Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. The study of Synaptotagmin's expression during Drosophila neurogenesis has been initiated in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin message is present in the cell bodies of all peripheral nervous system neurons, in mature neurons, and in many, if not all, central nervous system neurons during neurite outgrowth and synapse formation. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neuropil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, it is proposed that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes (Littleton, 1993a).
In wild-type ovaries Syx1A protein is detected in regions 2 and 3 of the germarium, outlining the membranes of germline cyst cells. Syx1A continues to be abundantly expressed in the nurse cell membranes of egg chambers during stages 1-8, and Syx1A levels fade during stages 8 and 9. Syx1A protein is present in the adult brain and the synaptic substations of the visual system. Syx1A is concentrated in the neuropil regions of the brain and is somewhat enriched at synaptic regions, such as in the lamina and medulla of the optic lobes where photoreceptor axons form synaptic contacts with second order neurons. Syx1A distribution in the brain differs from that of synaptic vesicle-specific proteins such as synaptotagmin, as Syx1A is also present in axons and cell bodies, whereas synaptotagmin is restricted to synaptic terminals (Schulze, 1996).
Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers. This study shows that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, it was found that the degree of daily structural plasticity exceeds that originally described, underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique, this study showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead to a proposal that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels (Gorostaza, 2014).
Circadian remodeling of the small ventral lateral neuron (sLNv) dorsal terminals was first described at the peak and trough levels of pigment-dispersing factor (PDF) immunoreactivity, that is at zeitgeber time 2 (ZT2) and ZT14 (2 hr after lights ON and lights OFF, respectively), as well as their counterparts under constant darkness (DD) (circadian time 2 [CT2] and CT14). For a more precise examination of the extent of structural remodeling, a time course was carried out. An inducible GAL4 version termed GeneSwitch restricted to PDF neurons (pdf-GS) combined with a membrane-tethered version of GFP (mCD8GFP) was used as control. As expected from the original observations, a significant reduction in complexity of the axonal arbor-measured as total axonal crosses-could be seen between CT2 and CT14 and between CT18 and CT22, which remained unchanged at nighttime. However, toward the end of the subjective night (CT22), the primary processes appeared to be shorter. To more precisely describe this additional form of plasticity, the length of the maximum projection was measure from the lateral horn toward the midbrain. This analysis revealed that toward the end of the subjective night (CT22), PDF projections are significantly shorter than at the beginning of the day (CT2). These observations imply that mechanisms other than the proposed changes in the degree of fasciculation are recruited during circadian plasticity. To get a deeper insight into the nature of the phenomena, the changes were monitored in brain explants kept in culture for 48 hr after dissection. Transgenic pdf-GAL4; UAS-mCD8RFP flies (referred to as pdf>RFP) were dissected under safe red light, and brains were maintained under DD. Imaging of individual brains at two different time points highlighted three types of changes experienced by axonal terminals: (1) changes in the degree of fasciculation/defasciculation, more common in primary branches, (2) the addition/retraction of new processes, mostly affecting those of secondary or tertiary order, and (3) positional changes of minor terminals, thus confirming and extending previous observations. Altogether, these results indicate that a rather complex remodeling process takes place on daily basis in the axonal terminals of PDF neurons (Gorostaza, 2014).
The level of structural remodeling occurring at the dorsal terminals suggested that synapses themselves could undergo changes in a time-dependent fashion. The presynaptic protein Synaptotagmin (SYT) was examined at different times across the day as an indicator of vesicle accumulation. A GFP-tagged version of SYT was expressed in PDF neurons (pdf >sytGFP), and both the number and area span by SYT+ puncta (most likely describing the accumulation of several dense core vesicles) were analyzed separately at the sLNv dorsal terminals. No statistical differences were observed in the number of SYT+ puncta (although there is a tendency for higher numbers in the early morning), perhaps as a result of the nature of the signal, which is too diffuse for precise identification of individual spots. On the other hand, SYT+ puncta were larger and, as a result, the area covered by SYT+ immunoreactivity was significantly different at CT2 compared to CT14, but not between CT22 and CT2, perhaps reflecting that vesicles started to accumulate at the end of the day in preparation for the most dramatic membrane change taking place between CT22 and the beginning of the following morning (Gorostaza, 2014).
The observation that a more complex structure correlated with a larger area covered by presynaptic vesicles reinforced the notion that indeed the number of synapses could be changing throughout the day and prompted analysis of Bruchpilot (BRP), a well-established indicator of active zones. Expressing a tagged version of BRP in PDF neurons, the number of BRP+ puncta was quantitated as a proxy for active zones at times when the most dramatic changes in structure had been detected (i.e., CT2, CT14, and CT22). Interestingly, the number of active zones was significantly larger at CT2 than at CT14 or CT22; in fact, no statistical differences were observed between the last two time points, underscoring that axonal remodeling can occur (i.e., pruning of major projections taking place toward the end of the night) without significantly affecting overall connectivity. Thus, circadian structural plasticity is accompanied by changes in the number of synapses. Not only are more vesicles recruited toward CT2, but also a higher number of active zones are being established (Gorostaza, 2014).
Circadian changes in the abundance of the presynaptic active zone BRP have also been shown in the first optic neuropil of the fly brain, although BRP abundance in the lamina increases in the early night under DD conditions, in contrast to the oscillations in BRP levels observed at the dorsal protocerebrum that peak in the early subjective day just described. In addition, rhythmic changes in the number of synapses have also been described in the terminals of adult motor neurons in Drosophila examined through transmission electron microscopy, as well as BRP+ light confocal microscopy, underscoring the validity of the approach employed herein. Interestingly, in different brain areas, the level of presynaptic markers (such as BRPRFP or SYTGFP) also changes in response to the sleep/wake 'state,' being high when the animals are awake and lower during sleep; this observation led to the proposal that sleep could be involved in maintaining synaptic homeostasis altered during the awaking state. This trend coincides with observation of higher levels during the subjective morning and lower levels at the beginning of the subjective night; however, no changes were detected through the night, suggesting that, at least in clock neurons, there is a circadian rather than a homeostatic control of synaptic activity. Given that clock outputs are predominantly regulated at the transcriptional level and that there is circadian regulation of MEF2, a transcription factor that turns on a program involved in structural remodeling, this correlation opens the provocative possibility that the circadian clock is controlling the ability of assembling novel synapses in particularly plastic neurons, which might become recruited and/or stabilized, or otherwise pruned (disassembled), toward the end of the day (Gorostaza, 2014).
Adult-specific electrical silencing of PDF neurons reduces the complexity of dorsal arborizations, although a certain degree of circadian remodeling of the axonal terminals still takes place. To examine whether electrical alterations could affect circadian changes in the number of active zones, either Kir2.1 or NaChBac was expressed (to hyperpolarize or depolarize PDF neurons, respectively). To avoid any undesired developmental defects, pdf-GS was used to drive expression of the channels only during adulthood. Interestingly, Kir2.1 expression abrogated circadian changes in the number of active zones. In fact, PDF neurons displayed a reduced number of active zones compared to controls at CT2 and remained at similar levels throughout the day, indistinguishable from nighttime controls. On the other hand, when neurons were depolarized through NaChBac expression, the number of active zones did not change along the day and was maintained at daytime levels even at CT14 and CT22 (Gorostaza, 2014).
It has recently been shown that MEF2, a transcription factor involved in activity-dependent neuronal plasticity and morphology in mammals, is circadianly regulated and mediates some of the remodeling of PDF dorsal terminals through the regulation of Fasciclin2. In contrast, adult-specific silencing (and depolarization) of PDF neurons abolishes cycling in the number of BRP+ active zones, despite the fact that it does not completely obliterate the remodeling of the axonal terminals, suggesting that some of the mechanisms underlying structural plasticity are clearly activity independent and are most likely the result of additional clock-controlled output pathways still to be identified (Gorostaza, 2014).
Since structural remodeling of PDF neurons results in the formation and disappearance of new synapses on daily basis, it was anticipated that not only the number but also the postsynaptic partners of these contacts could concomitantly be changing. To shed light on this possibility, GFP reconstitution across synaptic partners (GRASP), which labels contacts between adjacent membranes, was used. In brief, two complementary fragments of GFP tethered to the membrane are expressed in different cells. If those cells are in contact, GFP is reconstituted and becomes fluorescent. GRASP has previously been employed to monitor synapses in adult flies. Given the complex arborization at the dorsal protocerebrum, it was asked whether specific subsets of circadian neurons projecting toward that area could be contacting across the day. Perhaps not surprisingly, an extensive reconstituted GFP signal could be observed between the sLNv dorsal projections and those of the posterior dorsal neuron 1 cells (DN1ps, lighted up by the dClk4.1-GAL4 line, suggesting contacts along the entire area, which are detectable across all time points analyzed (ZT2, ZT14, and ZT22). Consistent with these observations, extensive physical contact between the sLNv projections and those of the DN1p neurons has just been reported at the dorsal protocerebrum with no clear indication of the time of day examined. Next the study examined whether a subset of dorsal LNs (LNds), projecting toward both the accessory medulla and the dorsal protocerebrum (through the combined expression of Mai179-GAL4; pdf-GAL80), could also contact the profuse dorsal arborization of sLNv neurons; this genetic combination enables expression of split-GFP in a restricted number of circadian cells (which are part of the evening oscillator, i.e., up to four LNds, including at least a CRYPTOCHROME-positive one, and the fifth sLNv), as well as others located within the pars intercerebralis (PI), a neurosecretory structure recently identified as part of the output pathway relevant in the control of locomotor behavior. In contrast to the extensive connections between DN1p and sLNv clusters, only very discreet reconstituted puncta were detected. Quite strikingly, the degree of connectivity appeared to change across the day, reaching a maximum (when almost every brain exhibited reconstituted signal) at ZT22. However, due to the nature of the signal, no quantitation of its intensity was attempted. Although a more detailed analysis is required to define the identity (i.e., whether it is one or several LNds, the fifth sLNv, or both groups that directly contact the sLNvs), this finding highlights a potentially direct contact between the neuronal substrates of the morning and evening oscillators. In sum, through GRASP analysis, this study has begun to map the connectivity within the circadian network; commensurate with a hierarchical role, the sLNvs appear to differentially contact specific subsets in a distinctive fashion (Gorostaza, 2014).
To address the possibility that PDF neurons could be contacting noncircadian targets at different times across the day, an enhancer trap screen was carried out employing a subset of GAL4 enhancers selected on the basis of their expression pattern in the adult brain, i.e., known to drive expression in the dorsal protocerebrum, and an additional requirement imposed was that none of the selected GAL4 lines could direct expression to the sLNv neurons to avoid internal GFP reconstitution. Reconstitution of the GFP signal at the sLNv dorsal terminals by recognition through specific antibodies was assessed at three different time points for each independent GAL4 line (ZT2, ZT14, and ZT22). Some of the GAL4 lines showed reconstituted GFP signal at every time point analyzed, suggesting that those neuronal projections are indeed in close contact across the day and might represent stable synaptic contacts. No GFP signal was detected in the negative parental controls. Despite the fact that several GAL4 drivers directed expression to the proximity of the PDF dorsal terminals, some of the selected lines did not result in reconstituted GFP signal (Gorostaza, 2014).
Quite remarkably, a proportion of the GAL4 lines showed GFP+ signal only at a specific time point. One such example is line 3-86, where reconstitution was detected in most of the brains analyzed at ZT2, but not at nighttime. Being able to identify putative postsynaptic contacts to the sLNvs in the early morning is consistent with the observation of a higher number of BRP+ active zones in the early day. This enhancer trap spans different neuropils, such as the mushroom body (MB) lobes and lateral horn, and directs expression to particularly high levels in the PI, a structure that has recently been implicated in the rhythmic control of locomotor activity. In fact, some yet unidentified somas in the PI appear to arborize profusely near the PDF dorsal terminals, underscoring a potential link between the two neuronal groups. These direct contacts are unlikely to be the ones reported by Mai179-GAL4; pdf-GAL80 since those connect to the sLNv neurons preferentially at night. Interestingly, a subset of neurons in the PI is relevant in mediating the arousal promoting signal from octopamine; in addition, sleep promoting signals are also derived from a different subset of neurons in the PI, opening the attractive possibility that both centers could be under circadian modulation (Gorostaza, 2014).
GRASP analysis also uncovered a different neuronal cluster (4-59) that contacts PDF neurons preferentially during the early night (ZT14), which is in itself striking, since this time point corresponds to that with fewer arborizations and an overall decrease in the number of synapses. This enhancer trap is expressed in the MBs, subesophagic ganglion, antennal lobes, and accessory medulla. Among those structures, the MBs are important for higher-order sensory integration and learning in insects. Interestingly, circadian modulation of short-term memory and memory retrieval after sleep deprivation has been reported; short-term memory was found to peak around ZT15-ZT17, coinciding with the window of GFP reconstitution, thus providing a functional connection to the synaptic plasticity observed. To corroborate whether there is a direct contact between the two neuronal clusters, the extensively used GAL4 driver OK107, which is expressed in the α'/β'and the γ lobes of the MBs and to a lower extent in the PI, was employed for GRASP analysis. Surprisingly, reconstituted GFP signal could be observed at every time point analyzed, suggesting that MB lobes contact PDF neurons throughout the day but that specific clusters (for example those highlighted by the 4-59 line) establish plastic, time-of-day-dependent physical contact with PDF neurons (Gorostaza, 2014).
It was next asked whether these prospective postsynaptic targets of PDF neurons could play a role in the output pathway controlling rhythmic locomotor activity. To address this possibility, the impact of adult-specific alteration of excitability of distinct neuronal groups was examined through expression of TRPA1. Interestingly, adult-specific depolarization of specific neuronal populations triggered a clear deconsolidation of the rhythmic pattern of activity, which resulted in less-rhythmic flies accompanied by a significant decrease in the strength of the underlying rhythm. These results lend support to the notion that the underlying neuronal clusters are relevant in the control of rest/activity cycles (Gorostaza, 2014).
Over the years, it has become increasingly clear that the circadian clock modulates structural properties of different cells. In fact, a number of years ago, it was reported that the projections of a subset of core pacemaker fly PDF+ and mammalian VIP+ neurons undergo structural remodeling on daily basis. The work presented in this study lends support to the original hypothesis that circadian plasticity represents a means of encoding time-of-day information. By changing their connectivity, PDF neurons could drive time-specific physiological processes. As new synapses assemble while others are dismantled, the information flux changes, allowing PDF neurons to promote or inhibit different processes at the same time. This type of plasticity adds a new level to the complex information encoded in neural circuits, where PDF neurons could not only modulate the strength in the connectivity between different partners, but also define which neuronal groups could be part of the circadian network along the day. Although further analysis of the underlying process is ensured, evidence so far supports the claim that structural plasticity is an important circadian output (Gorostaza, 2014).
Synaptotagmin is one of the major integral membrane proteins of synaptic vesicles. It has been postulated to dock vesicles to their release sites, to act as the Ca2+ sensor for the release process, and to be a fusion protein during exocytosis. To clarify the function of this protein, a genetic analysis of the synaptotagmin gene in Drosophila was undertaken. Five lethal alleles of synaptotagmin were identified, at least one of which lacks detectable protein. Surprisingly, however, many embryos homozygous for this null allele hatch and, as larvae, crawl, feed, and respond to stimuli. Electrophysiological recordings in embryonic cultures confirm that synaptic transmission persists in the null allele. Therefore, synaptotagmin is not absolutely required for the regulated exocytosis of synaptic vesicles. The lethality of synaptotagmin in late first instar larvae is probably due to a perturbation of transmission that leaves the main apparatus for vesicle docking and fusion intact (DiAntonio, 1993).
Synaptotagmin is a synaptic vesicle protein implicated in neurotransmitter release. Molecular characterization of four mutant alleles of this protein in Drosophila has permitted an investigation of Synaptotagmin's role in synaptic physiology and of some of the structural requirements for its function. Reduced levels of Synaptotagmin result in a substantial alteration in synaptic function in the eye and at larval neuromuscular junctions. Decreased neurotransmitter release causes smaller evoked synaptic potentials. However, the frequency, but not the size, of spontaneous quantal events is simultaneously increased. These abnormalities do not appear to be secondary to a detectable morphological change in the arborization of the synapse. The increased frequency of spontaneous events is insufficient to deplete significantly the vesicle supply and thereby account for reduced transmission (DiAntonio, 1994).
Synaptotagmin (Syt), a synaptic vesicle-specific protein known to bind Ca2+ in the presence of phospholipids, has been proposed to mediate Ca(2+)-dependent neurotransmitter release. The role of Syt in neurotransmitter release in vivo has been addressed by generating mutations in synaptotagmin in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca2+ and a 15-fold reduction in EJP amplitude in 1.0 mM Ca2+ when compared with heterozygous controls. In contrast, an increase in the frequency of spontaneous miniature EJPs is observed in the mutants. These results provide in vivo evidence that Syt plays a key role in Ca2+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release (Littleton, 1993b).
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Genetic and electrophysiological evidence is presented that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as the increase in rate of spontaneous vesicle fusion can be electrophysiologically separated from the decrease in evoked response. Some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis (Littleton, 1994).
Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that Synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. It is concluded that Synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. These data support a model in which Synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission (Broadie, 1994).
Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium-regulated fusion of synaptic vesicles with the presynaptic membrane. The possible requirement for synaptotagmin in axonal outgrowth has been examined by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. In wild-type embryos, synaptotagmin is expressed abundantly in axons and growth cones before synapse formation. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, it has been demonstrated that vesicle populations containing Synaptotagmin actively undergo exocytosis during axonogenesis. In synaptotagmin null mutations, immunocytochemical techniques were used to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein Syntaxin, and the synaptic vesicle protein Cysteine string protein. The distribution of these proteins is similar in wild-type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, it is suggested that the molecular mechanisms underlying vesicular-mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release (Littleton, 1995).
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, Synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for Synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Genetic and electrophysiological evidence is presented that Synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that Synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as the increase in rate of spontaneous vesicle fusion can be electrophysiologically separated from the decrease in evoked response. Some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a Synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis (Littleton, 1998).
Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. The relationship between synaptotagmin and the number of morphologically docked vesicles has been investigated by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal is reduced, although each active zone continues to have a cluster of vesicles in its vicinity. In addition, there is an increase in the number of large vesicles near synapses. Examining the clusters, it was found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, is reduced to 24% of control in the syt null mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, syt mutants were examined in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation results in the expected decrease in evoked release but suppresses the increase in spontaneous release frequency. Motor nerve terminals in this genotype contain more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones is still reduced (33% of control). These findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the known increase in spontaneous release that occurs in syt mutants. These results support the hypothesis that synaptotagmin stabilizes the docked state (Reist, 1998).
Genetic analysis of a Drosophila synaptotagmin (Syt) I mutant (AD3) has revealed that Tyr-334 within the C2B domain is essential for efficient Ca(2+)-dependent neurotransmitter release. However, little is known as to why a missense mutation (Tyr-334-Asn) disrupts the function of the C2B domain at the molecular level. Evidence is presented that a Tyr-312 to Asn substitution in mouse Syt II, which corresponds to the Drosophila AD3 mutation, completely impairs Ca(2+)-dependent self-oligomerization activity mediated by the C2B domain but allows partial interaction with wild-type proteins in a Ca(2+)-dependent manner. This observation is consistent with the fact that the AD3 allele is homozygous lethal but complements another mutant phenotype. The Ca(2+)-dependent C2B self-oligomerization is inhibited by inositol 1,3,4, 5-tetrakisphosphate, a potent inhibitor of neurotransmitter release. All of these findings strongly support the idea that self-oligomerization of Syt I or II is essential for neurotransmitter release in vivo (Fukuda, 2000).
Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Synaptotagmin can facilitate SNARE complex formation in vitro and synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and thus diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes (Littleton, 2001).
Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin [syt(null)] strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreases evoked transmitter release by >95%, and decreases the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission (Mackler, 2002).
To begin structure-function studies on synaptotagmin, the quantity and distribution of a number of synaptic proteins were examined in syt mutants. In the null sytAD4 mutant, no synaptotagmin could be detected by Western analysis or immunocytochemistry using anti-synaptotagmin antibodies. Both sytAD3 and sytAD1 mutants make and target the mutated synaptotagmin to synapses as assayed by immunocytochemistry and Western analysis. The levels of the AD3 mutant protein are unaltered from wild-type. Immunostaining for the AD1 mutant protein is reduced, but it is properly localized to synapses, suggesting the C2B domain is not required for synaptic targeting of synaptotagmin. The reduced staining in sytAD1 can be attributed in part to the loss of missing epitopes in the C2B domain, since the polyclonal anti-synaptotagmin antibody was made to both C2 domains (Littleton, 1993a). syt mutants do not affect neuronal pathfinding or synaptic morphology, ruling out a developmental role for synaptotagmin. In addition, Western analysis demonstrated that the mutants did not reduce the levels of the SNARE proteins Syntaxin or Synaptobrevin, or the postsynaptic glutamate receptor GluRIIA. The distribution of synaptobrevin at synapses was also normal in syt mutants (Yoshihara, 2002).
To assess the physiological consequences of disrupting Synaptotagmin, synaptic currents arising from activation of neuromuscular junctions on muscle fiber 6 were measured in mature Drosophila embryos. At this stage of development, the neuromuscular junctions on muscle fibers 6 and 7 contain approximately 8.5 ± 1.7 varicosities, each containing a few active zones. Recordings were done with syt alleles in trans to a null mutant of syt [Df(2L)N13] that specifically deletes the 5' end of the gene (Littleton, 1994). In control animals, nerve stimulation elicits rapid synchronous vesicle fusion that is completed in several milliseconds. In sytAD4 null mutants, synchronous release is abolished, with the appearance of residual delayed release that is not observed in controls. Asynchronous release in sytAD4 mutants following an action potential is clearly distinct from spontaneous fusion events (minis), since mini frequency is less than 0.01 Hz at these synapses. Delayed release is rarely observed in wild-type, indicating that Synaptotagmin functions to suppress asynchronous release and trigger rapid and transient fusion signals that contribute to the high temporal resolution of synaptic transmission. The absence of asynchronous release in wild-type animals is not due to vesicle depletion following an action potential, since wild-type synapses can continue to release neurotransmitter during stimulation frequencies as high as 50 Hz, as well as during high K+- or Ca2+-ionophore-induced release. The population time constant for delayed release latencies measured over many stimulation trials in sytAD4 was calculated from exponential fitting and is 115 ms, in dramatic contrast to the less than 6 ms time constant measured for synchronous release in wild-type. Latency histograms of delayed release in sytAD4 reveal that asynchronous release is not detected in 1 mM Ca2+, but is robust in 2 mM Ca2+, indicating a steep Ca2+ dependence. The time constant and fourth order Ca2+ cooperativity of delayed release in syt mutants in mice (Geppert, 1994; Goda, 1994) is similar to what has been measured in Drosophila, suggesting a conserved molecular mechanism for asynchronous release. The shape of the postsynaptic response to delayed release is identical to that elicited from synchronous release in wild-type, suggesting that once the fusion pore is triggered to open, fusion kinetics are identical with or without synaptotagmin. Thus, synaptotagmin is required to rapidly trigger vesicle fusion in response to Ca2+ entry, while suppressing asynchronous release. This contrasts with mutations in the t-SNARE syntaxin, that abolish both synchronous and asynchronous release, suggesting the SNARE complex is required for both vesicle fusion pathways. This analysis identifies two distinct phases of neurotransmitter release at Drosophila synapses -- a fast component completed within a few milliseconds, and a second phase of asynchronous release that has a decay time constant of 115 ms (Yoshihara, 2002).
Given that only asynchronous fusion is present in the complete absence of synaptotagmin, partial loss-of-function alleles were examined to determine the molecular features of synaptotagmin required to suppress asynchronous fusion and trigger the rapid synchronous release observed at wild-type synapses. In contrast to the complete absence of synchronous release in sytAD4, sytAD1 embryos show synchronous release, although it is greatly reduced compared to controls. The synchronous component of release observed in sytAD1 suggests that the C2A domain alone is capable of promoting rapid vesicle fusion, likely due to the preserved Ca2+-dependent binding of the C2A domain to phospholipids in the AD1 mutant (Littleton, 2001). A comparison of delayed release in sytAD1 and sytAD4 indicates that the presence of the C2A domain alone is able to suppress some, though not all, of the asynchronous release that is observed in the null mutant. Although the absolute number of asynchronous release events is reduced in AD1, the time constant for the remaining delayed release events is unchanged. Thus, both the synchronous and asynchronous phases of release coexist in the AD1 mutant. Whereas asynchronous release is still present in sytAD1, no asynchronous release was detected in sytAD3. Given the underlying biochemical defects in the AD1 and AD3 mutant proteins, the triggering of synchronous release and the suppression of asynchronous fusion events likely require synaptotagmin to engage both SNAREs and phospholipids during elevated Ca2+ transients, but do not require Ca2+-dependent conformational changes in C2B required for oligomerization (Yoshihara, 2002).
Next the properties of synchronous release remaining in sytAD1 and sytAD3 were compared. Wild-type embryonic synapses have a Ca2+ dependence of synchronous release with a cooperativity of 3.5 in nonsaturating Ca2+ ranges (between 0.2 mM Ca2+ and 0.5 mM Ca2+). This cooperative Ca2+ dependence is consistent with previous studies in mammals and Drosophila and is thought to arise from approximately 4 Ca2+ ions that cooperatively bind to the Ca2+ sensor(s). In sytAD3, Ca2+ cooperativity is not altered (3.5 between 0.5 mM Ca2+ and 1 mM Ca2+) despite a more than 10-fold reduction in quantal content. SNARE binding is not abolished in sytAD3 mutants, while Ca2+-dependent conformational changes required for oligomerization of synaptotagmin are severely disrupted (Littleton, 2001; Fukuda, 2000). Thus, Ca2+ binding by the C2B domain greatly enhances the sensitivity of vesicle fusion to Ca2+, but is not required for cooperativity. In stark contrast, the cooperative Ca2+ dependence of synchronous release is abolished in sytAD1 mutants. Mean quantal content is linearly dependent on Ca2+ between 0.5 mM Ca2+ and 2 mM Ca2+ (slope value of 0.77) and there is no increase in release between 2 mM and 4 mM Ca2+ in sytAD1 mutants. The loss of cooperativity in sytAD1 as opposed to sytAD3 suggests that the Ca2+ cooperativity of neurotransmitter release likely arises from synaptotagmin's Ca2+-dependent interaction with SNAREs. To rule out the possibility that the syt alleles caused dominant-negative effects on neurotransmission, nerve-evoked release was examined in 0.5 mM Ca2+ in heterozygotes of syt mutants and quantal contents were compared with homozygotes containing both copies of the wild-type allele. All heterozygotes showed approximately half the quantal content of controls. Single factor ANOVA and Scheffe's multiple comparision tests indicated significant differences between heterozygotes and controls (+/+), but no differences between the null alleles and sytAD3 or sytAD1. These findings indicate synaptotagmin is dose dependent for neurotransmitter release and that sytAD3 and sytAD1 do not have dominant-negative effects (Yoshihara, 2002). .
Synaptotagmin has also been proposed to function in vesicle recycling (Zhang, 1994; Jorgensen, 1995; Reist, 1998), suggesting that the reduction in quantal content in syt mutants could be confounded by a reduced pool size. To test this possibility, motor nerves were stimulated repetitively at 10 Hz to elevate and sustain Ca2+ concentrations to enhance release via the high-affinity Ca2+ sensor. In sytAD1, sytAD3, and sytAD4, large numbers of vesicles were released during repetitive stimulation. Although the total number of vesicles released was approximately 2-fold less than in wild-type, the reduction in mean quantal content in sytAD1 (more than 100-fold) and sytAD3 (more than 10-fold) cannot be accounted for by reductions in vesicle recycling, docking, or the generation of fusion-competent vesicles. As observed in the response to single stimuli, release during repetitive stimulation in sytAD4 is asynchronous to nerve stimulation. To further test vesicle availability, hypertonic stimulation (500 mM sucrose) was used to measure the readily releasable pool. Similar numbers of vesicles were released in sytAD3 and controls in response to hypertonic stimulation. This indicates sytAD3 has a normal readily releasable vesicle pool size and the decrease in quantal content following nerve stimulation is caused by reduced release probability. sytAD1 and sytAD4 both show a 3-fold reduction in vesicle fusion induced by hypertonic stimulation compared to wild-type. One interpretation of these results is that the readily releasable pool is slightly smaller in sytAD1 than wild-type and may partially contribute to the reduced probability of release (though a 3-fold reduction in pool size would only be a very minor contributor to a 100-fold reduction in release probability). However, repetitive stimulation in sytAD1 can induce the same number of fusion events as sytAD3, which has a normal vesicle pool size. A second interpretation that is favored is that the reduced SNARE binding by synaptotagmin in sytAD1 and sytAD4, as opposed to sytAD3, makes the fusion machinery unstable and the activation energy needed for hypertonic-induced fusion larger. This interpretation is consistent with recent studies indicating that synaptotagmin I stabilizes the fusion pore during dense core vesicle release (Wang, 2001) and that SNARE proteins are required (Fergestad, 2001) for hypertonic-induced vesicle fusion (Yoshihara, 2002).
In addition to triggering Ca2+-dependent fusion, synaptotagmin has also been postulated to function as a fusion clamp, inhibiting vesicle release in the absence of Ca2+. This model is supported by the increased frequency of spontaneous miniature synaptic currents (minis) in Drosophila hypomorphic mutants recorded late in larval development (Littleton, 1993b, 1994; DiAntonio, 1994) and by the suppression of acetylcholine release from fibroblast cells transfected with synaptotagmin I (Morimoto, 1995). This hypothesis was tested by examining miniature frequency in sytAD1, sytAD3, and sytAD4. No increase was found in mini frequency at embryonic synapses, consistent with recordings from young synapses in neuronal cultures from synaptotagmin mutant mice (Geppert, 1994). However, Drosophila synaptotagmin suppresses delayed asynchronous release. Since delayed release is induced by residual Ca2+, likely via a high-affinity Ca2+ sensor, the lack of detectable delayed release at wild-type synapses indicates a suppression of this release mechanism by synaptotagmin I. It is known that presynaptic Ca2+ concentrations remain elevated for several hundred milliseconds after an action potential, yet release events largely occur only during the early peak of the Ca2+ spike. The ability to restrict vesicle fusion temporally within milliseconds to the onset of a Ca2+ signal is an essential feature of brain function, yet the molecular machinery that mediates this critical task is unknown. These data indicate that Synaptotagmin performs this essential function by clamping asynchronous release during sustained Ca2+ influx. To test this hypothesis, presynaptic terminals were depolarized with high potassium (K+) and the frequency of fusion events that occur during sustained Ca2+ influx induced by depolarization was examined. Release properties under these conditions in the null mutant were compared with release in sytAD3, which has a normal vesicle pool size as assayed by hypertonic stimulation and shows no asynchronous fusion. A higher frequency of spontaneous release was observed in sytAD4 than in sytAD3 in solutions containing 60 mM K+ and 0.25 mM Ca2+, indicating that the lack of a synaptotagmin fusion clamp in the null mutant leads to excessive release activated through the high-affinity Ca2+ sensor. In addition, fusion induced by high K+ in the sytAD4 null mutant can exceed 10 Hz and continue unabated for over an hour during continuous recordings, confirming that a large recycling vesicle pool is present in syt nulls and can be triggered to fuse via the asynchronous high-affinity Ca2+ sensor. These data exclude an essential role for synaptotagmin in synaptic vesicle endocytosis and docking. It is possible that a subpopulation of the vesicle pool at nerve terminals is not recycling properly in syt nulls, but these data indicate that the recycling pathway remaining in syt mutants is capable of robustly resupplying the readily releasable vesicle pool (Yoshihara, 2002).
To further characterize the suppression of the asynchronous release mechanism by synaptotagmin, release properties were examined in sytAD4 and sytAD3 in preparations where sustained elevated Ca2+ concentrations were triggered using the Ca2+ ionophore, ionomycin. Since Ca2+ influx into presynaptic terminals occurs through ionomycin channels, as opposed to Ca2+ channels, synaptic vesicle release occurs through a general increase in nerve terminal Ca2+ concentrations. This provides a more appropriate experimental condition to examine the effects of sustained Ca2+ elevation than the high K+ experiments described above. Addition of 5 µM ionomycin results in a large gradual increase in vesicle release secondary to the influx of Ca2+ through ion-permeable channels in the membrane that bypass the presynaptic Ca2+ channel. Under conditions of sustained Ca2+ influx, sytAD4 null mutants show a ~10-fold higher frequency of release than in sytAD3 mutants at 1 and 2 min following ionomycin addition, suggesting that the ability of the AD3 mutant protein to function as a clamp suppresses asynchronous release responding to low Ca2+ concentrations via the high-affinity Ca2+ sensor. It is concluded that Synaptotagmin functions to restrict synaptic vesicle fusion temporally to the Ca2+ signal during an action potential and prevent further release that is asynchronous to the initial Ca2+ spike (Yoshihara, 2002).
A noninvasive technique has been demonstrated for protein photoinactivation using a transgenically encoded tag. A tetracysteine motif that binds the membrane-permeable fluorescein derivative 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) was engineered into synaptotagmin I (Syt I4C). Neuronally expressed Syt I4C rescues the syt I null mutation, can be visualized after FlAsH labeling, and is normally distributed at the Drosophila neuromuscular synapse. Illumination of FlAsH bound Syt I4C at 488 nm decreases evoked release in seconds demonstrating efficient fluorophore-assisted light inactivation (FlAsH-FALI) of Syt I. The inactivation of Syt I is proportional to the duration of illumination and follows first-order kinetics. In addition, Syt I FlAsH-FALI is specific and does not impair Syt I-independent vesicle fusion. This study demonstrates that Syt I is required for a post-docking step during vesicle fusion but does not function to stabilize the docked vesicle state (Marek, 2002).
The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. This study reports the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins alpha and beta). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. The results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse (Adolfsen, 2004).
Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. This study reports the development of supernumerary or 'satellite' boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. It is proposed that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals (Dictman, 2006).
A wide variety of endocytosis mutants produce 4–10 times more satellite boutons than wild-type controls and with comparable severity to that reported in dap160 mutants. The extent to which endocytosis was reduced in each genotype may differ. For example, synaptotagmin facilitates, but is not absolutely required for, endocytosis, and the rate of classical endocytosis is slowed, but not abolished, in null alleles of synj and endo. Further, AP180 mutants retain the ability to endocytose synaptic vesicles, even though vesicle size is altered, and shibire was studied at a temperature that does not fully block endocytosis. Indeed, because endocytosis is essential for viability, the satellite boutons arise from the impairment rather than the abolition of endocytosis in each mutant studied. Endocytosis mutations have also been shown to alter bouton number at the neuromuscular junction and synaptic development in the giant fiber system (Dictman, 2006).
Secondary changes in the amount of synaptic transmission are not likely to account for the anatomical changes reported in this study because neither a severe loss of postsynaptic sensitivity in DGluRIII mutants nor the poisoning of the fusion machinery for presynaptic release with tetanus toxin caused any increase in satellite boutons. Similarly, mutants were examined in eag, Sh, and napts, mutations that alter neuronal excitability, and no satellite boutons were observed. Thus, the mutant phenotype correlates with the loss of endocytosis per se (Dictman, 2006).
Mutations in none of the known signaling pathways at the Drosophila NMJ display phenotypes resembling the satellite boutons observed in this study. The BMP, highwire, and activity-dependent pathways seem to regulate the extent of the muscle surface covered by the neuronal terminal and the size and strength of synaptic boutons, but satellite boutons have not been reported in these mutants. However, mutations in some synaptic proteins give rise to altered synaptic morphologies that resemble, at least in part, the satellite boutons described in this study. It is possible, therefore, that these molecules (Appl, Spastin, Shaggy, and Dlar) are elements of a developmental process that is disrupted by endocytosis mutants. The increased density of active zones and dense bodies in endocytosis mutants also represents an unusual phenotype. In fasciclin II mutants, for example, the spacing of active zones remains normal despite alterations in the morphology and number of boutons. However, changes in postsynaptic sensitivity can cause increases in the density of presynaptic release sites, and a similar compensatory mechanism may explain the increased density of active zones observed in endophilin and synaptojanin mutants. Thus, the phenotype linked to endocytic defects at these synapses cannot readily be tied to any of the previously known signaling pathways, and the mechanism for satellite bouton formation may be distinct from that which regulates active zone number and spacing (Dictman, 2006).
To explain the generation of satellite boutons, three hypotheses have been considered: that they arise (1) as an indirect consequence of excess surface membrane; (2) from the direct involvement of endocytic proteins in cytoskeletal organization, and (3) from a defect in a signaling pathway, regulated by endocytosis, that normally controls bouton growth and spacing. The first of these hypotheses is the most straightforward, but is difficult to reconcile with the details of the anatomical phenotypes. The satellite boutons are not simple infoldings of the membrane, but are highly organized, functional, and connected by stalks to the axial boutons. In addition, a net shift of synaptic vesicles to the plasma membrane could not explain increases in active zone number and the density of active zones and dense bodies. Thus, an inability to recover synaptic vesicle membrane might contribute to the phenotype; however, considerable remodeling of the synapse, including the addition of new active zones, would have to follow as a consequence (Dictman, 2006).
The second hypothesis, stipulating a direct ability of these endocytic proteins to organize the cytoskeleton, has been proposed as an explanation of the phenotype of dap160 mutants, an endocytic protein that may interact with Cdc42/N-WASP and its effectors and has been suggested to serve as a regulator of the actin cytoskeleton. A similar explanation was also invoked in nervous wreck mutants, which alter a WASP binding protein that may regulate the cytoskeleton and whose synaptic branching phenotype may or may not be related to satellite boutons. However, most of the proteins studied are not known to interact directly with the cytoskeleton or its regulators (Dictman, 2006).
The third hypothesis is highly speculative -- that endocytosis is crucial to one or more signaling pathways in the nerve terminal that regulate bouton growth and active zone formation at the neuromuscular junction. The satellite boutons and altered active zone spacing, however, are unlike the phenotypes associated with known ligands and receptors at the neuromuscular junction; this hypothesis therefore suggests the presence of a novel pathway controlling the development of this synapse. Plausibly, this could be a factor secreted by either the nerve or muscle that promotes the formation of side branches and whose surface receptor is inactivated by endocytosis. Alternatively, it could be a factor that suppresses bouton and active zone formation and whose receptor requires internalization, perhaps into a signaling endosome, for efficacy. These possibilities must remain conjectural pending the identification of the hypothesized signaling pathways (Dictman, 2006).
A previous analysis indicated that active zones in various genotypes conform to a regular spacing function, which ensures that neighboring active zones are spaced between 0.84 μm and 1.05 μm apart in the boutons of Axon 1. This spacing was also confirmed for the wild-type boutons examined in this study (Ra = 0.94 μm) and suggests that each active zone exerts an inhibitory influence on nearby synaptogenesis. In synj boutons, however, it was found that active zones are more densely packed (Ra = 0.75 μm) and have more dense bodies than control boutons. Active zones are slightly smaller in synj than in wild-type boutons, so that the percentage of the bouton plasma membrane covered by active zone remains roughly constant, with 38%–46% of the surface area in both mutant and control terminals falling outside the active zone region. It is in this region, the periactive zone, that proteins of the endocytic pathway are concentrated, each active zone being surrounded by an annulus of endocytic machinery to form an integral zone of recycling. The persistence of this region in synj is consistent with immunocytochemical observations that other endocytotic proteins remain localized to periactive zones in synj terminals and that the classical endocytotic pathway endures in synj mutants (Dictman, 2006).
The increased number of dense bodies per active zone in synj mutant boutons resembles that seen after neuromuscular transmission is strengthened by raising wild-type larvae at 29°C, relative to those raised at 18°C. The correlation of dense-body number with synaptic strength suggests that this aspect of the synj phenotype may be part of a compensatory mechanism to offset any decreases in exocytosis resulting from vesicle depletion. At low frequencies of stimulation, synj synapses release normal levels of quanta despite a greatly decreased pool of vesicles in the terminal. The anatomical phenotype of synj may, at least in part, account for this release: the increased number of boutons and the spacing density of dense bodies at active zones increases the number of release sites at least 2-fold per neuromuscular junction; this change in dense-body number may reflect an increase in release probability per active zone (Dictman, 2006).
The analysis of mutations in endocytic proteins has illuminated the process by which the fine features of the synapse are sculpted during development. Endocytosis per se is needed for the normal organization of the neuromuscular junction and this process is hypothesized to be required as part of a signaling system organizing synaptic growth. Interestingly, the endocytic machinery is localized to periactive zone regions, areas in which many signaling molecules that regulate synaptic development also reside. This juxtaposition of the apparatus for synaptic endocytosis with one for developmental signaling is consistent with a role for endocytosis in regulating development, in addition to its established role in synaptic vesicle recycling. Moreover, the ultrastructural changes in synj terminals suggest a homeostatic, compensatory mechanism to adapt active zone spacing and synaptic strength in response to diminished vesicle recycling (Dictman, 2006).
Synapses lacking functional synaptotagmin I (Syt I), the primary Ca2+ sensor for synaptic vesicle exocytosis, have a decreased rate of synaptic vesicle endocytosis. Beyond this, the function of Syt I during endocytosis remains undefined. This study demonstrates that a decreased rate of endocytosis in sytnull mutants correlates with a stimulus-dependent perturbation of membrane internalization, assayed ultrastructurally. The mechanisms that control endocytic rate and vesicle size were separated by mapping these processes to discrete residues in the Syt I C2B domain. Mutation of a poly-lysine motif alters vesicle size but not endocytic rate, whereas the mutation of calcium-coordinating aspartate residues (syt-D3,4N) alters endocytic rate but not vesicle size. Finally, slowed endocytic rate in the syt-D3,4N animals, but not sytnull animals, can be rescued by elevating extracellular calcium concentration, supporting the conclusion that calcium coordination within the C2B domain contributes to the control of endocytic rate (Poskanzer, 2006).
Endocytosis rate can proceed normally while vesicles of inappropriate size are generated. Conversely, endocytosis rate can be slowed without perturbing the mechanisms that control vesicle size. Thus, the mechanisms that govern the rate and size of synaptic vesicle re-formation can be broken down into independent processes that are normally coordinated by Syt I (Poskanzer, 2006).
Two important comparisons should be made with previous work in which a function was demonstrated for Syt I during endocytosis. In synapto-pHluorin (SpH) experiments examining sytnull animals, muscle contraction occluded a portion of the data during and shortly after nerve stimulation and prevented an accurate calculation of endocytic rate. By including a postsynaptic receptor antagonist in the current experiments, muscle contraction was prevented, allowing uninterrupted visualization of fluorescence change over time. The current data now agree with a report examining vesicle recycling at sytnull central neurons in which endocytosis is significantly slowed but not blocked. It was also previously reported that FlAsH photoinactivation of Syt I prevented subsequent quenching of SpH following the release of a temperature-sensitive blockade of endocytosis. The data now demonstrate that, in this protocol, membrane is internalized in the form of unusually large vesicles. Why was there no evidence of quenched SpH signal in the previous study? It was previously shown, by FM4-64 staining, that membrane internalization was impaired following FlAsH photoinactivation and that a significant fraction of SpH remains at the cell surface. If reacidification is also impaired within the large vesicle structures, then the combined effect of these three deficits may prevent significant SpH quenching during this protocol (Poskanzer, 2006).
Mutation of a C2B domain poly-lysine motif, previously shown to be important for Syt I-AP-2 binding, causes a defect in vesicle size. This defect in vesicle size is less severe than that observed in the sytnull animal, indicating that these residues contribute to this role of Syt I, but may not be the only residues important for this process. It is remarkable, however, that the mutation of the C2B poly-lysine motif causes a selective defect in vesicle size without altering endocytosis rate. The specificity of this endocytic defect is surprising, considering that altered rate and altered membrane retrieval generally go hand in hand in genetic studies of synaptic vesicle recycling in vivo (Poskanzer, 2006).
How can the specific defect in the size of recovered synaptic vesicles be explained? Previous studies have described endosomal cisternae that may represent normal intermediates in a vesicle recovery pathway, similar to those observed in non-neuronal cells. One possibility is that perturbing the Syt I-AP-2 interaction slows the progression through an acidified endosomal intermediate (perhaps before a clathrin-dependent budding step) that is normally rapidly resolved. Syt I-inositol polyphosphate interactions mediated by these residues could also be important for this process. However, no budding events from large vesicular structures were observed in micrographs, whereas budding from larger endosomal/cisternal membranous structures has been described at other synapses following stimulation. An alternate possibility is that Syt I controls the size of synaptic vesicle membrane retrieval at the plasma membrane. Syt I could demarcate the boundaries of new vesicles and thereby control the amount, but not the speed, of membrane internalization. Finally, Syt I could control the integrity of the clathrin lattice, which could help to confine the proper extent and curvature of newly internalized membrane (Poskanzer, 2006).
Mutation of Ca2+-coordinating residues in the C2B domain of Syt I slows endocytosis rate without obviously altering any other aspect of endocytosis. It should be emphasized that when Ca2+-coordinating residues in the C2B domain are mutated, the endocytosis rate slows to the same extent as that observed in sytnull mutations, demonstrating that these residues are sufficient to account for the function of Syt I in specifying endocytic rate. Since raising extracellular Ca2+ significantly restores endocytosis rate in this mutation, it indicates that the Ca2+ coordination by these residues is important for Syt I function during endocytosis. Importantly, no change in endocytic rate is observed in sytnull animals, demonstrating that this is a Syt I-dependent effect, likely mediated by the binding of Ca2+ to either the C2A or C2B domains of Syt I. Because the C2A domain does not appear to play a significant role in endocytosis rate, the conclusion is favored that Ca2+ coordination in the C2B domain of Syt I is essential for endocytosis. Finally, these data are in contrast to most other genetic studies of endocytosis molecules where a slower endocytic rate is accompanied by other effects, such as altered membrane internalization or vesicle size. Therefore, the current data suggest that Ca2+-coordinating residues in the C2B domain of Syt I are involved in a step of the endocytic process directly related to the endocytic rate (Poskanzer, 2006).
There are several possibilities for how Syt I could participate in the control of endocytic rate. First, it is possible that these Ca2+-coordinating residues are involved in an interaction controlling the uncoating of synaptic vesicles prior to vesicle reacidification. Disrupting this process would slow the rate of SpH decay, even though vesicles would be endocytosed correctly. No obvious accumulation of clathrin-coated vesicles is seen in EM images, although it is difficult to resolve clathrin coats on vesicles at this synapse. Another possible explanation is that the Ca2+-coordinating residues are involved in the sorting of synaptic vesicle proteins to newly internalized vesicles. In this case, some SpH molecules may be deposited in the plasma membrane during stimulation, but not reinternalized. If this were true, it might be expected that SpH molecules would accumulate on the plasma membrane over time. No evidence was seen that baseline SpH fluorescence levels are higher in the UAS-D3,4N rescue animals than controls, suggesting that this possibility is unlikely. A third possibility is that Ca2+-coordinating residues within the C2B domain are specifically involved in a rate-limiting process that normally governs how fast vesicles are retrieved from the synaptic plasma membrane. If the initiation of endocytosis were delayed or became variable in these animals, the SpH decay rate would appear slower because SpH measurements reflect the state of an entire population of vesicles. Support for the idea that the initiation of endocytosis is delayed comes from the observation that endocytic intermediates attached to the plasma membrane are not found at the UAS-D3,4N rescue synapses. This hypothesis is attractive because it has long been hypothesized that Syt I functions to couple exo- and endocytosis at the neuronal synapse (Poskanzer, 2006).
If Syt I does indeed function at such an early stage in the endocytic process, two models may explain its role. In one model, Syt I could establish new protein interactions—or take on a new conformation—following vesicle fusion that are required to initiate endocytosis. In a second model, Syt I could assume a conformation or participate in a binding interaction during exocytosis that is required for subsequent vesicle endocytosis. In other words, Syt I would be primed to initiate rapid endocytosis via molecular interactions established during exocytosis. Since mutating residues implicated in Syt I oligomerization does not alter endocytic rate even though exocytosis is perturbed, the data argue against oligomerization of Syt I as being the essential interaction for control of endocytic rate. On the other hand, the importance of phospholipid regulation during endocytosis has been suggested by recent work showing altered endocytic rate in a PI(4,5)P2 kinase null mutation, making this option an attractive possibility (Poskanzer, 2006).
Syt I has been proposed to couple exo- and endo-cytosis because it is required for both processes. This study has identified mutations in Syt I that uncouple exo- and endocytosis, for example, perturbing exocytosis but not endocytosis in the sytAD3 mutations. Mutations were identified in Syt I that separate two properties of compensatory synaptic vesicle endocytosis that are normally coordinated: endocytosis rate and the size of internalized membrane. Thus, different regions of Syt I may normally couple exo- and endocytosis as well as distinct aspects of the endocytic process. These data argue that Syt I is a molecular linchpin that enables rapid, high-fidelity endocytosis at the neuronal synapse (Poskanzer, 2006).
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