasm unusually far, not matching postsynaptic GluR fields in either planar or vertical views. Organization of the AZ cytomatrix in drbp mutants was clearly affected in electron microscopy (EM) of both conventionally embedded samples and high-pressure frozen- and freeze-substituted (HPF) samples. The drbp hypomorph (drbpMinos/Df) still formed structures resembling T-bar platforms, but they resided on thinner pedestals. At AZ membranes of drbp nulls, abnormally shaped electron-dense material was found, but no regular T bars. In three-dimensional electron tomography reconstructions, it became obvious that the entire cytomatrix was severely misshapen. Free-floating electron-dense material detached from the AZ plasma membrane was occasionally observed in drbp nulls. These atypical electron densities still tethered SVs, a function mediated by the C-terminal end of Bruchpilot (Hallermann, 2010). With EM, overall SV density in NMJ boutons appeared unaltered. The number of membrane-proximal SVs (up to a 5-nm distance) counted over the whole AZ, however, were reduced in drpbSTOP1 animals. For functional analysis, two-electrode voltage-clamp recordings were performed at larval NMJs. In drbp hypomorphs, evoked excitatory junctional current (eEJCs) amplitudes were reduced by about half. In drbp nulls, synaptic transmission was practically abolished. Synaptic release was facilitated by recording in elevated extracellular Ca2+. In 2 mM Ca2+, eEJC amplitudes were reduced to ~10% of control levels in all three null alleles. One copy of the genomic drbp transgene completely rescued this phenotype. Frequency and amplitude of miniature excitatory junctional currents (mEJCs) were unchanged in drbpSTOP1, despite enlarged GluR fields. Thus, the number of quanta (i.e., SVs) released per individual action potential (AP) (quantal content) was dramatically reduced in the absence of DRBP (Liu, 2011).
At paired-pulse stimulation, drbpSTOP1 synapses exhibited an unusually strong facilitation. During a 10-Hz stimulation train, a protocol that leads to marked depression of eEJCs in controls, drbpSTOP1 mutants showed sustained facilitation and reached a steady-state level more than double the initial amplitude. When stimulated with five consecutive pulses at 100 Hz, drbpSTOP1 eEJCs exhibited substantial recovery and almost reached absolute control levels on the fifth pulse. Altogether, these results suggest that drbp null mutants suffer from a severely reduced release probability. However, as SV release can be substantially elevated under high-frequency stimulation, it is concluded that the core fusion machinery is still operational in drbp mutants, and it is hypothesized that the reduced SV release evoked by single APs is mainly due to defects upstream of the SV fusion process (Liu, 2011).
The eEJC rise times were slightly but significantly increased. Thus, evoked SV fusion events in drbp mutants appeared desynchronized with the invasion of the presynaptic terminal by an AP. Because this synchronization depends on the spatiotemporal pattern of AP-triggered Ca2+ influx into the nerve terminal, changes in the abundance of Ca2+ channels might contribute to the observed defect. Indeed, Ca2+ channel signals, as evaluated by expression of CacGFP, were slightly but consistently reduced by ~25% measured over the whole NMJ in all three drbp null alleles. For the drbp hypomorph, Ca2+ channel signal was reduced by about 25% as well, whereas the evoked response was only reduced to 44%. Measuring CacGFP intensity per postsynaptic density (PSD) yielded a slightly stronger 36% decrease in drbpSTOP1 animals compared with controls. This decrease in Ca2+ channel signal intensity was reflected by a ~30% decrease in calcium influx in response to single APs as determined by the ΔF/F amplitude of spatially averaged Ca2+ signals recorded from drbpSTOP1 mutant boutons (Liu, 2011).
Consistent with previous findings in mammals this study found a biochemical interaction between DRBP and both Drosophila Ca2+ channel α1 subunit Cac and the Drosophila homolog of AZ protein RIM. Interactions were specifically mediated by highly homologous PXXP motifs that bind to the third DRBP SH3 domain. Recently, RIM has been suggested to tether Ca2+ channels to the AZ membrane by means of (1) a direct interaction with the Ca2+ channel α1 subunit and (2) an interaction with RBP (Kaeser, 2011). Mouse rim-1 and rim-2 double knockouts (DKOs), however, showed a rather moderate reduction in SV release in comparison with the dramatic drbp null phenotype. Thus, DRBP might bundle multiple interactions that are not necessarily downstream of RIM. Mechanistically, loss of DRBP leads to defects in Ca2+ channel abundance, Ca2+ influx, SV docking, and cytomatrix organization that probably all contribute to the severity of the drbp-release deficit (Liu, 2011).
Of note, in Bruchpilot mutants, cytomatrix and Ca2+ channel clustering defects are more pronounced than in drbp nulls. Functionally, drbp and bruchpilot phenotypes appear similar: Both demonstrate decreased and desynchronized evoked SV release with atypical short-term facilitation. However, the deficits in evoked SV release are much more severe in drbp nulls than in bruchpilot nulls [i.e., release occurs at 5% versus 30% of the respective wild-type level]. DRBP levels were clearly reduced in bruchpilot mutants, whereas gross Bruchpilot levels were not altered in drbp mutants. Given that even a partial loss of DRBP causes marked reduction in SV release, deficits in bruchpilot mutants might be explained, at least in part, by a concomitant loss of DRBP, and DRBP probably serves functions beyond the structural and Ca2+ channel-clustering roles of Bruchpilot (Liu, 2011).
Taken together, this study identified DRBP as a central part of the AZ cytomatrix. How, in detail, DRBP functionally integrates into this protein network is subject to future analyses. Notably, the short-term plasticity phenotype of drbp mutants is reminiscent of mammalian munc13-1 KO and caps-1 and caps-2 DKO mutants, which implicates functional links between priming factors and DRBP. Consistent with the functional importance of the DRBP protein family suggested by this study, human genetics recently identified two rbp loci associated with autism with high confidence (Liu, 2011).
Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). This study demonstrates axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. RBP C-terminal SH3 domains were shown in atomic detail to bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Point mutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes (Siebert, 2015).
Large multi-domain scaffold proteins such as BRP/RBP are ultimately destined to form stable scaffolds, characterized by remarkable tenacity and a low turnover, likely due to stabilization by multiple homo- and heterotypic interactions simultaneously. How these large and 'sticky'; AZ scaffold components engage into axonal transport processes to ensure their 'safe'; arrival at the synaptic terminal remains to be addressed. This study found that the AZ scaffold protein RBP binds the transport adaptor Aplip1 using a 'classic'; PxxP/SH3 interaction. Notably, the same RBP SH3 domain (II and III) interaction surfaces are used for binding the synaptic AZ ligands of RBP, that is, RIM and the voltage gated Ca2+ channel, though with clearly lower affinity than for Aplip1. A point mutation which disrupts the Aplip1-RBP interaction provoked a 'premature'; capture of RBP and the co-transported BRP at the axonal membrane, thus forming ectopic but, concerning T-bar shape and BRP/RBP arrangement, WT-like AZ scaffolds. The Aplip1 orthologue Jip1 has been shown to homo-dimerize via interaction of its SH3 domain. Thus, the multiplicity of interactions, with Aplip1 dimers binding to two SH3 domains of RBP as well as to KLC, might form transport complexes of sufficient avidity to ensure tight adaptor–cargo interaction and prevent premature capture of the scaffold components (Siebert, 2015).
Intravital imaging experiments showed that within axons RBP and BRP are co-transport in shared complexes together with Aplip1, whereas, despite efforts, no any co-transport of other AZ scaffold components, that is, Syd-1 or Liprin-α with BRP/RBP, were detected. In addition, STED analysis of axonal aggregates in srpk79D mutants showed BRP/RBP in stoichiometric amounts, but also failed to detect other AZ scaffold components. Moreover, BRP and RBP co-aggregated in the axoplasm of several other transport mutants tested (acsl, unc-51, appl, unc-76), consistent with both proteins entering synaptic AZ assembly from a common transport complex. Of note, during AZ assembly at the NMJ, BRP incorporation is invariably delayed compared to the 'early assembly'; phase which is driven by the accumulation of Syd-1/Liprin-α scaffolds. As the early assembly phase is, per se, still reversible, the transport of 'stoichiometric RBP/BRP complexes'; delivering building blocks for the 'mature scaffold'; might drive AZ assembly into a mature, irreversible state, and seems mechanistically distinct from early scaffold assembly mechanisms (Siebert, 2015).
Previous work suggested that AZ scaffold components (Piccolo, Bassoon, Munc-13 and ELKS) in rodent neurons are transported to assembling synapses as 'preformed complexes';, so-called Piccolo-Bassoon-Transport Vesicles (PTVs). The PTVs are thought to be co-transported with SV precursors anterogradely mediated via a KHC(KIF5B)/Syntabuli/Syntaxin-1 complex and retrogradely via a direct interaction between Dynein light chain and Bassoon. Since their initial description, however, further investigations of PTVs have been hampered by the apparent relative scarcity of PTVs, and by the lack of genetic or biochemical options for specifically interfering with their transport or final incorporation into AZs (Siebert, 2015).
A direct interaction of Aplip1 and BRP was not detected although their common transport can be uncoupled from the presence of RBP. One possible explanation could be a direct interaction of Aplip1 to other AZ proteins that are co-transported together with BRP and RBP. It is interesting that the very C-terminus of BRP is essential for SV clustering around the BRP-based AZ cytomatrix. Thus, it is tempting to speculate that adaptor/transport complex binding might block premature AZ protein/SV interactions before AZ assembly, but further analysis will have to await more atomic details than were obtained for the RBP::Aplip1 interaction (Siebert, 2015).
The down-regulation of the motor protein KHC also provoked severe axonal co-accumulations of BRP and RBP but per se should leave the adaptor protein-AZ cargo interaction intact. In contrast to aplip1, the axonal aggregations in khc mutants adapted irregular shapes most of the time, likely not representing T-bar-like structures. Thus, the data suggest a mechanistic difference when comparing the consequences between eliminating adaptor cargo interactions with a direct impairment of motor functions. Still, it cannot be excluded that trafficking of AZ complexes naturally antagonizes their ability to assemble into T-bars (Siebert, 2015).
The idea that proteins/molecules are held in an inactive state till they reach their final target has been observed in many other cell types. For example, in the context of local translation control, mRNAs are shielded or hidden in messenger ribonucleoprotein particles during transport so that they are withheld from cellular processing events such as translation and degradation. Shielding is thought to operate through proteins that bind to the mRNA and alter its conformation while at the correct time or place the masking protein is influenced by a signal that alleviates its shielding effect. As another example, hydrolytic enzymes, for example, lysosomes, are transported as proteolytically inactive precursors that become matured by proteolytic processing only within late endosomes or lysosomes. Particularly relevant in the context of AZ proteins involved in exocytosis, the Habc domain of Syntaxin-1 folds back on the central helix of the SNARE motif to generate a closed and inactive conformation which might prevent the interaction of Syntaxin-1 with other AZ proteins during diffusion (Siebert, 2015).
Previous genetic analysis of C. elegans axons forming en passant synapses suggested a tight balance between capture and dissociation of protein transport complexes to ensure proper positioning of presynaptic AZs. In this study, overexpression of the kinesin motor Unc-104/KIF1A reduced the capture rate and could suppress the premature axonal accumulations of AZ/SV proteins in mutants of the small, ARF-family G-protein Arl-8. Interestingly, large axonal accumulations in arl-8 mutants displayed a particularly high capture rate. Similarly, both aplip1 alleles exhibited enlarged axonal BRP/RBP accumulations. Thus, the capture/dissociation balance for AZ components might be shifted towards 'capture'; in these mutants, consistent with the ectopic axonal T-bar formation. It is tempting to speculate that loss of Aplip1-dependent scaffolding and/or kinesin binding provokes the exposure of critical 'sticky'; patches of scaffold components such as RBP and BRP. Such opening of interaction surfaces might increase 'premature'; interactions of cargo proteins actually destined for AZ assembly, thus increase overall size of the cargo complexes by oligomerization between AZ proteins and, finally, promote premature capture and ultimately ectopic AZ-like assembly. On the other hand, the need for the system to unload the AZ cargo at places of physiological assembly (i.e., presynaptic AZ) might pose a limit to the 'wrapping'; of AZ components and ask for a fine-tuned capture/dissociation balance (Siebert, 2015).
Several mechanisms for motor/cargo separation such as (1) conformational changes induced by guanosine-5′-triphosphate hydrolysis, (2) posttranslational modification as de/phosphorylation, or (3) acetylation affecting motor-tubulin affinity, have been suggested for cargo unloading. Notably, Aplip1 also functions as a scaffold for JNK pathway kinases, whose activity causes motor-cargo dissociation. JNK probably converges with a mitogen-activated protein kinase (MAPK) cascade (MAPK kinase kinase Wallenda phosphorylating MAPK kinase Hemipterous) in the phosphorylation of Aplip1, thereby dissociating Aplip1 from KLC. Thus, JNK signaling, co-ordinated by the Aplip1 scaffold, provides an attractive candidate mechanism for local unloading of SVs and, as shown in this study, AZ cargo at synaptic boutons. This study further emphasizes the role of the Aplip1 adaptor, whose direct scaffolding role through binding AZ proteins might well be integrated with upstream controls via JNK and MAP kinases. Intravital imaging in combination with genetics of newly assembling NMJ synapses should be ideally suited to further dissect the obviously delicate interplay between local cues mediating capturing and axonal transport with motor-cargo dissociation (Siebert, 2015).
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).
Ultrafast neurotransmitter release requires tight colocalization of voltage-gated Ca2+ channels with primed, release-ready synaptic vesicles at the presynaptic active zone. RIM-binding proteins (RIM-BPs) are multidomain active zone proteins that bind to RIMs and to Ca2+ channels. In Drosophila, deletion of RIM-BPs dramatically reduces neurotransmitter release, but little is known about RIM-BP function in mammalian synapses. This study generated double conditional knockout mice for RIM-BP1 and RIM-BP2, and analyzed RIM-BP-deficient synapses in cultured hippocampal neurons and the calyx of Held. Surprisingly, it was found that in murine synapses, RIM-BPs are not essential for neurotransmitter release as such, but are selectively required for high-fidelity coupling of action potential-induced Ca2+ influx to Ca2+-stimulated synaptic vesicle exocytosis. Deletion of RIM-BPs decelerated action-potential-triggered neurotransmitter release and rendered it unreliable, thereby impairing the fidelity of synaptic transmission. Thus, RIM-BPs ensure optimal organization of the machinery for fast release in mammalian synapses without being a central component of the machinery itself (Acuna, 2015).
Synaptic transmission is remarkably fast. At physiological temperatures, a presynaptic action potential elicits a postsynaptic response in less than a millisecond, which means that neurotransmitter release must proceed in only a few hundred microseconds! To achieve such speed, nerve terminals efficiently couple Ca2+ entry to Ca2+-triggered vesicle fusion at the active zone by precisely localizing Ca2+ channels adjacent to release-ready synaptic vesicles. The present study demonstrated that active zone proteins called RIM-BPs are required for precise coupling of Ca2+ entry to Ca2+-triggered vesicle fusion at murine synapses, and thereby are essential for faithful transmission of presynaptic information to postsynaptic neurons (Acuna, 2015).
This study systematically dissected the functions of RIM-BPs by generating constitutive and conditional KO mice for RIM-BP1 and RIM-BP2, the only RIM-BPs expressed at significant levels at murine synapses. In constitutive double KO mice, deletion of RIM-BPs produced a modest but significant impairment in survival without major changes in brain structure or composition. Morphological, ultrastructural, and electrophysiological studies were performed on conditional double KO mice in two preparations, synapses of hippocampal neurons in culture and synapses of the calyx of Held synapse in acute slices. In these synapses, RIM-BPs are selectively essential for reliable triggering of release by an action potential. Based on detailed measurements of synaptic transmission in calyx synapses, this phenotype is most plausibly explained by a loss of Ca2+ channel colocalization with release-ready, docked, and primed synaptic vesicles (Acuna, 2015).
Recent studies using immunogold EM revealed that Ca2+ channels are distributed nonrandomly in the active zone and colocalize with RIMs which in turn are attached to vesicles. The data suggest that the RIM-BP1,2 DKO phenotype impairs this colocalization and clustering of Ca2+ channels with vesicles. Thus, it is proposed that, consistent with the known biochemical interactions of RIM-BPs with RIMs and Ca2+ channels, RIM-BPs perform a discrete function in mammalian brain to control the speed and precision of Ca2+-dependent synaptic vesicle fusion by coupling Ca2+ channels to synaptic vesicles. An alternative interpretation of the data is that the RIM-BP deletion dramatically increases the Ca2+-buffering capacity of presynaptic terminals. However, how a deletion of an active zone protein would cause recruitment of a large number of Ca2+ buffers to the terminals is difficult to imagine; moreover, such an increase in Ca2+-buffering capacity would have produced larger changes in the amplitude of release. Thus, this interpretation is felt to be highly unlikely (Acuna, 2015).
The evidence for these conclusions can be summarized as follows: first, the quantitatively most significant phenotype in RIM-BP-deficient hippocampal and calyx synapses consisted of a dramatic increase in the variability of action potential-triggered release, consistent with an increase in stochastic release failures due to a larger distance of Ca2+-channels to release sites. Measurements of precise release parameters in cultured neurons were enabled by the development of channelrhodopsin-assisted paired recordings, which may be generally useful for analysis of cultured neurons but are still less accurate than paired recordings in calyx synapses. Second, RIM-BP1,2-deficient calyx synapses exhibited a significant decrease in the amount of action potential-induced release and the release probability, again consistent with an increased distance of Ca2+ channels to release sites. Only a trend toward decreased release in hippocampal synapses was detected, possibly because of the relatively lower resolution of electrophysiological measurements in hippocampal synapses and/or because the overall phenotype of the RIM-BP1,2 DKO is smaller in cultured hippocampal neurons than in calyx synapses. Third, the RIM-BP1,2 DKO did not decrease the RRP size or the rate of priming of vesicles into the RRP, but strongly decelerated release triggered from the RRP. Fourth, the RIM-BP1,2 DKO did not alter Ca2+ current amplitudes or kinetics both in vivo and in vitro. Fifth, release at RIM-BP1,2-deficient synapses was significantly more sensitive to the slow Ca2+ buffer EGTA than release at control synapses, suggesting that the average residence time of Ca2+ in the terminal before triggering release is longer. Sixth, measurements of the reliability with which a presynaptic action potential triggers a postsynaptic action potential in calyx synapses demonstrated a major decrease in transmission fidelity during high-frequency spike trains in RIM-BP1,2-deficient synapses (Acuna, 2015).
Mechanistically, RIM-BPs likely tether Ca2+ channels to the active zone in collaboration with RIMs, another essential component of the presynaptic active zone. Like RIM-BPs, RIM proteins directly bind to Ca2+ channels, although different from RIM-BPs, RIM proteins only bind to N- and P/Q-type but not to L-type Ca2+ channels. Interestingly, RIM- and RIM-BP-deficient synapses differ significantly in their Ca2+-influx phenotype. RIM-deficient synapses exhibit a physical loss of Ca2 channels from presynaptic terminals and a decrease in overall presynaptic Ca2+ influx, whereas RIM-BP1,2-deficient synapses display no change in presynaptic Ca2+ influx. This phenotypic difference suggests that RIMs perform a more central function in recruiting Ca2+ channels to active zones, and may perform such a function via multiple mechanisms, including but not restricted to tethering of RIM-BPs to the active zone (Acuna, 2015).
The current results are broadly consistent with previous studies. RIM-BPs are known to biochemically bind to both L-type and N- and P/Q-type Ca2+ channels, and are coimmunoprecipitated with N- and P/Q-type Ca2+ channels from brain. Rescue of the impairment in presynaptic Ca2+ influx in RIM-deficient hippocampal neurons requires the RIM-BP-binding sequence of RIMs. This rescue experiment supported the conclusion that RIM-BP binding to RIMs may be important for localizing Ca2+ channels to active zones, but did not actually show that RIMs operate by binding RIM-BPs, since other SH3-domain-containing proteins may have also bound. In Drosophila neuromuscular junctions, deletion of RIM-BP produces a major phenotype that, consistent with the current data, includes a looser coupling of vesicles to Ca2+ influx. However, the effect of the RIM-BP deletion in Drosophila is much more severe than the phenotype observed in murine synapses. Specifically, deletion of Drosophila RIM-BP abolishes the presynaptic T-bars that are characteristic of active zones in Drosophila neuromuscular junctions, mislocalizes the protein Bruchpilot that is specific for these T-bars, and causes major changes in most parameters of neurotransmitter release (Liu, 2011, Müller, 2015). Comparing the relative phenotypes of RIM and RIM-BP mutations in mouse and Drosophila synapses suggests that in mammals, RIM proteins are functionally dominant, whereas RIM-BPs play a more ancillary role. In Drosophila, by contrast, RIM-BPs appear to be central to the organization of the active zone, whereas RIMs may perform a lesser role (Liu, 2011, Graf, 2012, Müller et al., 2012, Müller et al., 2015). This interesting evolutionary difference may be related to the emergence in Drosophila of Bruchpilot, which evolved from the active zone protein ELKS and is specific to arthropods. C. elegans which lacks Bruchpilot but retains an ELKS homolog similar to mammals seems to exhibit a similarly severe RIM loss-of-function phenotype as mammals. Viewed together, these studies thus suggest that RIM and RIM-BPs may act in partly overlapping functions with distinct degrees of redundancy in different organisms, raising the possibility that elimination of both RIMs and RIM-BPs may provide a more decisive manipulation than deletion of either protein family alone (Acuna, 2015).
Ca2+ channel tethering by RIM-BPs has important functional consequences for the reliability and timing of synaptic transmission and thereby for the input/output relations of neural circuits. A deceleration of release and a decrease in the reliability of release during high-frequency bursts of action potential trains, as caused by deletion of RIM-BPs, would render vulnerable circuits that include such bursts as a regular coding feature. Notably, RIM-BP1 mutations were associated with autism-spectrum disorder and schizophrenia, raising the possibility that brain dysfunction in patients with such diseases might be, at least in part, due to abnormal timing of information transfer at relevant brain circuits. Further studies should aim to determine if RIM-BP deletions lead to behavioral abnormalities related to these brain disorders, and whether this is linked to synaptic dysfunctions in specific brain regions such as the cerebellum or striatum. With the availability of RIM-BP double KO mice, these important questions can now be addressed (Acuna, 2015).
Voltage-dependent Ca2+ channels (CaVs) represent the principal source of Ca2+ ions that trigger evoked neurotransmitter release from presynaptic boutons. Ca2+ influx is mediated mainly via CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels, which differ in their properties. Their relative contribution to synaptic transmission changes during development and tunes neurotransmission during synaptic plasticity. The mechanism of differential recruitment of CaV2.1 and CaV2.2 to release sites is largely unknown. This study shows that the presynaptic scaffolding protein Bassoon localizes specifically CaV2.1 to active zones via molecular interaction with the RIM-binding proteins (RBPs). A genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca2+ current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. These data establish Bassoon as a major regulator of the molecular composition of the presynaptic neurotransmitter release sites (Davydova, 2014).
Synaptic transmission consists of fast and slow components of neurotransmitter release. This study shows that these components are mediated by distinct exocytic proteins. The Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM (a protein associated with calcium channels), anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13's spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release (Hu, 2013).
The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca2+ entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. This study shows that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone (Zhou, 2013).
RIM-binding proteins (RIM-BPs) were identified as binding partners of the presynaptic active zone proteins RIMs as well as for voltage-gated Ca2+-channels. They were suggested to form a functional link between the synaptic-vesicle fusion apparatus and Ca2+-channels. This study shows that the RIM-BP gene family diversified in different stages during evolution, but retained their unique domain structure. While invertebrate genomes contain one, and vertebrates include at least two RIM-BPs, an additional gene, RIM-BP3, has been identified that is exclusively expressed in mammals. RIM-BP3 is encoded by a single exon of which three copies are present in the human genome. All RIM-BP genes encode proteins with three SH3-domains and two to three fibronectin III repeats. The flanking regions diverge in size and sequence and are alternatively spliced in RIM-BP1 and -2. Quantitative real-time RT-PCR and in situ hybridization analyses revealed overlapping but distinct expression patterns throughout the brain for RIM-BP1 and -2, while RIM-BP3 was detected at high levels outside the nervous system. The modular domain structure of RIM-BPs, their expression pattern and the conservative expansion during evolution shown in this study support their potential role as important molecular adaptors (Mittelstaedt, 2007).
Search PubMed for articles about Drosophila
Acuna, C., Liu, X., Gonzalez, A. and Sudhof, T. C. (2015). RIM-BPs Mediate Tight Coupling of Action Potentials to Ca2+-Triggered Neurotransmitter Release. Neuron 87: 1234-1247. PubMed ID: 26402606
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
Davydova, D., Marini, C., King, C., Klueva, J., Bischof, F., Romorini, S., Montenegro-Venegas, C., Heine, M., Schneider, R., Schroder, M. S., Altrock, W. D., Henneberger, C., Rusakov, D. A., Gundelfinger, E. D. and Fejtova, A. (2014). Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein. Neuron 82: 181-194. PubMed ID: 24698275
Frank, C. A., Kennedy, M. J., Goold, C. P., Marek, K. W. and Davis, G. W. (2006). Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis. Neuron 52: 663-677. PubMed ID: 17114050.
Graf, E. R., Valakh, V., Wright, C. M., Wu, C., Liu, Z., Zhang, Y. Q. and DiAntonio, A. (2012). RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction. J Neurosci 32: 16586-16596. PubMed ID: 23175814
Hallermann, S., Kittel, R. J., Wichmann, C., Weyhersmuller, A., Fouquet, W., Mertel, S., Owald, D., Eimer, S., Depner, H., Schwarzel, M., Sigrist, S. J. and Heckmann, M. (2010). Naked dense bodies provoke depression. J Neurosci 30: 14340-14345. PubMed ID: 20980589
Hibino, H., Pironkova, R., Onwumere, O., Vologodskaia, M., Hudspeth, A. J. and Lesage, F. (2002). RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca(2+) channels. Neuron 34: 411-423. PubMed ID: 11988172
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
Kaeser, P. S., Deng, L., Wang, Y., Dulubova, I., Liu, X., Rizo, J. and Sudhof, T. C. (2011). RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144: 282-295. PubMed ID: 21241895
Liu, K. S., Siebert, M., Mertel, S., Knoche, E., Wegener, S., Wichmann, C., Matkovic, T., Muhammad, K., Depner, H., Mettke, C., Buckers, J., Hell, S. W., Muller, M., Davis, G. W., Schmitz, D. and Sigrist, S. J. (2011). RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334: 1565-1569. PubMed ID: 22174254
Mittelstaedt, T. and Schoch, S. (2007). Structure and evolution of RIM-BP genes: identification of a novel family member. Gene 403: 70-79. PubMed ID: 17855024
Müller, M., Pym, E. C., Tong, A. and Davis, G. W. (2011). Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release. Neuron 69: 749-762. PubMed ID: 21338884
Müller, M., Genc, O. and Davis, G. W. (2015). RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles. Neuron 85: 1056-1069. PubMed ID: 25704950
Siebert, M., et al. (2015). A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones. Elife 4 [Epub ahead of print]. PubMed ID: 26274777
Spangler, S. A. and Hoogenraad, C. C. (2007). Liprin-alpha proteins: scaffold molecules for synapse maturation. Biochem Soc Trans 35: 1278-1282. PubMed ID: 17956329
Weyhersmüller, A., Hallermann, S., Wagner, N. and Eilers, J. (2011). Rapid active zone remodeling during synaptic plasticity. J Neurosci 31: 6041-6052. PubMed ID: 21508229
Younger, M. A., Muller, M., Tong, A., Pym, E. C. and Davis, G. W. (2013). A presynaptic ENaC channel drives homeostatic plasticity. Neuron 79: 1183-1196. PubMed ID: 23973209
Zhou, K., Stawicki, T. M., Goncharov, A. and Jin, Y. (2013). Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics. Elife 2: e01180. PubMed ID: 24220508
date revised: 20 October 2016
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