InteractiveFly: GeneBrief

RIM-binding protein: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

RIM-binding protein couples synaptic vesicle recruitment to release site

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. This study shows in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites (Petzoldt, 2020).

Chemical synapses are the fundamental building blocks of neuronal communication, allowing for a fast and directional exchange of chemical signals between a neurotransmitter releasing presynaptic and receiving postsynaptic target cells. To couple synaptic vesicle (SV) release to electrical stimulation by action potentials, Ca2+ ions entering the cell through voltage-gated Ca2+ channels activate the Ca2+ sensor synaptotagmin that is anchored on SVs to trigger fusion events. The presynaptic site of SV fusion ('active zone' [AZ]) is covered by an electron-dense scaffold ('cytomatrix') formed by a set of large conserved multidomain proteins. How individual AZ scaffold proteins intersect mechanistically with the SV cycle still remains largely enigmatic, though such knowledge was of importance to properly model synapse function in healthy and diseased circuits (Petzoldt, 2020).

On the level of the individual AZs, recent data from both cultivated rodent neurons and Drosophila in vivo neurons suggest that the AZ cytomatrix provides stable SV release sites or 'fusion slots' via the clustering of the critical release factor (m)Unc13. The number of such release sites seems to be determined independently of the mechanisms controlling release probability. How SV release sites might be coupled and integrated with additional processes organized by the AZ scaffold, such as the recruitment of SVs under high demand periods of resupply (e.g., high action potential frequencies), remains a largely open question (Petzoldt, 2020).

The physically extended ELKS family protein (glutamic acid [E], leucine [L], lysine [K],and serine [S]-rich protein) Bruchpilot (BRP) not only operates as the fundamental building block of the Drosophila AZ scaffold but was also shown to promote SV recruitment depending on a binding motif at its extreme, AZ membrane-distal C terminus. Adaptor proteins physically and functionally connecting such extended scaffold proteins, which sample the cytoplasm, and the SV release sites at the AZ membrane might well be relevant in this regard. Rab3-interacting molecule (RIM)-binding proteins (RIM-BPs) are an evolutionarily conserved family of extended AZ scaffold proteins, obviously critical for SV release at Drosophila and mammalian central synapses. RIM-BP family proteins with the C-terminal SH3-II/III domains bind to Ca2+ channels, release factor Unc13A, and RIM, another critical AZ scaffold protein. At Drosophila neuromuscular junction (NMJ) synapses, rim-bp null alleles provoke a most severe functional phenotype, stronger than for rim and rim-bp single mutants at most mammalian synapses. This study exploits the severity of the synaptic Drosophila rim-bp phenotype for a detailed structure-function analysis at Drosophila larval neuromuscular (NMJ) synapses. Elimination of the individual RIM-BP SH3 domains affected transport to AZs severely; however, the low levels of properly locating RIM-BP-ΔSH3 variants were still able to rescue the 'nanoscopic' defects of rim-bp null mutants AZ scaffolds. Crystallographic analysis of the RIM-BP fibronectin III-like (FN-III) domain cluster describes an extended 'hinge' in the protein center, which likely plays conformational roles. Finally, the N-terminal region (NTR) of RIM-BP was required to properly organize the overall nanoscopic architecture of the AZ scaffold. While SV docking and numbers of (m)Unc13 clusters remained at normal levels, the NTR was specifically needed for efficient SV recruitment. Thus, it is suggested that RIM-BP family proteins evolved as adaptors physically and functionally connecting SV release sites with BRP/ELKS-dependent SV recruitment processes (Petzoldt, 2020).

The current view of AZ scaffolds emphasizes their role in providing a 'smart catalytic surface' integrating sub-functionalities that facilitate and control the SV cycle. Providing SV release sites with a proteinaceous 'nano-environment' defining release probability, by precisely defining their spatial distance to voltage-operated Ca2+ channels, is probably one fundamental function using these highly conserved, 'ancient' protein architectures. The SV release sites probably also have to be coupled to processes retrieving SVs from more membrane-distant positions. Concerning protein architectures harvesting SVs, evolutionary solutions might have been somewhat more flexible, obviously adopted to the synapse-type specific needs in physiologically relevant fusion rates. Identifying proteins that might be involved in structurally and functionally coupling recruitment protein architectures with release sites is an emerging topic. This study provides evidence that RIM-BP with its interactions obviously plays a role in both 'nanoscopic locations.' First, through its SH3 domains, it binds RIM, the intercellular C-terminus of the voltage-operated Ca2+ channel and also Unc13A, the release factor whose nanoscale positioning has been recently identified as critical for SV release definition (Reddy-Alla, 2017). Second, this study found that it also binds conserved regions of BRP in the core of the AZ central scaffold. Deleting this interaction surface, though not reducing AZ protein levels, obviously undermines the proper bundling of BRP filaments, at least for a major fraction of AZs. Though it is not easy to prove that individual RIM-BP protein molecules span this distance physically, it is tempting to hypothesize that RIM-BP might literally connect the recruitment of SVs from more membrane distant pools, probably starting at the distal end of the BRP filaments, with their integration into SV release sites (see RIM-BP NTR localizes to the center of the AZ to stabilize the BRP scaffold and promote SV recruitment) (Petzoldt, 2020).

At mammalian synapses, recent multi-loci genetics have demonstrated most severe deficits when eliminating combinations of AZ scaffold proteins between RIM, ELKS, and RIM-BP family members. In this study, quadruple knockouts of RIM1/2, together with RIM-BP1/2 proteins in mice, exhibit a total loss of neurotransmitter release from severe impairments in SV priming and docking, a dramatic loss of AZ scaffold density, with a trans-synaptic effect that impairs the organization of the postsynaptic density. These severe synthetic 'catastrophic' phenotypes, eliminating ultrastructural specializations and release factor targeting, demonstrate a principal functional redundancy between RIM-BPs, RIMs, and ELKS family proteins. Similar results were also retrieved from work on mice in which RIM1αβ and RIM2αβγ, together with ELKS1α and ELKS2α isoforms, have been completely eliminated. Cultured hippocampal synapses of these mutant mice consequently lose Munc13, Bassoon, Piccolo, and RIM-BP, following a mass disassembly of the AZ scaffold (Wang, 2016). Similarly, this study observed that RIM seemingly operates synergistically with RIM-BP in establishing the AZ nanoscopic architecture (Petzoldt, 2020).

Despite the obvious importance of these findings for understanding the collective role of the AZ scaffold, the fact that the domain organization for all specific scaffold proteins has been individually conserved over hundreds of millions of years motivated a search for ways to demonstrate specific functions of these AZ core scaffold proteins. This study has molecularly isolated an additional sub-functionality for the so-far functionally nonconsidered RIM-BP NTR, and provide evidence that its protein architecture might have been conserved for the reason that it operates as an adaptor coupling SV recruitment processes with the membrane-associated SV release sites (Petzoldt, 2020).

Mechanistic analysis of RIM-BPs so far has largely focused on SH3 domains II and III, which bind to Ca2+ channels and RIM in both mammals and Drosophila, and Unc13A in Drosophila. Analysis of RIM-BP at Drosophila NMJ synapses was the first to demonstrate a major role of the protein family in neurotransmission, characterized by a severe reduction in release probability and signs of defective Ca2+ channel clustering. This study has exploited the glutamatergic NMJ synapses for a stringent genetic analysis with multimodal readouts, also including an analysis of transport and nanoscale integration into the AZ scaffold. Deletion of individual SH3 domains majorly affected RIM-BP transport, probably directly reflecting their high affinity binding to JIP-1 homologue Aplip1, whose deletion also interferes with effective axonal transport of BRP/RIM-BP 'packages'. Somewhat surprisingly, however, moderately or even strongly reduced levels of RIM-BPΔSH3-II or RIM-BPΔSH3-III, respectively, still restored RIM-BP functionality when expressed in a null background. It obviously might be argued that only combined elimination of SH3-II together with SH3-III might uncover their function collectively. Indeed, Aplip1 binds both SH3-II and III, and AZ levels were decreased even more strongly in the RIM-BPΔSH3-II/III double-deleted variant. However, at least concerning the role of Ca2+ channel binding, the AZ scaffold can seemingly compensate for the absence of RIM-BP-SH3 mediated interactions, as Ca2+ channels with the highly conserved PXXP RIM-BP binding motif deleted still retained full activity in a genetic rescue assay (instead of resembling the rim-bp null phenotype). It is suggested that redundant interactions stabilize Ca2+ channel: AZ scaffold contacts at NMJ synapses, an idea that might be relevant for other synapses as well. Indeed, it was shown previously that the N terminus of BRP also binds the Ca2+ channel α1 subunit intracellular C terminus. Given the essential character of SH3-III for survival, however, it appears likely that slightly different rules and 'vulnerabilities' apply to other synapse types, for example, in the Drosophila brain. In fact, it was found recently that synapses in the Drosophila brain differ substantially in their BRP content, with, for example, interneuron synapses largely lacking BRP (Fulterer, 2018). This might result in different efficacy of compensation via BRP and be responsible for the essential character of SH3-III. Put differently, it still appears plausible that the C-terminal close SH3 domains might be of truly essential character dependent on synapse type given the results retrieved at mammalian synapses (Petzoldt, 2020).

Finally, the presence of three FN-III domains, despite their invariable character, remained and remains an enigma. FN-III modules are common in extracellular but rarer in intracellular proteins, such as RIM-BP, here found chiefly as the main components of a group of intracellular proteins associated with the contractile apparatus of muscles. The first x-ray structure of the RIM-BPs, which is presented in this study, depicts a typical FN-III-type organization; however, it is oriented in an extended hinge-like arrangement. It is tempting to speculate that the hinge-like architecture might support RIM-BP conformations or conformational change when connecting release sites with recruitment processes (Petzoldt, 2020).

Physiological analysis points toward a discrete function of the RIM-BP N-terminal domain. Analysis of SV recruitment based on the rim-bp null allele is complicated due to the severe release probability deficits dominating the physiological scenario. Nonetheless, a careful analysis, using high Ca2+ concentrations to milden the influence of release probability differences, indeed identified recruitment defects. Remarkably, similar to the NTR-specific deletions, a previous study analyzing the rim-bp null mutant for RIM-BP suggested a rate-limiting function for the replenishment of high release probability SVs following vesicle depletion at NMJ synapses. Moreover, in mouse rim-bp2 knockouts, recruitment/replenishment deficits at auditory hair cells were reported. In rim-bp null mutants, UNC13A levels are strongly reduced, while for the dNTR-2 construct, UNC13A levels and consequently release site number were not reduced (instead rather slightly increased). Thus, the data apparently uncouple the role of RIM-BP for release site organization (via Unc13A clustering) from its role in overall scaffold organization where the NTR plays an important role (Petzoldt, 2020).

The extended BRP filaments in Drosophila probably operate as 'antennae' to harvest SVs from the reserve pool, a process facilitated by the C-terminal amino acids of BRP, made evident by the defects of the brp nude allele lacking only the last 17 amino acids. BRP in its first half is highly homologous to ELKS/CAZ-associated structural proteins (CAST) AZ proteins; however, its C-terminal half is specific for insects and obviously evolved for harvesting SVs. The large vertebrate-specific AZ protein Bassoon was shown to help SV replenishment at the central synapse under conditions of heavy stimulation. Notably, Bassoon binds to the first SH3 domain of RIM-BP, suggestive of convergent evolution where different molecular antennae were used to finally target RIM-BP, the generic 'old' adaptor coupling to the membrane close release sites (Petzoldt, 2020).

In its current form, static STED microscopy as used here only samples average epitope distributions, while the relevant proteins likely are dynamically switched in the course of the SV cycle. As an indication of this, the RIM-BP C-terminal SH3 domains were found to bind both the Ca2+ channel C-terminus and Unc13A N terminus. As these binding events can hardly be accomplished by a single Unc13A molecule at a single time point, future analysis will have to address the underlying conformational dynamics likely involved here (Petzoldt, 2020).

Taken together, RIM-BPs might take a generic role of SV replenishment, apart from their obvious role in coclustering of release machinery and Ca2+ channels at the AZ membrane. Indeed, the highly conserved domain architecture might have evolved for exactly this reason, to guide SVs into the proper release environment (Petzoldt, 2020).

RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release

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 Cac^GFP (Liu, 2011).

The drbp locus was subjected to genetic analysis. A strain with a transposon inserted between exons 6 and 7 (MB02027, drbp^Minos) 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 (drbp^STOP1, drbp^STOP2, and drbp^STOP3). 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 drbp^STOP1-3 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 (drbp^Minos/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 drpb^STOP1 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 drbp^STOP1, 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, drbp^STOP1 synapses exhibited an unusually strong facilitation. During a 10-Hz stimulation train, a protocol that leads to marked depression of eEJCs in controls, drbp^STOP1 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, drbp^STOP1 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 Cac^GFP, 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 Cac^GFP intensity per postsynaptic density (PSD) yielded a slightly stronger 36% decrease in drbp^STOP1 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 drbp^STOP1 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).

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

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 H_abc 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).

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).

Vesicle tethering on the surface of phase-separated active zone condensates from rat brains

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. This study discovered that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca(2+) channels anchored on membranes. Thus, this study has reconstituted a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates (see Drosophila Synapsin). The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy (Wu, 2020).

Active zones in presynaptic terminal boutons of synapses are intimately involved in neurotransmitter release, because active zones are responsible for tethering and docking synaptic vesicles (SVs) to be near release sites at plasma membranes, priming SVs for action potential-evoked fusion and clustering voltage-gated Ca2+channels (VGCCs) for precise spatiotemporal control of Ca2+level at the release sites. Electron microscopy and super-resolution optical imaging experiments have shown that active zone proteins, including RIM, RIM-BP, ELKS, Munc13, and Liprin, are located within a narrow space of <100 nm from the presynaptic terminal membranes. Biochemically, these active zone proteins are all multi-domain scaffold proteins without intrinsic enzymatic activities, and these proteins can form an intricate molecular network by specific protein-protein interactions, suggesting that active zones are large and dense protein-based molecular assemblies. Such active zone assemblies were proposed to be anchored to presynaptic plasma membranes by directly binding to lipids or transmembrane proteins, such as LAR and VGCCs, but none of them alone has been proved to be essential. Genetic and cell biology studies have shown that these proteins function together and in redundant ways in organizing active zones and in modulating neurotransmitter releases. For example, removal of each active zone component in rodents individually showed little impact on the active zone structures and vesicle tethering to active zones, except that removal of RIM led to 50% reduction of tethered vesicles. Combined removals of RIM with RIM-BP or with ELKS led to disintegration of active zone molecular assemblies, near-total loss of vesicle tethering and docking, and dramatic impairments of synaptic release. It had been recently shown that RIM and RIM-BP, when mixed in vitro, can autonomously form condensed molecular assembly via phase separation. The RIM/RIM-BP condensates can cluster the cytosolic tail of VGCC (VGCC-CT) tethered to lipid membranes, forming highly concentrated RIM/RIM-BP/VGCC-CT assembly on the surface of membranes. The formation of condensed RIM/RIM-BP/VGCC-CT on the surface of lipid membranes via phase separation provides an explanation to how condensed active zone protein assemblies, which are not enclosed by any physical barriers, can form at presynaptic boutons. The highly dynamic nature of the RIM/RIM-BP/VGCC-CT condensates may also explain why the dense projection structures in active zones can be observed by electron microscopy (EM) only when synapses are chemically fixed. The presence of dense RIM clusters within the active zone has also been observed in living neurons. Phase separation-mediated formation of condensed molecular assemblies has also been suggested to form the postsynaptic density and for AMPA receptor synaptic clustering and transmission (Zeng, 2019). Formation of condensed molecular assemblies via phase separation is likely to be advantageous for synapse formation and plasticity, although such research is in its early stage (Wu, 2020).

Although tethering of SVs to release sites of presynaptic membranes requires the active zone, how SVs are physically tethered to active zones is not known. Recently, Milovanovic (2018) showed that lipid vesicles can be clustered by coacervation with the synapsin condensates, providing a mechanism for maintaining large reserve pool SVs away from active zones. However, numerous EM studies have shown that tethered SVs are discretely distributed within active zone sheets and the number of tethered SVs is linearly proportional to the areas of active zone sheets. In EM images of chemically fixed synapses, SVs are evenly separated by the dense projections, which are grid-like structures on active zone sheets. In addition, perturbation of synapsin affects only the reserve pool SVs, and the tethered pool remains unchanged. Conversely, disruption of active zone scaffolds impairs only the tethered pool SVs without changing the reserve pool. Thus, tethering of SVs to the active zones is likely mediated by mechanisms that are very different from that seen in the reserve pool SV clustering by synapsin. This study demonstrates that negatively charged liposomes, as well as SVs purified from rat brains, coat on the surface of the phase-separated RIM/RIM-BP condensates. By tethering VGCC-CT to the giant unilamellar vesicle (GUV) membranes, this study shows that the RIM/RIM-BP condensates can bind to and cluster VGCC-CT on GUV membranes on one hand and link SUVs on the other hand, resembling active zone-mediated tethering of SVs to presynaptic membrane release sites. Remarkably, the synapsin-clustered SUV condensates and the SUV-coated RIM/RIM-BP condensates form phase-in-phase assemblies, which recapitulate coexistence of reserved and tethered pools of SVs in presynaptic boutons. The finding that the RIM/RIM-BP condensates can simultaneously link two totally different types of membrane-based cellular organelles suggests a wide range of unanticipated cellular roles of membraneless biological condensates formed by phase separation (Wu, 2020).

Tethered SVs are typically defined by their physical distance(30 nm or less) to the presynaptic plasma membranes. Decades of past research have identified molecules, including RIM, RIM-BP,ELKS, and Munc13, that are critical for SVs to tether and dock to presynaptic membranes. However, how SVs are retained at the active zone remains elusive. This study discovered that SVs coat on the surface of condensed droplets formed by the active zone proteins, including RIM, RIM-BP, and ELKS, via phase separation. This finding may explain several observations made in past decades of studies. First, the number of tethered SVs is proportional to the surface area of the active zone. Second, SVs coated on the surface of RIM/RIM-BP condensates are highly dynamic but are very close to both fusion machineries and presynaptic plasma membranes; thus, such active zone tethered SVs are primed for rapid fusion. Third, the RIM/RIM-BP condensates can cluster Ca2+ channels on the membranes. The direct coating of SVs on the surface of the RIM/RIM-BP droplets can physically link clustered Ca2+ channels with SVs for Ca2+-regulated fusion reactions. Fourth, formation of the RIM/RIM-BP condensates can greatly enhance the weak interaction between RIM/RIM-BP and negatively charged lipids on SVs, and thus allow stable interaction between the active zone molecular assembly and SVs. Fifth, the binding of SVs to the liquid-like active zone may shape the active zone into grid-like structures observed by EM under chemical fixing conditions. The condensed droplets formed by proteins, including RIM, RIM-BP, and ELKS, are highly dense but still very dynamic. Chemical fixation may capture such dynamic but dense assemblies referred to as dense projections. The coating of SVs on the surfaces of active zones likely involves proteins in addition to RIM, RIM-BP, and ELKS examined in this study. Proteins such as Munc13, Munc18, Liprin, Piccolo, Bassoon,SNAREs, the Rab family small GTPases, synaptotagmins, and so on can modulate SV docking and priming. These proteins may also participate in the regulation of active zone assembly and SVs coating. It is noted that the SV-coated RIM/RIM-BP condensates are not physically insulated by SVs. Proteins can readily enter in or escape from the SV-coated active zone protein condensates. Thus, such SV-coated active zone protein condensates can connect with molecular components both near and distal to the presynaptic membranes (Wu, 2020).

Another major finding of this study is that interactions of SVs with different protein condensates formed via phase separation can be radically different. SVs co-phase separate with synapsin, leading to clustering and concentration of SVs. Given that synapsin is massively concentrated in presynaptic boutons, the protein is ideally suited for maintaining the vast majority of SVs as the reserve pool. Strikingly, SVs do not enter the condensed phase formed by the active zone proteins, but instead coat on the surface of the condensates. Remarkably, the synapsin/SUV condensates encapsulate the SUV-coated RIM/RIM-BP droplets when the two phase-separated condensates are mixed, suggesting that the synapsin-clustered reserve SV pool can co-exist with the active zone-tethered SVs, and the two pools of SVs can exchange with each other. Finally, this study has been able to reconstitute a minimalistic presynaptic bouton-like structure, in which the active zone-coated vesicles are attached to the presynaptic plasma membrane and the synapsin-clustered reserve pool SV condensates are situated more distal to the presynaptic plasma membrane but directly interact with the active zone condensates. This reconstituted system recapitulates the basic features of the SV clustering and tethering in presynaptic boutons. In real synapses, the presynaptic plasma membranes together with various membrane proteins likely serve as the starting layer structure to organize the subsequent active zone layer, tethered SVs, and synapsin-clustered reserve pool SVs, forming the elaborate and polarized multiphase organizations. It should be noted that the scale/size of the reconstituted, presynaptic bouton-like assemblies here is much larger than the sizes of real presynaptic boutons. The awkward size of pre-synaptic boutons has presented challenges to investigate whether the layered structures observed in EM studies are indeed formed via the multiphase organization seen in this in vitro study. New technologies will need to be developed to answer this question in the future (Wu, 2020).

In a broader cell biology context, distinct mode of interactions between membraneless organelles and various membrane demarcated organelles, similar to what is observed in this study, may be broadly adopted by cells to modulate processes such as organelle biogenesis, vesicle formation and trafficking, autophagy, and membrane-associated signaling assembly formation (Wu, 2020).

RIM-BPs Mediate Tight Coupling of Action Potentials to Ca²⁺-Triggered Neurotransmitter Release

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).

Bassoon specifically controls presynaptic P/Q-type Ca²⁺ channels via RIM-binding protein

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).

UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans

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).

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics

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).

Structure and evolution of RIM-BP genes: identification of a novel family member

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 Rim-binding protein

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.

Fulterer, A., Andlauer, T. F. M., Ender, A., Maglione, M., Eyring, K., Woitkuhn, J., Lehmann, M., Matkovic-Rachid, T., Geiger, J. R. P., Walter, A. M., Nagel, K. I. and Sigrist, S. J. (2018). Active zone scaffold protein ratios tune functional diversity across brain synapses. Cell Rep 23(5): 1259-1274. PubMed ID: 29719243

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

Milovanovic, D., Wu, Y., Bian, X. and De Camilli, P. (2018). A liquid phase of synapsin and lipid vesicles. Science 361(6402): 604-607. PubMed ID: 29976799

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

Petzoldt, A. G., Gotz, T. W. B., Driller, J. H., Lutzkendorf, J., Reddy-Alla, S., Matkovic-Rachid, T., Liu, S., Knoche, E., Mertel, S., Ugorets, V., Lehmann, M., Ramesh, N., Beuschel, C. B., Kuropka, B., Freund, C., Stelzl, U., Loll, B., Liu, F., Wahl, M. C. and Sigrist, S. J. (2020). RIM-binding protein couples synaptic vesicle recruitment to release sites. J Cell Biol 219(7). PubMed ID: 32369542

Reddy-Alla, S., Bohme, M. A., Reynolds, E., Beis, C., Grasskamp, A. T., Mampell, M. M., Maglione, M., Jusyte, M., Rey, U., Babikir, H., McCarthy, A. W., Quentin, C., Matkovic, T., Bergeron, D. D., Mushtaq, Z., Gottfert, F., Owald, D., Mielke, T., Hell, S. W., Sigrist, S. J. and Walter, A. M. (2017). Stable positioning of Unc13 restricts synaptic vesicle fusion to defined release sites to promote synchronous neurotransmission. Neuron 95(6): 1350-1364 e1312. PubMed ID: 28867551

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

Wang, S. S. H., Held, R. G., Wong, M. Y., Liu, C., Karakhanyan, A. and Kaeser, P. S. (2016). Fusion competent synaptic vesicles persist upon active zone disruption and loss of vesicle docking. Neuron 91(4): 777-791. PubMed ID: 27537483

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

Wu, X., Ganzella, M., Zhou, J., Zhu, S., Jahn, R. and Zhang, M. (2020). Vesicle tethering on the surface of phase-separated active zone condensates. Mol Cell. PubMed ID: 33202250

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

Zeng, M., Diaz-Alonso, J., Ye, F., Chen, X., Xu, J., Ji, Z., Nicoll, R. A. and Zhang, M. (2019). Phase separation-mediated TARP/MAGUK complex condensation and AMPA receptor synaptic transmission. Neuron 104(3): 529-543 e526. PubMed ID: 31492534

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: 10 July 2021

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