InteractiveFly: GeneBrief

Rab3 interacting molecule: Biological Overview | References

BIOLOGICAL OVERVIEW

The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. Drosophila Fife, a Piccolo-RIM homolog. has been shown to regulate neurotransmission and motor behavior through an unknown mechanism. This study demonstrates that Fife localizes and interacts with RIM (Rab3 interacting molecule) at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, a decrease was found in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, Fife was found to be essential for the homeostatic modulation of neurotransmission. It is proposed that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release (Bruckner 2016).

The strength of synaptic transmission is a critical determinant of information processing in neural circuits. Evoked neurotransmission depends on localized Ca2+ influx triggering neurotransmitter release from synaptic vesicles at specialized domains of presynaptic terminals called active zones. At the active zone membrane, synaptic vesicles are docked and molecularly primed to respond to a rise in Ca2+ concentration by fusing with the membrane to release neurotransmitter. A conserved complex of active zone-associated proteins makes up the active zone cytomatrix. In Drosophila melanogaster, these proteins include the ELKS family protein Bruchpilot, Rab3-interacting molecule (RIM), RIM-binding protein, Unc13, and Fife. Active zone cytomatrix proteins contain many lipid- and protein-binding domains that mediate diverse interactions with key players in synaptic transmission, leading to the model that the active zone cytomatrix spatially organizes presynaptic terminals for the millisecond coupling of neurotransmitter release to action potentials (Bruckner, 2016).

The specific neurotransmitter release properties of an active zone are determined by two key parameters acting in concert: (1) the number of synaptic vesicles docked at the membrane and molecularly primed for Ca2+-triggered release, termed the readily releasable pool, and (2) the release probability of these vesicles. Vesicle release probability is established by multiple parameters, including Ca2+ channel levels, localization and function at active zones, the spatial coupling of Ca2+ channels and release-ready vesicles, and the intrinsic Ca2+ sensitivity of individual vesicles. The observation that the presynaptic parameters of synaptic strength vary significantly even between the synapses of an individual neuron indicates that neurotransmitter release properties are determined locally at active zones and raises the question of how this complex regulation is achieved. Genetic studies in Drosophila, Caenorhabditis elegans, and mice are revealing a key role for the active zone cytomatrix in determining the functional parameters underlying synaptic strength. A mechanistic understanding of how the active zone cytomatrix achieves local control of synaptic release properties will yield fundamental insights into neural circuit function (Bruckner, 2016).

Fife, a Piccolo-RIM-related protein is required for proper neurotransmitter release and motor behavior. This study demonstrates that Fife localizes to the active zone cytomatrix, where it interacts with RIM to promote neurotransmitter release. The active zone cytomatrix is diminished and molecularly disorganized at Fife mutant synapses, and Fife is critical for vesicle docking at the active zone membrane. Not only are the number of release-ready vesicles reduced in the absence of Fife, but their probability of release is also significantly impaired because of disrupted coupling to calcium channels. These results suggest that Fife promotes high-probability neurotransmitter release by organizing the active zone cytomatrix to create vesicle release sites in nanometer proximity to clustered Ca2+ channels. Finally, it was found that in addition to its role in determining baseline synaptic strength, Fife plays an essential role in presynaptic homeostatic plasticity. Together, these findings provide mechanistic insight into how synaptic strength is established and modified to tune communication in neural circuits (Bruckner, 2016).

This study demonstrates that Fife plays a key role in organizing presynaptic terminals to determine synaptic release properties. Fife functions with RIM at the active zone cytomatrix to promote neurotransmitter release. Functional and ultrastructural imaging studies demonstrate that Fife regulates the docking of release-ready synaptic vesicles and, through nanodomain coupling to Ca2+ channels, their high probability of release. It was further found that Fife is required for the homeostatic increase in neurotransmitter release that maintains circuit function when postsynaptic receptors are disrupted. These findings uncover Fife's role as a local determinant of synaptic strength and add to understanding of how precise communication in neural circuits is established and modulated (Bruckner, 2016).

The finding that Fife interacts with RIM provides insight into how Fife functions within the network of cytomatrix proteins. RIM is a central active zone protein that was recently shown to facilitate vesicle priming at mammalian synapses by relieving autoinhibition of the priming factor Munc13. Like Fife, Drosophila RIM promotes Ca2+ channel accumulation at active zones and exhibits EGTA-sensitive neurotransmitter release at the Drosophila NMJ. This suggests that Fife and RIM may promote high-probability neurotransmitter release by acting together to dock and prime synaptic vesicles in close proximity to Ca2+ channels clustered at the cytomatrix. The findings are consistent with previous work in pancreatic β cells, where it was found that Piccolo and RIM2α form a complex that promotes insulin secretion through an unknown mechanism. To date, functional studies at mammalian synapses have focused on investigating interactions between Piccolo and Bassoon, which bind through their common coiled-coil regions. Thus, it will be of interest to investigate the functional relationship between Piccolo and RIM in promoting neurotransmitter release in mouse models. Piccolo also binds CAST1 through coiled-coil domain interactions. Although the Piccolo coiled-coil region is not present in Fife, future studies to determine whether this interaction is preserved through distinct interacting domains will be important, as Fife and Drosophila CAST-related Bruchpilot carry out overlapping functions. Similarly, although neither Fife nor Piccolo contains the conserved SH3-binding domain that mediates the interaction between RIM and RIM-binding protein, the overlap between Fife and RIM-binding protein phenotypes raises the possibility of functional interactions that will also be important to investigate in future experiments (Bruckner, 2016).

Significant alterations to active zone cytomatrix size and structure were observed in Fife mutants, whereas none have been detected in RIM mutants, indicating that Fife carries out this function independently of RIM. Previous ultrastructural analysis in aldehyde-fixed samples revealed occasional free-floating electron-dense structures that resemble active zone cytomatrix material and cluster synaptic vesicles. These unanchored electron-dense structures have also been observed at low frequency in Drosophila RIM-binding protein mutants, which, like Fife mutants, exhibit smaller active zone cytomatrices, and at higher frequency in rodent ribbon synapses lacking Bassoon. These structures were not visible in HPF/FS-prepared electron microscopy samples, likely because protein components of the synapse are not cross-linked upon fixation. That these structures are not observed in control synapses but have been found in multiple active zone cytomatrix mutants argues that the extensive cross-linking of proteins in chemically fixed preparations may enable the visualization of biologically relevant complexes missed with cryofixation. This supports the idea that the two fixation methods may offer different advantages for ultrastructural studies of synapses. In any case, the active zone cytomatrix is significantly reduced in size at Fife active zones in both HPF/FS- and aldehyde-fixed electron micrographs (Bruckner, 2016).

Although diminished or absent cytomatrices have been observed in electron micrographs of RIM-binding protein and Bruchpilot mutants, this phenotype has not been observed in electron micrographs of other active zone cytomatrix mutants, suggesting it represents more than the loss of a single component protein. Rather, the reduced complexity visible in electron micrographs likely reflects a broader underlying molecular disorganization. Further support for this model comes from superresolution imaging, which reveals molecular disorganization at Fife active zones as indicated by the loss of the characteristic ring-shaped localization pattern of Bruchpilot's C terminus. Similar disorganization of Bruchpilot was observed at active zones lacking RIM-binding protein. Superresolution imaging was used to investigate the localization of active zone proteins Cacophony and RIM-binding protein and, although the levels of Cacophony are reduced at Fife active zones, no apparent differences were observed in the patterns of these proteins. Cacophony and RIM-binding protein both localize in smaller puncta than Bruchpilot, so future studies with higher resolution imaging modalities such as stimulated emission depletion microscopy may reveal more subtle abnormalities. Correlations between active zone molecular composition and release probability have been observed at diverse synapses. At the Drosophila NMJ, functional imaging with genetically encoded Ca2+ indicators has demonstrated that active zones display a wide range of release probabilities. Active zones with high release probability contain higher levels of Bruchpilot, which may in turn correlate with higher Ca2+ channel levels. At mouse hippocampal synapses, Bassoon and RIM levels directly correlate with neurotransmitter release probability. Consistently, synaptic probability of release is significantly decreased in Fife mutants (Bruckner, 2016)

Through a combination of morphological and functional studies, this study found that Fife acts to promote the active zone docking of synaptic vesicles and regulates their probability of release. Because the number of readily releasable vesicles appears to scale with active zone cytomatrix size and molecular composition at diverse synapses, a conserved function of the active zone cytomatrix may be to establish release sites for synaptic vesicles. Consistent with this view, the number of release-ready vesicles is also reduced in Drosophila RIM-binding protein-null mutants and isoform-specific bruchpilotΔ170 and bruchpilotΔ190 mutants, which share similar active zone structural abnormalities with Fife. By combining rapid preservation of intact Drosophila larvae by HPF/FS fixation, electron tomography, and extensive segmentation of active zone structures, this study obtained a detailed view of the 3D organization of active zones in near-native state that allowed further dissection of Fife's role in determining the size of the readily releasable vesicle pool. Membrane-docked vesicles are significantly decreased in Fife mutants, whereas more distant vesicles attached to the membrane by long tethers appear unaffected. Correlating physiological and morphological parameters of neurotransmission is an ongoing challenge in the field. It has been proposed that docking and priming are not separable events in the establishment of the readily releasable vesicle pool, but rather the morphological and physiological manifestations of a single process. Although approximately one third of docked vesicles in these preparations lack obvious short connections to the membrane, which are thought to represent priming factors, the possibility cannot be excluded that these filaments are present but obscured, perhaps because the vesicles are more tightly linked to the membrane. As this proportion is unchanged in Fife mutants, it is concluded that Fife acts to promote vesicle docking and may simultaneously facilitate molecular priming-possibly through its interactions with RIM (Bruckner, 2016).

The data indicate that neurotransmitter release at Fife synapses is highly sensitive to EGTA, a slow Ca2+ chelator that has been used to investigate the coupling of Ca2+ influx at voltage-gated Ca2+ channels and Ca2+ sensors on synaptic vesicles. At synapses with high release probability, including inhibitory synapses in the mammalian hippocampus and cerebellum, excitatory synapses of the mature Calyx of held, and the Drosophila NMJ, molecularly primed synaptic vesicles and Ca2+ channel clusters are thought to be positioned within ~100 nm of one another to ensure the tight coupling of Ca2+ influx and Ca2+ sensors that explains observed release properties. The EGTA sensitivity of release at Fife, but not wild-type, NMJs indicates that Fife likely regulates the probability that a docked vesicle is released by positionally coupling release-ready vesicles to Ca2+ channels clustered beneath the active zone cytomatrix. The trend toward fewer docked vesicles associated with the active zone cytomatrix in tomograms of Fife synapses provides morphological support for this model. Building on detailed tomographic studies of the Drosophila NMJ to visualize how Ca2+ channels and vesicles are spatially organized at active zones in different genetic backgrounds will be an important step in advancing understanding of the geometry of release probability and how it is established (Bruckner, 2016).

Finally, this study found that Fife is required for presynaptic homeostasis. In response to decreases in glutamate receptor levels or function, Drosophila motoneurons rapidly increase synaptic vesicle release to maintain postsynaptic excitation. This homeostatic increase in presynaptic neurotransmission is accompanied by an increase in the number of dense projections per active zone and Bruchpilot levels. Cytomatrix proteins RIM, RIM-binding protein, and now Fife have all been shown to function in presynaptic homeostasis, indicating a critical role for the active zone cytomatrix as a substrate for synaptic plasticity. These studies provide insight into the molecular mechanisms through which the active zone cytomatrix determines neurotransmitter release parameters to modulate how information flows in neural circuits (Bruckner, 2016).

An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling

The homeostatic modulation of neurotransmitter release, termed presynaptic homeostatic potentiation (PHP), is a fundamental type of neuromodulation, conserved from Drosophila to humans, that stabilizes information transfer at synaptic connections throughout the nervous system. This study demonstrates that α2δ-3 (straitjacket), an auxiliary subunit of the presynaptic calcium channel, Cacophony, is required for PHP. The α2δ gene family has been linked to chronic pain, epilepsy, autism, and the action of two psychiatric drugs: gabapentin and pregabalin. Loss of α2δ-3 blocks both the rapid induction and sustained expression of PHP due to a failure to potentiate presynaptic calcium influx and the RIM-dependent readily releasable vesicle pool. These deficits are independent of α2δ-3-mediated regulation of baseline calcium influx and presynaptic action potential waveform. α2δ proteins reside at the extracellular face of presynaptic release sites throughout the nervous system, a site ideal for mediating rapid, transsynaptic homeostatic signaling in health and disease (Wang, 2016).

Presynaptic homeostatic potentiation (PHP) can be initiated by disruption of postsynaptic neurotransmitter receptors and is expressed as a change in presynaptic vesicle release. As such, PHP requires retrograde, transsynaptic signaling. The homeostatic potentiation of presynaptic release is mediated by increased presynaptic calcium influx without a change in the presynaptic action potential waveform. A remarkable property of presynaptic homeostatic plasticity is that it can be induced in a time frame of seconds to minutes and can be stably maintained throughout the life of an organism -- months in Drosophila and decades in humans. Equally remarkable, presynaptic homeostasis can precisely offset the magnitude of postsynaptic perturbations that vary widely in severity. This implies the existence of profoundly stable and remarkably precise homeostatic modifications to the presynaptic release apparatus. Transsynaptic signaling systems that are capable of achieving the rapid, accurate, and persistent control of presynaptic vesicle release are generally unknown (Wang, 2016).

In a large-scale forward genetic screen for homeostatic plasticity genes, mutations were identified in the α2δ-3 auxiliary subunit of the Ca_V2.1 calcium channel. α2δ genes encode a family of proteins that are post-translationally processed into a large glycosylated extracellular α2 domain that is linked through disulfide bonding to a short, membrane-associated δ domain. Existing loss-of-function data are consistent with the primary function of α2δ being the trafficking and synaptic stabilization of pore-forming α1 calcium channel subunits, with which they associate in the ER. There is also evidence that α2δ subunits control calcium channel kinetics in a channel-type- and cell-type-specific manner. However, the function of the α2δ gene family extends beyond calcium channel trafficking and membrane stabilization, including activities related to synapse formation and stability. As such, the large, glycosylated extracellular domain in α2δ may have additional, potent signaling activities at the active zone (Wang, 2016).

Importantly, the α2δ gene family is associated with a wide range of neurological diseases, including autism spectrum disorders (ASDs), neuropathic pain, and epilepsy. The α2δ-1 and α2δ-2 proteins are the primary targets of gabapentin and pregabalin, two major drugs used to treat neuropathic pain and epilepsy. This study demonstrates that α2δ-3 is essential for PHP. Thus, while α2δ-3 is an extracellular component of the extended presynaptic calcium channel complex (Davies, 2010), it nonetheless has a profound ability to modulate the intracellular neurotransmitter release mechanism. It is proposed that α2δ-3 relays signaling information from the synaptic cleft to the cytoplasmic face of the presynaptic active zone during PHP, an activity that could reasonably be related to the function of α2δ-3 during neurological disease (Wang, 2016).

This study demonstrates that α2δ-3 is essential for the rapid induction and sustained expression of presynaptic homeostatic potentiation (PHP). α2δ-3 encodes a glycosylated extracellular protein known to interact with matrix proteins that reside within the synaptic cleft. As such, it is proposed that α2δ-3 mediates homeostatic, retrograde signaling by connecting signaling within synaptic cleft to effector proteins within the presynaptic terminal, such as RIM. Since α2δ-3 associates with the pore-forming α1 subunit of calcium channels, it is ideally positioned to relay signaling to the site of high-release probability vesicle fusion adjacent to the presynaptic calcium channels (Wang, 2016).

It was previously demonstrated that PHP requires not only potentiation of presynaptic calcium influx but also a parallel homeostatic expansion of the readily releasable pool (RRP). Several lines of evidence argue against the possibility that the homeostatic potentiation of presynaptic calcium influx fully accounts for the observed potentiation of the RRP. First, it is well established in mammalian systems and the Drosophila NMJ (Müller, 2015) that the calcium-dependence of the RRP is sub-linear. Therefore, the relatively small change in presynaptic calcium influx that occurs during PHP (12%-25%) would not be sufficient to account for the observed doubling of the RRP, an effect that has been quantified across a wide range of extracellular calcium (0.3-15 mM [Ca²⁺]_e) (Müller, 2015). Second, the homeostatic increase of presynaptic calcium influx and the homeostatic expansion of RRP are genetically separable processes (Harris, 2015). Since loss of α2δ-3 completely blocks the homeostatic expansion of the RRP, it appears that α2δ-3 has an additional activity that is directed at the homeostatic modulation of the RRP (Wang, 2016).

Collectively, these data argue that α2δ-3 functions with Rab3 interacting molecule (rim), either directly or indirectly, to achieve a homeostatic potentiation of the RRP. First, the loss of function phenotype of α2δ-3 is strikingly similar to that observed in rim mutants. Both mutations cause a deficit in presynaptic release that is associated with diminished baseline presynaptic calcium influx, diminished size of the baseline RRP, and enhanced sensitivity to application of EGTA-AM. Second, this study demonstrates a strong trans-heterozygous interaction between rim/+ and α2δ-3/+, suggesting that both genes function to control the same presynaptic processes during PHP. Since the rim mutation selectively disrupts the homeostatic modulation of the RRP, this genetic interaction could reflect a failure to homeostatically modulate the RRP (Wang, 2016).

Both RIM and α2δ-3 bind the pore-forming α1 subunit of the Ca_V2.1 calcium channel. As such, signaling could be relayed from α2δ-3 to RIM through molecular interactions within the extended Ca_V2.1 calcium channel complex. However, not all evidence is consistent with this possibility. For example, RNAi-mediated depletion of Ca_V2.1 channels, sufficient to decrease release by ∼80%, does not prevent presynaptic homeostasis. Thus, loss of α2δ-3 blocks PHP, whereas loss of the Ca_V2.1 α1 subunit does not. In addition, the double-heterozygous mutant of rim/+ and α2δ-3/+ blocks PHP but does not disrupt baseline vesicle release, arguing that this genetic interaction is not due to a decrease in the number or organization of presynaptic α1 calcium channel subunits. Thus, it is speculated that α2δ-3 conveys signaling through a co-receptor on the plasma membrane to participate in the homeostatic modulation of the RRP. There are very few extracellular proteins known to establish baseline levels of primed, fusion-competent synaptic vesicles. Since α2δ proteins should reside at chemical synapses throughout the nervous system, this signaling could reasonably be related to the neurological and psychiatric diseases associated with α2δ genes (Wang, 2016).

RIM controls homeostatic plasticity through modulation of the readily-releasable vesicle pool

Rab3 interacting molecules (RIMs) are evolutionarily conserved scaffolding proteins that are located at presynaptic active zones. In the mammalian nervous system, RIMs have two major activities that contribute to the fidelity of baseline synaptic transmission: they concentrate calcium channels at the active zone and facilitate synaptic vesicle docking/priming. This study confirms that RIM has an evolutionarily conserved function at the Drosophila neuromuscular junction and then defines a novel role for RIM during homeostatic synaptic plasticity. Loss of RIM disrupts baseline vesicle release, diminishes presynaptic calcium influx, and diminishes the size of the readily-releasable pool (RRP) of synaptic vesicles, consistent with known activities of RIM. However, loss of RIM also completely blocks the homeostatic enhancement of presynaptic neurotransmitter release that normally occurs after inhibition of postsynaptic glutamate receptors, a process termed synaptic homeostasis. It is established that synaptic homeostasis requires enhanced presynaptic calcium influx as a mechanism to potentiate vesicle release. However, despite a defect in baseline calcium influx in rim mutants, the homeostatic modulation of calcium influx proceeds normally. Synaptic homeostasis is also correlated with an increase in the size of the RRP of synaptic vesicles, although the mechanism remains unknown. This study demonstrates that the homeostatic modulation of the RRP is blocked in the rim mutant background. Therefore, RIM-dependent modulation of the RRP is a required step during homeostatic plasticity. By extension, homeostatic plasticity appears to require two genetically separable processes, the enhancement of presynaptic calcium influx and a RIM-dependent modulation of the RRP (Muller, 2012b).

Throughout the nervous system, homeostatic signaling systems are thought to stabilize neural function through the regulation of ion channel density, neurotransmitter receptor abundance, and presynaptic neurotransmitter release. Previous work consisted of a large-scale effort to identify genes involved in the homeostatic modulation of presynaptic release at the Drosophila neuromuscular junction (NMJ) (Dickman, 2009; Müller, 2011). In brief, inhibition of postsynaptic glutamate receptors at the Drosophila NMJ induces a retrograde signaling system that causes an increase in presynaptic neurotransmitter release. The increase in release precisely offsets the postsynaptic perturbation and restores muscle excitation in the continued presence of the perturbation, evidence of a homeostatic signaling system. The homeostatic modulation of presynaptic release is blocked by point mutations in the α1 subunit of the presynaptic calcium channel Ca_V2.1. A recent calcium imaging study has extended these genetic data, demonstrating that the homeostatic enhancement of release requires increased calcium influx through the Ca_V2.1 calcium channel (Müller, 2012a). Mechanistically, it has been shown that Rab3-GAP, which acts in concert with the presynaptic vesicle-associated protein Rab3, is also necessary for homeostatic synaptic plasticity (Müller, 2011). Based on the involvement of both Rab3 signaling and the Ca_V2.1 calcium channel, it was hypothesized that the Rab3 interacting molecule (RIM), which biochemically interacts with both Rab3 and calcium channels, might be centrally involved in homeostatic plasticity (Muller, 2012b).

RIMs are evolutionarily conserved scaffolding proteins that are located at presynaptic active zones. Electrophysiological analyses of mammalian synapses lacking RIM1 or RIM2 isoforms demonstrate a role for RIMs in the control of synaptic transmission. Most recently, analysis of RIM1/2 double knock-out mice has shown that RIMs concentrate calcium channels at the presynaptic active zone and facilitate synaptic vesicle docking at the presynaptic release site. In addition to the involvement of RIM in synaptic baseline transmission, different RIM isoforms are required for LTP and LTD at various synapses (Muller, 2012b).

Drosophila is predicted to encode a single rim gene, facilitating a loss-of-function genetic analysis of rim function in Drosophila (Graf, 2012). This study provides a genetic dissection of rim function during homeostatic regulation of transmitter release at the Drosophila NMJ. rim is shown to have an evolutionarily conserved function to promote baseline presynaptic calcium influx, vesicle release, and readily-releasable pool (RRP) size. rim is specifically required during homeostatic synaptic plasticity by acting on the RRP and not on the homeostatic modulation of presynaptic calcium influx. The data not only define a novel activity for RIM but also provide a novel molecular mechanism for the homeostatic control of neurotransmitter release (Muller, 2012b).

This study demonstrates that RIM is required for the retrograde, homeostatic enhancement of presynaptic neurotransmitter at the Drosophila NMJ. Three independent mutations in the RIM gene, including a small internal deletion, block the homeostatic increase of presynaptic release. In addition, neuronal expression of UAS-rim RNAi blocks synaptic homeostasis. The defect in homeostatic potentiation does not appear to result from major changes in synapse or active zone morphology. Furthermore, the defect in homeostatic potentiation is observed over a 10-fold range of [Ca²⁺]_e (0.3-3 mm.Evidence that RIM has two independent functions, only one of which is required for homeostatic plasticity. First, evidence is provided that RIM is necessary for normal presynaptic calcium influx and presynaptic release probability. This is consistent with a well-established role for RIM in binding to presynaptic calcium channels (Kiyonaka, 2007; Kaeser, 2011) and concentrating calcium channels to the active zone (Han, 2011; Kaeser, 2011). However, the regulation of presynaptic calcium channel density does not appear to be the function that RIM contributes to the mechanisms of synaptic homeostasis. The homeostatic modulation of presynaptic calcium influx is still observed in the rim mutant, implying that the homeostatic, retrograde signaling system remains functional and is able to modulate calcium channel number or function in the absence of rim (Muller, 2012b).

This study provides evidence that the required function of RIM during homeostatic plasticity is the modulation of the RRP. This study confirmed that synaptic homeostasis is correlated with an increase in the RRP. It was then demonstrated that the homeostatic increase in the RRP is blocked in the rim mutation. This observation is significant for several reasons. First, these data provide evidence that a change in the RRP is not just correlated with homeostatic plasticity but is required. Second, these data imply that the homeostatic modulation of RRP size can be controlled independently of, or in parallel with, the homeostatic modulation of presynaptic calcium influx that persists in the rim mutant. Third, the data provide evidence that RIM imparts an essential activity that is required for the homeostatic modulation of the RRP during homeostatic plasticity. Finally, the data indicate that the precise homeostatic modulation of presynaptic neurotransmitter release occurs at the intersection of two independently regulated processes, calcium influx and the RRP size. This suggests new complexity underlying the expression of homeostatic plasticity within the presynaptic nerve terminal (Muller, 2012b).

One of the remarkable features of homeostatic plasticity is that it is a quantitatively accurate form of synaptic modulation. It has been shown that the change in presynaptic neurotransmitter release precisely offsets the change in postsynaptic glutamate receptor function. More specifically, a 20% decrease in receptor sensitivity is offset by a 20% increase in presynaptic release, whereas a 50% decrease in receptor sensitivity is offset by a 50% increase in release (Frank, 2006). This is precisely what is expected of a true homeostatic signaling system that restores a system to baseline, or set-point functionality. However, this capacity of a homeostatic signaling system also places an unusual demand on the signaling system controlling the modulation presynaptic neurotransmitter release. It makes intuitive sense that a change in presynaptic calcium influx is involved in homeostatic plasticity. However, the highly cooperative nature of calcium-dependent vesicle fusion would necessitate that calcium influx be controlled very precisely during homeostatic plasticity to prevent overshoot and inappropriate potentiation of vesicle release. Therefore, it might also make sense for the expression of synaptic homeostasis to require a parallel change within the presynaptic nerve terminal, in addition to a modulation of presynaptic calcium influx. The current data suggest that the RIM-dependent homeostatic modulation of RRP size is one such mechanism that is independent of calcium influx and necessary, in parallel, for the expression of homeostatic plasticity. If both mechanisms can limit the amount of vesicle release, this would provide an additional opportunity to control or limit the extent of homeostatic plasticity (Muller, 2012b).

How might RIM participate in the regulated modulation of RRP size? First, synaptic homeostasis at the Drosophila NMJ requires a retrograde signal from muscle to nerve. It has been hypothesized that this retrograde signal is able to directly influence presynaptic calcium channel number or function, and genetic evidence has been provided that Eph-Ephrin signaling could impinge on the regulation of presynaptic Ca_V2.1 calcium channels. However, it remains unknown whether Eph-Ephrin signaling functions during the rapid induction of homeostatic plasticity and whether this signaling system participates in the modulation of the RRP. It is formally possible that an independent retrograde signal targets RIM and the regulation of the RRP (Muller, 2012b).

In mammalian systems, RIM has been shown to interact with numerous proteins, including Rab3 and the presynaptic calcium channel. The zinc finger domain of RIM has also been shown to interact with Munc13 and has been implicated in the vesicle priming/docking function of RIM. Rab3-GAP and Rab3-dependent signaling have been implicated in synaptic homeostasis. It is currently unknown how Rab3 signaling might intersect with RIM to participate in homeostatic plasticity, and this is something that could be addressed in the future. However, it is interesting to speculate that RIM-dependent regulation of vesicle docking/priming via Munc13 could be a function of RIM that is required for the homeostatic modulation of RRP size during homeostatic plasticity. Mutations in the Drosophila homolog of Munc13 are late embryonic lethal, preventing a straightforward analysis during homeostatic plasticity. This could be addressed in the future through protein knockdown and a molecular dissection of the RIM-Dunc13 protein interaction (Muller, 2012b).

RIM binding protein (RBP) function was analyzed recently at the Drosophila NMJ. RBP mutations cause a deficit in calcium influx and calcium channel abundance that are quantitatively similar to the changes observed here in rim mutants. Although RBP mutations show a more severe defect in baseline transmission in response to a single AP, the RBP mutant synapse shows dramatic facilitation similar to that observed in rim mutants, indicating that the synapse is capable of substantial vesicle release provided sufficient presynaptic calcium entry during a stimulus train. Because the defect in calcium influx is similar when comparing rim and RBP mutants, it seems that additional deficits contribute to the RBP release phenotype, which could include the observed active zone disorganization. It remains unknown whether RBP is required for homeostatic synaptic plasticity, but the existing data are consistent with these two proteins functioning in concert to control active zone function and, potentially, homeostatic synaptic plasticity (Muller, 2012b).

RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. This study has identified Drosophila RIM (Rab3 interacting molecule) and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission because of a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Because RIM has traditionally been studied as an effector of Rab3 function, this study investigated whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild-type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites (Graf, 2012).

Synaptic vesicle exocytosis occurs at specialized regions of the presynaptic membrane, termed active zones, in which presynaptic release machinery proteins cluster opposite postsynaptic neurotransmitter receptors. The complement of presynaptic proteins associated with each active zone is a determinant of the release properties at each release site. In particular, proteins that determine the number of Ca²⁺ channels at each release site control synaptic efficacy (Graf, 2012).

At the Drosophila larval neuromuscular junction (NMJ), Bruchpilot (Brp) and RIM-binding protein (RIM-BP) regulate Ca²⁺ channel accumulation. RIM (Rab3 interacting molecule) enhances Ca²⁺ channel levels at mammalian synapses (Han, 2011; Kaeser, 2011); however, RIM has not been characterized previously in flies. RIM is an active zone protein that acts as an organizer of the presynaptic release apparatus via its interactions with multiple core active zone components, including α-liprins, Munc-13, RIM-BPs, and Ca²⁺ channels. RIM also interacts with CAST/ERC in mice and ELKS in Caenorhabditis elegans (Deken, 2005), the mammalian and worm orthologs of Brp. Hence, RIM may have a similar function in Drosophila (Graf, 2012).

In rodents and C. elegans, RIM binds to and is an effector of the small GTPase Rab3. Previous studies have shown that Rab3 dynamically controls the presynaptic protein composition of individual active zones at the Drosophila NMJ. At wild-type (WT) NMJs, release machinery proteins are distributed across all active zones, resulting in the formation of hundreds of low probability release sites peppered with higher probability sites. Conversely, in the rab3 mutant, key constituents of the presynaptic active zone, including Brp and Ca²⁺ channels, are enriched at a subset of active zones, leaving the remaining release sites apparently devoid of such proteins. The molecular mechanisms by which Rab3 mediates this function remain essentially unknown; however, RIM is a potential effector for Rab3 in this process (Graf, 2012).

To test the role of RIM at Drosophila active zones, the single Drosophila ortholog of RIM was cloned and RIM excision mutants were generated (Müller, 2012b). This study shows that Drosophila RIM localizes to active zones and that its distribution requires Rab3. Mutant analysis demonstrates that full-length RIM is not necessary for the proper localization of Brp or for the altered distribution of Brp across active zones in rab3 mutants. Rather, RIM enables robust Ca²⁺-dependent synaptic release by promoting the accumulation of Ca²⁺ channels at release sites and synaptic vesicles in the readily releasable pool (RRP). Hence, WT RIM is not an essential effector of Rab3 for the control of protein composition across active zones but instead promotes synaptic efficacy by enhancing Ca²⁺ channel density and the size of the RRP at the Drosophila NMJ (Graf, 2012).

This study shows that RIM localizes to active zones at the Drosophila NMJ and is required for the normal accumulation of Ca²⁺ channels. In RIM excision mutants, Ca²⁺ channel levels are significantly reduced at release sites, and there is a reduction in the size of the RRP of synaptic vesicles and a modest decrease in active zone number. As a result, RIM mutants show a dramatic impairment of evoked synaptic vesicle release. Conversely, the synaptic ultrastructure and localization and distribution of the release machinery protein Brp remains unaffected by the lack of WT RIM. Furthermore, the reduction in Ca²⁺ channels with no apparent effect on Brp is observed in both a WT background and in a rab3 mutant background that exhibits an altered distribution of active zone components across potential release sites (Graf, 2012).

These findings are consistent with recent studies in rodents that show decreased accumulation of Ca²⁺ channels and reduced Ca²⁺ currents at synapses of conditional knock-out RIM mutant mice (Han, 2011; Kaeser, 2011), indicating an evolutionarily conserved role for RIM in regulating Ca²⁺ channel density at release sites. In rodents, this role requires the direct interaction of the PDZ domain of RIM with the C termini of N- and P/Q-type Ca²⁺ channels (Kaeser, 2011). In these studies, the entire central region of the Drosophila RIM gene is excised in both the RIM^Ex73 and RIM^Ex98 mutants, including the genomic sequence that encodes for the PDZ domain. It is unknown whether an evolutionarily conserved interaction exists between the PDZ domain of Drosophila RIM and Cacophony; however, the complete removal of the PDZ domain in RIM^Ex73 and RIM^Ex98 ensures that such direct interactions cannot exist in the mutant, even if truncated versions of RIM protein are potentially expressed in RIM^Ex73 and RIM^Ex98. Thus, Ca²⁺ channel accumulation is likely reduced in RIM^Ex73 and RIM^Ex98 mutants because of the inability of RIM and Ca²⁺ channels to directly interact (Graf, 2012).

Several proteins have now been identified in Drosophila as regulators of Ca²⁺ channel accumulation, including Brp and the recently reported Drosophila RIM-binding protein (DRBP). Disruption of any one of the three proteins fails to completely disrupt Ca²⁺ channel localization to release sites, suggesting that they may play partially overlapping roles. Evidence in Drosophila, rodents, and C. elegans indicates that all three proteins directly interact with Ca²⁺ channels but also interacting with each other and several other components to form the active zone cytomatrix. Consistent with protein binding studies, this study shows that GFP-tagged RIM concentrates at release sites and colocalizes with Brp. Interestingly, whereas DRBP levels are reduced in brp mutants, RIM::GFP localizes in an active zone-like pattern even in the absence of Brp. This finding is consistent with studies in C. elegans (Deken, 2005) and suggests that Brp is unnecessary for the localization of RIM to release sites. Furthermore, Brp localization to active zones is maintained in both drbp (Liu, 2011) and RIM^Ex73 and RIM^Ex98 mutant neurons. Together, these studies indicate that the large protein complex that forms the active zone release machine is at least partially maintained in the absence of any one of these proteins, which may explain why the absence of only a single component results in the partial but not complete reduction of Ca²⁺ channels (Graf, 2012).

These studies further indicate that Drosophila RIM function is not limited to Ca²⁺ channel accumulation but rather plays multiple roles at the active zone, including regulation of the size of the RRP. This finding is also consistent with decreases in RRP size observed in conditional knock-out RIM mutant mice and indicates that multiple functions of RIM are evolutionarily conserved (Graf, 2012).

It was originally hypothesized that RIM may act as an effector of Rab3 to regulate active zone protein composition across release sites. RIM has been studied previously in rodents and C. elegans as an important effector in the Rab3-mediated docking of synaptic vesicles to active zones during the synaptic vesicle cycle. The active zone localization and extensive interactions of RIM with multiple active zone proteins place it in an attractive position to potentially mediate this newly described role for Rab3 in the control of active zone protein composition. Moreover, the observation that RIM can localize in an active zone-like manner in the absence of Brp suggests that RIM can potentially act upstream of other presynaptic cytomatrix proteins to regulate the formation of the presynaptic release machine (Graf, 2012).

Brp is a central component of Drosophila active zones and is dramatically redistributed in rab3 mutant NMJs, it would be expected that a downstream effector of Rab3 would control Brp localization to active zones. However, analysis of Brp in the RIM mutant reveals a localization pattern indistinguishable from WT. It is important to note that, in certain cases, confocal microscopy is unable to distinguish fine alterations in Brp morphology that can be observed with higher-resolution microscopes. Nevertheless, the observations reveal no obvious difference in Brp staining between WT and RIM mutant NMJs, indicating that WT RIM is unnecessary for general Brp localization and that Rab3 does not act through RIM to regulate active zone protein composition. Moreover, disruption of RIM in the rab3 mutant background has no effect on the altered distribution of Brp that results after rab3 disruption, demonstrating that RIM is not involved in the molecular mechanisms that control Brp redistribution in the rab3 mutant. The reduction of Ca²⁺ channels in the rab3--RIM double mutant does suggest that RIM may be required for the enhanced efficacy of 'super active zones' formed in the rab3 mutant; however, the molecular mechanism by which Rab3 controls the general distribution of active zone components across release sites remains unknown (Graf, 2012).

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).

C-terminal splice variants of P/Q-type Ca2+ channel Ca_V2.1 alpha1 subunits are differentially regulated by Rab3-interacting molecule proteins

Voltage-dependent Ca²⁺ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α₁ and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α₁ subunit Ca_V2.1 gene generates major variants of the Ca_V2.1 C-terminal region, known for associating with presynaptic proteins, this study focused on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by Ca_V2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the Ca_V2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the Ca_V2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the Ca_V2.1 CTD with RIMs enable Ca_V2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents (Hirano, 2017).

A trans-synaptic nanocolumn aligns neurotransmitter release to receptors

Synaptic transmission is maintained by a delicate, sub-synaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorders. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, while it has long been recognized that this spatial relationship modulates synaptic strength, it has not been precisely described, owing in part to the limited resolution of light microscopy. Using localization microscopy, this study shows that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometre-scale subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses in cultured rat hippocampal neurons, this study found that action-potential-evoked fusion is guided by this protein gradient and occurs preferentially in confined areas with higher local density of Rab3-interacting molecule (RIM) within the active zones. These presynaptic RIM nanoclusters closely align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of a trans-synaptic molecular 'nanocolumn'. It is thus proposed that the nanoarchitecture of the active zone directs action-potential-evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by trans-synaptic nanoscale realignment. This architecture suggests a simple organizational principle of central nervous system synapses to maintain and modulate synaptic efficiency (Tang, 2016).

RIM1α SUMOylation is required for fast synaptic vesicle exocytosis

The rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. This study shows that posttranslational modification of the active zone protein Rab3-interacting molecule 1alpha (RIM1alpha) by the small ubiquitin-like modifier 1 (SUMO-1) functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1alpha SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca(2+) influx necessary for vesicular release, whereas non-SUMOylated RIM1alpha participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1alpha is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated 'switching' of RIM1alpha between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction (Girach, 2013).

RIM determines Ca(2)+ channel density and vesicle docking at the presynaptic active zone

At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca(2)+ channels close to docked vesicles. The mechanisms that enrich Ca(2)+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. This study has established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca(2)+ channel density, revealing a role of RIM proteins in Ca(2)+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca(2)+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca(2)+ channel density and vesicle docking at the active zone (Han, 2011).

RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction

At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, this study identified a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, conditional knockout mice were generated lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. It is proposed that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse (Kaeser, 2011).

Redundant localization mechanisms of RIM and ELKS in Caenorhabditis elegans

Active zone proteins play a fundamental role in regulating neurotransmitter release and defining release sites. The functional roles of active zone components are beginning to be elucidated; however, the mechanisms of active zone protein localization are unknown. Studies have shown that glutamine, leucine, lysine, and serine-rich protein (ELKS), a recently defined member of the active zone complex, acts to localize the active zone protein Rab3a-interacting molecule (RIM) and regulates synaptic transmission in cultured neurons. This study tested the function of ELKS in vivo. Like mammalian ELKS, Caenorhabditis elegans ELKS is an active zone protein that directly interacts with the postsynaptic density-25/Discs large/zona occludens (PDZ) domain of RIM. However, RIM protein localizes in the absence of ELKS and vice versa. In addition, elks mutants exhibit neither the behavioral nor the physiological defects associated with unc-10 RIM mutants, indicating that ELKS is not a critical component of the C. elegans release machinery. Interestingly, expression of the soluble PDZ domain of RIM disrupts ELKS active zone targeting, suggesting a tight association between the two proteins in vivo. RIM truncations containing only the PDZ and C2A domains target to release sites in an ELKS-dependent manner. Together, these data identify ELKS as a new member of the C. elegans active zone complex, define the role of ELKS in synaptic transmission, and characterize the relationship between ELKS and RIM in vivo. Furthermore, they demonstrate that multiple different protein-protein interactions redundantly anchor both ELKS and RIM to active zones and implicate novel proteins in the formation of the active zone (Deken, 2005).

Search PubMed for articles about Drosophila Rab3 interacting molecule

Bruckner, J. J., Zhan, H., Gratz, S. J., Rao, M., Ukken, F., Zilberg, G. and O'Connor-Giles, K. M. (2016). Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release. J Cell Biol [Epub ahead of print]. PubMed ID: 27998991

Davies, A., Kadurin, I., Alvarez-Laviada, A., Douglas, L., Nieto-Rostro, M., Bauer, C. S., Pratt, W. S. and Dolphin, A. C. (2010). The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function. Proc Natl Acad Sci U S A 107: 1654-1659. PubMed ID: 20080692

Deken, S. L., Vincent, R., Hadwiger, G., Liu, Q., Wang, Z. W. and Nonet, M. L. (2005). Redundant localization mechanisms of RIM and ELKS in Caenorhabditis elegans. J Neurosci 25(25): 5975-5983. PubMed ID: 15976086

Dickman, D. K. and Davis, G. W. (2009). The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis. Science 326(5956): 1127-1130. PubMed ID: 19965435

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(4): 663-677. PubMed ID: 17114050

Graf, E. R., Valakh, V., Wright, C. M., Wu, C., Liu, Z., Zhang, Y. Q. and DiAntonio, A. (2012). RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction. J Neurosci 32(47): 16586-16596. PubMed ID: 23175814

Girach, F., Craig, T. J., Rocca, D. L. and Henley, J. M. (2013). RIM1α SUMOylation is required for fast synaptic vesicle exocytosis. Cell Rep 5(5): 1294-1301. PubMed ID: 24290762

Han, Y., Kaeser, P. S., Sudhof, T. C. and Schneggenburger, R. (2011). RIM determines Ca(2)+ channel density and vesicle docking at the presynaptic active zone. Neuron 69(2): 304-316. PubMed ID: 21262468

Harris, N., Braiser, D. J., Dickman, D. K., Fetter, R. D., Tong, A. and Davis, G. W. (2015). The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity. Neuron 88: 1157-1164. PubMed ID: 26687223

Hirano, M., Takada, Y., Wong, C. F., Yamaguchi, K., Kotani, H., Kurokawa, T., Mori, M. X., Snutch, T. P., Ronjat, M., De Waard, M. and Mori, Y. (2017). C-terminal splice variants of P/Q-type Ca2+ channel Ca_V2.1 alpha1 subunits are differentially regulated by Rab3-interacting molecule proteins. J Biol Chem 292(22): 9365-9381. PubMed ID: 28377503

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(2): 282-295. PubMed ID: 21241895

Kiyonaka, S., Wakamori, M., Miki, T., Uriu, Y., Nonaka, M., Bito, H., Beedle, A. M., Mori, E., Hara, Y., De Waard, M., Kanagawa, M., Itakura, M., Takahashi, M., Campbell, K. P. and Mori, Y. (2007). RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels. Nat Neurosci 10(6): 691-701. PubMed ID: 17496890

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

Muller, 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(4): 749-762. PubMed ID: 21338884

Muller, M. and Davis, G. W. (2012a). Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release. Curr Biol 22(12): 1102-1108. PubMed ID: 22633807

Müller, M., Liu, K. S., Sigrist, S. J. and Davis, G. W. (2012b). RIM controls homeostatic plasticity through modulation of the readily-releasable vesicle pool. J Neurosci 32: 16574-16585. PubMed ID: 23175813

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

Tang, A. H., Chen, H., Li, T. P., Metzbower, S. R., MacGillavry, H. D. and Blanpied, T. A. (2016). A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature 536(7615): 210-214. PubMed ID: 27462810

Wang, T., Jones, R. T., Whippen, J. M. and Davis, G. W. (2016). α2δ-3 is required for rapid transsynaptic homeostatic signaling. Cell Rep 16: 2875-2888. PubMed ID: 27626659

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

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

date revised: 30 December 2020

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