InteractiveFly: GeneBrief

unc-104: Biological Overview | References

The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. This study isolated mutations in immaculate connections (imac; FlyBase name unc-104), encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila has implicated Kinesin-1 in transporting synaptic vesicle precursors, this study found that Imac/Unc-104 is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. The arrest of synaptogenesis is not a secondary consequence of the absence of transmission. These data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting (Pack-Chung, 2007).

When a neuronal growth cone reaches its target field and contacts appropriate cells, the axon terminal undergoes synaptic morphogenesis. This transformation is a complex process by which the filopodial specializations of the growth cone and apparatus for axon extension and navigation are replaced by stable varicose nerve terminals specialized for releasing neurotransmitter. At present, the molecular and cellular mechanisms that guide the transition of growth cones to mature synapses are not well defined (Pack-Chung, 2007).

Growth cones and synapses are structurally distinct. Networks of actin cytoskeleton support the outer edge of the growth cone, and the extension of microtubules can direct axonal growth. By contrast, mature synapses contain machinery for stable interactions with postsynaptic specializations, distinct cytoskeletal arrangements, and the apparatus for transmitter release and recycling. This apparatus includes synaptic vesicles and the active zone, an electron-dense membrane region that contains Ca²⁺ channels and protein complexes necessary for transmitter release. Thus, there is a distinction between the components of a growth cone and a mature synapse that parallels their distinct morphologies (Pack-Chung, 2007).

Synaptic components are primarily synthesized in the neuronal cell body and therefore, to be available for forming synapses, need to be transported down the axon. Synapse formation, however, can occur within 30 min of initial axodendritic contact. To permit this rapid transition, synaptic materials are present in axons before the onset of synaptogenesis and are subsequently captured by incipient synaptic contacts. Transported as aggregates of membrane-bound organelles, these vesicular structures, which are sometimes called 'packets', include synaptic vesicle proteins (synaptotagmin-I, synaptobrevin, SV2 and synapsin-1) and plasma membrane proteins (Ca²⁺ channels). These cargos become immobilized at axo-dendritic contacts as part of a rapid process that leads to functional connectivity. One transport organelle is known as a PTV (Piccolo-Bassoon transport vesicle) and is thought to provide many components of active zones. Both the packets and the PTVs show bidirectional movement in axons, but no motor has been shown to mediate this movement (Pack-Chung, 2007).

These transport organelles occur in both peripheral and central synapses of vertebrates, and analogous systems are probably needed at Drosophila neuromuscular junctions (NMJs). At this synapse, motor neuron growth cones transform to functional but immature NMJs within an hour of initial contact with target muscles. Morphological maturation into synaptic boutons, however, requires 3-5 h. As in mammalian systems, synaptotagmin-I and other synaptic components are present in axons before they reach their targets. Bouton formation correlates with increases in synaptotagmin immunoreactivity, in clear- and dense-cored vesicles, and in active zones. Transport of these components during NMJ development is thus implied, although the motors that are required remain unknown (Pack-Chung, 2007).

This study reports the identification of a genetic locus encoding a kinesin that is required for presynaptic maturation. Mutations in the gene immaculate connections prevent nerve endings from transforming to synaptic boutons: growth cones become constricted but remain within contact fields as thin processes that lack varicosities or boutons. In addition to lacking synaptic and dense-core vesicle components, imac mutant nerve endings also contain very few active zones. Characterization of imac thus implicates a particular motor in the process of presynaptic maturation and provides insight into the relationships between motors and synapse formation and pre- and postsynaptic differentiation (Pack-Chung, 2007).

A forward genetic screen was performed to isolate genes affecting synapses. In this screen, the EGUF-hid method was used to produce homozygous mutant eyes in an otherwise heterozygous fly, and synaptic function was assessed by selecting for blindness and abnormal electroretinograms (ERGs). One complementation group consisted of eight independently derived alleles. Homozygous mutant eyes externally appeared normal and their ERGs showed robust responses to light; however, the 'on-off transients' of the ERG, which require synchronous synaptic transmission from the photoreceptors to second-order neurons, were absent in eyes that were homozygous for any of the mutant alleles. Homozygous flies of each allele died as unhatched late-stage embryos. The gross morphology of these embryos was comparable to that of a wild-type fly that has completed embryogenesis 20-22 h after egg-laying (AEL). The mutants, however, were paralyzed and lacked the coordinated muscle peristalsis required for hatching. This paralysis suggested a neurological deficit, and therefore the developmental pattern of individual neurons was examined (Pack-Chung, 2007).

Specifically, the embryonic NMJ, a system in which the timing and anatomy of innervation and synaptogenesis have been described in detail, was explored. For the intersegmental nerve b (ISNb), focus was placed on branches which innervate the ventral longitudinal muscles (muscles 6, 7, 11 and 12). The growth cones arrive by 12 h AEL. These contacts rapidly mature and, by ~14 h AEL (stage 16), the neurites extend along boundaries between adjacent muscle fibers. These contacts then transform into synapses that have characteristic fine branches with varicosities or boutons that resemble 'beads on a string' (Pack-Chung, 2007).

In mutant embryos from this complementation group, including those homozygous for null alleles, ISNb axons had contacted their muscles by 14 h AEL. Despite normal axonal outgrowth and targeting, the contacts lacked extended branches. Filipodia-like structures were sometimes observed, but were not restricted to muscle boundaries. At 21 h AEL, the nerve was visible in its target regions, but the contacts had not transformed into mature synapses; the nerves lacked the bead-like boutons seen in wild-type embryos. Instead, the endings and their branches were more constricted than those at 14 h AEL and frequently retained some filipodia-like processes. The failure to form boutons was completely penetrant; it was observed in every abdominal segment of all genotypes examined (alleles imac⁵², imac¹⁰², imac¹⁶⁰, imac¹¹⁶ and imac¹⁷⁰ as homozygotes or in combination with Df(2R)DAlk21) (Pack-Chung, 2007).

At times, axons at the boundary of muscles 12 and 13 were not observed, raising the possibility that some had retracted. Occasionally, ectopic projections were observed from neighboring nerves. These inputs form as a result of denervation of the target muscle fibers and were therefore consistent with the absence of synapses on these muscles. Unlike the mature boutons formed by ectopic branches in other mutants, however, those that formed in imac mutant embryos did not have boutons. Consequently, both the normal innervation and the occasional ectopic projection were incapable of normal morphogenesis. Thus, axonal outgrowth and targeting of mutant motor neurons occurred but subsequent synapse formation was arrested. The gene was named immaculate connections (imac) to indicate the complete lack of synaptic boutons in the mutant (Pack-Chung, 2007).

Polymerase chain reaction (PCR) length polymorphism (PLP) mapping placed imac between 53C and 54E. A deficiency, Df(2R)DAlk21, that uncovered the region 53C7 to 53D did not complement the imac alleles. In this region, a predicted gene encoding a kinesin homolog (FlyBase symbol Klp53D) was identified as a plausible candidate gene. Sequencing of genomic DNA in all eight alleles of imac identified point mutations that altered the predicted protein product of this gene. Because these mutations were induced on an isozygous genetic background and because nearby genes showed no sequence differences between one another or in comparison to the parental chromosome, the predicted kinesin was identified as Imac. This identification was confirmed by rescuing the embryonic lethality by expression of an imac cDNA transgene under control of either a da-GAL4 or elav-GAL4 driver. With restored neuronal expression of the kinesin, mutant larvae emerged with synaptic boutons at their NMJs (Pack-Chung, 2007).

The predicted Imac product represents a kinesin isoform that has not previously been studied in Drosophila but contains a forkhead-associated (FHA) domain, a characteristic of the Kinesin-3 family of motors. Imac shows most homology to Unc-104, a Kinesin-3 from Caenorhabditis elegans (51% amino acid identity), and the murine kinesins KIF1A (53%) and KIF1Bβ (51%). Although the Drosophila genome predicts the existence of other members of the Kinesin-3 family, Imac is the only member that includes a carboxy terminus pleckstrin homology (PH) domain, a feature shared with Unc-104, KIF1A and KIF1Bβ. imac¹⁷⁰ has an early stop codon at Trp58 and therefore is likely to be a null allele. This allele was therefore used for most of the phenotypic analysis, either as a homozygote or over Df(2R)DAlk21 to avoid the effects of possible second-site mutations. Two of the alleles contain amino acid substitutions in the conserved motor domain. One of these, imac⁵², has a serine substitution at Gly97, an essential residue in the ATP-binding site of all kinesin motors. The arrest of presynaptic bouton formation in the imac⁵² mutant was comparable to that in the null allele mutant, indicating that the developmental defect resulted from loss of an ATP-dependent motor function of Imac (Pack-Chung, 2007).

Polyclonal antibodies were generated to a portion of the stalk domain of Imac. Immunostaining of wild-type embryos at 21 h AEL showed enrichment of Imac in the nervous system, in particular in the synapse-rich regions. Imac staining was absent in homozygous mutant embryos, verifying the specificity of the antiserum for Imac. In whole-mount embryos, neural expression of Imac was first detected at stages 11-12 and remained enriched in the nervous system for the remainder of embryogenesis. The onset of Imac expression corresponds to the expression of many presynaptic proteins. Thus, the temporal and spatial expression of Imac precludes a significant maternal contribution of the protein and instead is suggestive of a function late in neuronal development (Pack-Chung, 2007).

The identification of Imac as a kinesin suggested that the arrest of synaptogenesis might result from a failure to transport necessary cargos. Homologs of Imac in C. elegans (Unc-104) and mouse (KIF1A) are implicated in the transport of synaptic vesicle-associated proteins (Hall, 1991; Okada, 1995; Klopfenstein, 2004; Yonekawa; 1998; Zhao, 2001). In Drosophila neurons, by contrast, the conventional kinesin (Kinesin-1 or KHC) has been reported to transport synaptic vesicles on the basis of the appearance of axonal aggregates of synaptic vesicles in partial loss-of-function mutants of kinesin heavy chain (khc). Therefore the location of synaptic components was examined in imac mutants. NMJs of wild-type embryos at 21 h AEL showed clusters of various presynaptic components at synaptic boutons. For example, synaptotagmin-I, a synaptic vesicle membrane protein, was restricted to discrete zones in the synaptic varicosities. Little or no synaptotagmin-I was observed along the axons of wild-type embryos. In imac mutants, however, synaptotagmin-I staining was lacking both in the nerve endings and in the axon trunk. Synaptotagmin-I immunoreactivity was restored in the mutants by expressing imac cDNA in neurons. Similarly, terminal and axonal staining in the mutants was negligible with antibodies to the vesicular glutamate transporter (VGlut) (Pack-Chung, 2007).

Axon terminals can also release peptides that are stored in dense-core vesicles. These organelles are synthesized in the cell body and undergo kinesin-mediated, microtubule-dependent transport to the site of secretion. To examine whether transport of dense-core vesicles was affected in imac mutants, the expression of a transgene encoding green fluorescent protein (GFP) fused to rat atrial natriuretic factor precursor (ANF) was monitored. This transgene (UAS-ANF-GFP) undergoes processing, transport and release in a manner similar to other neuropeptides in Drosophila. Robust expression of GFP puncta was observed at the synaptic terminals of control lines. ANF-GFP was not detected in either the motor axons or their terminals in imac mutants. Thus, similar to the involvement of C. elegans Unc-104 in dense-core vesicle movement (Jacob, 2003), Imac probably mediates axonal transport of dense-core vesicles in Drosophila (Pack-Chung, 2007).

The lack of synaptic and dense-core vesicle components at the nerve terminals could be attributed to a failure to synthesize these proteins, an inability to export them from the cell bodies, or an inability to retain them at nascent synapses. To explore these possibilities, the localization of the synaptic molecules in the CNS was examined. In the CNS of Drosophila embryos, the neuropil defines a cell body-free region that is enriched in synaptic contacts, axons and glia. The cortex, a region surrounding the neuropil, primarily contains the neuronal cell bodies. Cross-sections of the CNS can thus be used to monitor the concentrations of proteins in cell bodies relative to the axonal and synaptic regions in a broad sampling of neuronal types. Labeling the plasma membranes of neurons with antibodies to horseradish peroxidase (HRP), a neuronal membrane marker, revealed the overall architecture of the CNS. The density of membrane in the neuropil causes this structure to be more intensely labeled than the cortex. This pattern was not detectably altered in imac mutants, indicating that the mutants had no gross anatomical defect (Pack-Chung, 2007).

Components of synaptic and dense-core vesicles were, however, markedly redistributed in imac mutants. Synaptotagmin-I immunoreactivity, for example, is normally intensely concentrated in the neuropil and scant in the cortex. In imac mutants this pattern was reversed: synaptotagmin-I accumulated in cell bodies and was low in the neuropil. Synaptotagamin-I-GFP, expressed in the imac mutant nervous system by an elav-GAL4 driver, showed the same redistribution. VGlut, normally found at a few CNS synapses that are glutamatergic, was similarly redistributed from neuropil to cell bodies in imac mutants, as was cysteine string protein and ANF-GFP. The concentration of the vesicular proteins in imac mutant cell bodies indicates that these proteins are synthesized in the mutants. The loss of these markers from axon tracts and synaptic regions indicates a defect in their transport from the cell bodies. Absence of immunoreactivity of these molecules in the peripheral axons of motor neurons likewise supports the idea that Imac has a direct role in the transport of synaptic materials. In addition, live-cell imaging of vesicle precursors in the segmental nerves of an imac mutant has revealed defects in the anterograde movement of these cargos (R. Barkus and W. Saxton, personal communication to Pack-Chung, 2007), further substantiating the idea that Imac has a motor function (Pack-Chung, 2007).

To determine whether Imac is involved in the transport of other proteins and organelles, the distribution of various intracellular components was examined. The normal axonal growth and guidance observed in imac mutants implies that the transport of post-Golgi vesicles with new membrane and cell-surface proteins persists. The neuronal membrane marker, HRP, did not reveal any significant differences between wild type and imac mutants. Post-Golgi membrane trafficking was examined in imac mutants by using a fusion of the extracellular and transmembrane domains of CD8 to a cytoplasmic GFP, a construct that serves as a nonspecific reporter of the constitutive transport of membrane proteins to the cell surface, including axons and terminals. The distribution of CD8-GFP was not affected in imac mutants. Similarly, Fasciclin II (FasII), a Drosophila homolog of vertebrate neural cell-adhesion molecules and an essential regulator of motor neuron growth and guidance, remained concentrated in the axon tracts and synaptic regions in imac mutants. Another plasma membrane protein, syntaxin, is needed for exocytosis and addition of membrane to the cell surface. No change in the distribution of syntaxin, visualized with the monoclonal antibody 8C3, was detected in imac-null embryos. These data are consistent with the observation that axon outgrowth and targeting proceeds normally in imac mutants, and they demonstrate that the vesicles required for membrane extension and axon targeting are not conveyed by Imac. In addition, the data support the current model wherein mechanisms regulating membrane outgrowth are distinct from mature synaptic vesicle exocytosis (Pack-Chung, 2007).

Synaptic terminals are also enriched in mitochondria, an organelle transported by kinesin-1. Mitochondria were visualized using Mito-GFP, a mitochondrially targeted GFP variant. Wild-type and imac mutant nerves had similar distributions of mitochondria, indicating their proper transport in motor neuron axons (Pack-Chung, 2007).

Because of the absence of bead-like boutons in imac mutants, the neuronal cytoskeletal component Futsch, a Drosophila protein with homology to the vertebrate microtubule-associated protein MAP1B, was examined. Loops of bundled, Futsch-containing microtubules are present in many wild-type synaptic boutons at NMJs of third-instar larvae and may contribute to the rounded structure of the bouton. By contrast, unbundled microtubules characterize growth cones, and Futsch is likely to contribute to the transition from unbundled to bundled microtubules. In embryos, however, the significance of Futsch at the NMJ is not known (Pack-Chung, 2007).

In wild-type embryos, Futsch immunoreactivity was abundant in the axons of motor neurons and in synapses at the NMJ, although Futsch-immunoreactive microtubule loops were discernible only occasionally in boutons. In imac mutants, Futsch was similarly detected in motor axons and at the NMJ, although the immunoreactivity was more discontinuous than in wild type. Consistent with the absence of boutons, no loops of Futsch immunoreactivity were seen in imac mutants. Although the distribution of Futsch suggests that changes have occurred in the cytoskeleton of imac mutants, the presence of Futsch at the endings indicates that this cytoskeletal protein is transported independently of Imac. This finding is consistent with the known movement of many cytoskeletal components by slow axonal transport. In addition, the normal distribution of mitochondria and trans-Golgi-derived vesicle markers indicates that the failure to transport synaptic components cannot be secondary to microtubule defects. Likewise, the absence of boutons in imac mutants cannot be attributed to the absence of Futsch (Pack-Chung, 2007).

The localization was examined of two synaptic proteins that are cytoplasmic rather than organelle associated or cytoskeletal. Both proteins, LAP (also known as AP180) and endophilin, are involved in the endocytosis of synaptic vesicles. Their distribution in the nervous system was enriched in, but not strictly limited to, the neuropil in both wild type and imac mutants. Thus, the mislocalizations observed in imac mutants seem to be specific to a subset of synaptic proteins (Pack-Chung, 2007).

The components of the active zone may arrive at nascent synapses, at least in part, through PTVs, although this vesicle class has not been described in Drosophila. To determine the extent of active-zone transport and assembly in imac mutants, an active-zone marker, monoclonal antibody nc82, was examined which has been shown to recognize Brp, a member of the ELKS/CAST/ERC protein family and a probable component of the T-bar, a dense body that projects back into the cytoplasm from the electron density at the plasma membrane. Wild-type NMJs at 21 h AEL showed clusters of nc82 puncta in boutons. imac mutants showed only 11% of the puncta of wild type and these puncta were also fainter. This reduction could not be explained by a decrease in the surface area of nerve-muscle contacts in the mutant: the amount of nc82 puncta per HRP area was only 23% of that of wild type. To verify that puncta in imac mutants were not being missed owing to a decrease in the intensity of the nc82 immunoreactivity, puncta were recounted with a higher confocal gain setting that could not be used on wild-type NMJs because of saturation and consequent optical merging of puncta. Even with this bias toward overcounting in the mutant, only 24% of the puncta seen in wild type were observed. In addition, within the area counted, many of the puncta in imac mutants occurred along the axons rather than at the endings, where they are normally located. In the CNS, the ratio of nc82 staining in the cell body region relative to the neuropil was also higher in imac mutants than in wild type (Pack-Chung, 2007).

Because the shift in nc82 immunoreactivity was not as absolute as the difference observed for synaptic vesicle markers, the emergence of nc82 immunoreactivity at the NMJs of ISNb was examined quantitatively. In wild type, a few puncta of nc82 immunoreactivity were detected at 13 and 14 h AEL, when the growth cone first contacts the muscle and processes begin to elongate along the muscle boundaries. At 15 h AEL, however, the number of puncta increased sharply, and modest further increases subsequently occurred until hatching. At 13 and 14 h AEL, imac embryos, like wild-type embryos, had only a few nc82 puncta. In imac mutants, however, the large increase at 15 h AEL did not take place. Instead, the number of puncta declined modestly for the remainder of embryonic development. Even taking into account the smaller surface area of the nerve-muscle contacts in imac mutants, nc82 puncta were sparser in imac mutants than in wild type. Despite their paucity, the presence of some nc82 puncta (but not synaptic vesicle proteins) in the mutants at early stages suggested that some active-zone protein can diffuse or be transported into the axons independently of imac, at least at this early stage. The lack of increase in nc82 staining, however, is an early phenotype of the mutants that is apparent before the stage at which varicosities fail to form (Pack-Chung, 2007).

To determine the nature of the mutant nerve-muscle contacts and to look for signs of active-zone formation or vesicle accumulation that might not have been detected by immunocytochemistry, electron microscopy was performed. In a given cross-section through a wild-type embryo, on average ten NMJs were identified, among which five active zones and three T-bars were seen. Identification of wild-type NMJs was relatively straightforward in that terminals contained uniformly sized synaptic vesicles. In imac mutants, however, most nerve-muscle contacts lacked vesicles and were therefore recognized on the basis of the close apposition of the electron-lucent nerve ending to the muscle fibers and the presence of the basement membrane overlying the nerve ending. By using these criteria, 0-2 nerve-muscle contacts were typically identified per section. The absence of synaptic vesicles made identification of the nerves more difficult in imac mutants and consequently some contacts may have been missed (Pack-Chung, 2007).

To systematize this characterization of these nerve-muscle contacts, serial electron microscopy of cross-sections of imac embryos was undertaken. Active-zone and T-bar counts were normalized to the measured surface area of the neurite to take into account the paucity of recognizable contacts in imac mutants and changes in their size. Active zones were indeed recognized in imac mutant nerve endings, but at ~60% of the frequency encountered in wild type, when either the active-zone number or the active-zone area was normalized to the total neuronal surface area in the sections. imac mutants showed a greater reduction (17% of control) in T-bar frequency. In addition, imac T-bars were often smaller than wild-type T-bars. Occasionally, they were surrounded by diffuse electron-dense material that did not resemble membrane vesicles. On average, 70 synaptic vesicle profiles were counted per section of wild-type boutons. Thus, as expected from the immunocytochemistry, imac nerve endings were less extensive, rarely if ever contained synaptic vesicles, and had few active zones and very few T-bars (Pack-Chung, 2007).

These studies have revealed that a molecular motor has a role in presynaptic maturation and provide insight into the formation of synapses. Specifically, this phenotypic analysis demonstrates, first, that Imac transports components required for the morphological transformation of axonal growth cones to mature boutons; second, that the membrane and proteins that support axon outgrowth, axon guidance and target recognition do not rely on the same kinesin for their transport as the one that supports synaptogenesis; and third, that postsynaptic differentiation does not depend on synaptic vesicle clustering, synaptic transmission or presynaptic bouton formation. In addition, the finding that Imac is essential for the transport of synaptic vesicle precursors suggests that this is their primary motor, rather than KHC. Other components of the synapse can be classified as either dependent or independent of Imac on the basis of their transport phenotype (Pack-Chung, 2007).

The phenotype of imac mutants is unprecedented despite extensive genetic analysis of the Drosophila NMJ as a model system for synapse development. Prior studies have uncovered molecules that are crucial to axon pathfinding, and yet, despite mistargeting, axons in those mutants still form boutons if they reach a muscle. By contrast, mutation of imac affects all of the NMJs in the embryo that were analyzed and prevents varicosity formation despite signs of appropriate outgrowth, guidance and nerve-target recognition. Many signaling pathways, including those mediated by electrical activity, Wingless, bone morphogenetic proteins, Highwire and fasciclins, can regulate the growth and plasticity of these synapses. These pathways influence bouton number and the size, length and branching of endings, but they do not prevent bouton formation outright. Likewise, mutants that affect synapse specificity in C. elegans (for example, syg-1 and syg-2) form structurally normal synapses (Pack-Chung, 2007).

Despite evidence from C. elegans and mammalian systems implicating Kinesin-3 motors (Unc-104 and KIF1A) in axonal transport of synaptic vesicle precursors, mammalian Kinesin-1 motors have also been associated with synaptic vesicle transport. In addition, KHC, a Kinesin-1, has been the predominant candidate for this transport in Drosophila. Key evidence implicating KHC has been the presence in khc mutant larvae of axonal swellings filled with various vesicles and organelles, including synaptic vesicle markers. These accumulations, sometimes called 'traffic jams', are distinct from the imac mutant phenotype: that is, the categorical absence of synaptic vesicle markers from motor neuron axons and the persistent transport of other organelles (Pack-Chung, 2007).

The absence of vesicles from axons in imac mutants would seem to indicate that no other motor can substitute for Imac during NMJ formation, although KHC is present in these motor neurons and transports mitochondria in the absence of Imac. The accumulation of synaptic vesicle proteins in axonal swellings in khc mutants might arise indirectly from the failure of other classes of transport, potentially snarling traffic in the axons. Alternatively, KHC-mediated transport may supplement Imac in older Drosophila, despite the essential role of Imac in transport of synaptic vesicles during de novo embryonic synaptogenesis (Pack-Chung, 2007).

PTVs are thought to carry many constituents of the mammalian active zone, but nothing is known about active-zone transport in Drosophila. Thus, the change in Brp (nc82) distribution and the ultrastructural phenotype of imac reported in this study represent an initial investigation into that process. These phenotypes are not as absolute, however, as those observed for synaptic and dense-cored vesicle markers; therefore, the delivery of active-zone proteins may be more complex. The paucity of these proteins at imac mutant nerve endings might occur because Imac has a direct role in their transport or because there may be a failure to trap or concentrate them at the terminal (Pack-Chung, 2007).

In contrast, the persistence of some Brp in axons and their endings, although sharply reduced at NMJs, suggests that transport or diffusion of Brp may persist but that its capture at synapses may be impaired by the lack of synaptic maturation. In contrast, the reductions in nc82-immunoreactive puncta and active-zone counts were greater than could be explained by the decrease in size of the nerve-muscle contacts. In addition, the dearth of nc82 puncta was apparent by 15 h AEL, before the stage when boutons form. Thus, the failure to form sufficient active zones and T-bars is unlikely to be secondary to the failure to form boutons. Rather, it suggests that Imac is directly responsible either for the transport of a portion of the active-zone proteins or for the transport of a protein needed to retain them at muscle contacts (Pack-Chung, 2007).

In imac mutants, postsynaptic differentiation was assessed by two methods: first, the presence of postsynaptic densities by electron microscopy; and second, the clustering of glutamate receptors by immunocytochemistry. By both criteria, specialization of the postsynaptic membrane at the site of nerve contact proceeded in the absence of synaptic vesicles and without the morphological transition to a synaptic bouton. Postsynaptic structural changes, as indicated by the clustering of receptors, are thought to be induced by contact with the nerve. The importance of transmitter release in these processes, however, has been controversial, in part because no mutants have been available that cleanly prevent both evoked release and the spontaneous release of synaptic vesicles without also compromising the survival of the axon. Axons in imac mutants lack components essential for neurotransmitter release including VGlut, which is required to load vesicles with glutamate, and indeed lack synaptic vesicles themselves. Despite this, glutamate receptor subunits cluster on the muscle beneath the neuronal membranes, indicating that vesicular transmitter release per se is not needed for postsynaptic differentiation (Pack-Chung, 2007).

The possibility cannot be excluded that non-vesicular glutamate induces the postsynaptic response or that other molecules are released from the growth cone. Pre- and postsynaptic development cannot be completely independent because release sites and receptor clusters need to be aligned. In imac mutants, this alignment is preserved, despite the reduced number of active zones and diminished nc82 immunoreactivity. Thus, the machinery involved in the matching of pre- and postsynaptic structures persists, at least in part, in imac mutants. Glutamate receptor clusters were not reduced in number or intensity as markedly as recognizable active zones in electron microscopy images or as the intensity of nc82 puncta in nerve endings. Thus, the low concentration of nc82 immunoreactivity opposite most of the glutamate receptor clusters may represent incompletely assembled active zones (Pack-Chung, 2007).

This analysis of imac mutants has facilitated a dissection of the transport mechanisms that function during a crucial phase of neuronal development. The existence of a motor for presynaptic maturation that is distinct from that for axon outgrowth and guidance may reflect the different regulatory needs for distributing the molecules that mediate these events. New membrane, for example, is consistently brought to the growing tip of the axon, whereas synaptic precursors travel in both directions in the axon, scanning for signals from target cells that will determine where they will form a functional connection. Further understanding of synaptogenesis will require identification of both the factors that regulate these motors and the particular cargos that alter the morphology of nerve endings (Pack-Chung, 2007).

Reference names in red indicate recommended papers.

Hall, D. H. and Hedgecock, E. M. (1991) Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell 65: 837-847. Medline abstract: 1710172

Jacob, T. C. and Kaplan, J. M. (2003). The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions. J. Neurosci. 23: 2122-2130. Medline abstract: 12657671

Klopfenstein, D. R. and Vale, R. D. (2004). The lipid binding pleckstrin homology domain in UNC-104 kinesin is necessary for synaptic vesicle transport in Caenorhabditis elegans. Mol. Biol. Cell 15(8): 3729-39. Medline abstract: 15155810

Okada, Y., Yamazaki, H., Sekine-Aizawa, Y. and Hirokawa, N. (1995). The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. Cell 81: 769-780. Medline abstract: 7539720

Pack-Chung, E., Kurshan, P. T., Dickman, D. K. and Schwarz, T. L. (2007). A Drosophila kinesin required for synaptic bouton formation and synaptic vesicle transport. Nat. Neurosci. 10(8): 980-9. Medline abstract: 17643120

Yonekawa, Y., et al. (1998). Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice. J. Cell Biol. 141(2): 431-41. Medline abstract: 9548721

Zhao, C. et al. (2001). Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1B. Cell 105: 587-597. Medline abstract: 11389829

date revised: 26 October 2007

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