unc-104: Biological Overview | References
Gene name - unc-104
Synonyms - Klp53D, Immaculate connections, Imac
Cytological map position-53D6-53D7
Function - cytoskeleton
Symbol - unc-104
FlyBase ID: FBgn0267002
Genetic map position -2R :12,639,001..12,660,002 [-]
Classification - Kinesin motor domain, KIF1_like proteins. KIF1A (Unc104), forkhead-associated (FHA) domain
Cellular location - cytoplasmic
|Recent literature||Zhang, Y. V., Hannan, S. B., Stapper, Z. A., Kern, J. V., Jahn, T. R. and Rasse, T. M. (2016). The Drosophila KIF1A homolog unc-104 is important for site-specific synapse maturation. Front Cell Neurosci 10: 207. PubMed ID: 27656128
Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia (HSP), hereditary and sensory autonomic neuropathy type 2 (HSAN2) and non-syndromic intellectual disability (ID). Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. An hypomorphic allele of unc-104, unc-104bris), was used to investigate impact on synapse maturation at the Drosophila neuromuscular junction (NMJ). Unc-104bris) mutants exhibit structural defects where a subset of synapses at the NMJ lack all investigated active zone (AZ) proteins, suggesting a complete failure in the formation of the cytomatrix at the active zone (CAZ) at these sites. Modulating synaptic Bruchpilot (Brp) levels by overexpression or RNAi suggests that the loss of AZ components such as Ca(2+) channels and Liprin-α is caused by impaired kinesin-3 based transport rather than due to the absence of the key AZ organizer protein, Brp. In addition to defects in CAZ assembly, unc-104bris mutants display further defects such as depletion of dense core and synaptic vesicle (SV) markers from the NMJ. Notably, the level of Rab3, which is important for the allocation of AZ proteins to individual release sites, was severely reduced at unc-104bris mutant NMJs. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104bris larvae, suggesting that lack of presynaptic Rab3 contributes to defects in synapse maturation.
|Voelzmann, A., Okenve-Ramos, P., Qu, Y., Chojnowska-Monga, M., Del Caño-Espinel, M., Prokop, A. and Sanchez-Soriano, N. (2016). Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking. Elife 5. PubMed ID: 27501441
The mechanisms regulating synapse numbers during development and aging are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, this study demonstrates roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, it was found that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and aging (synapse maintenance) alike. Therefore, these findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease.
| Zhang, Y.V., Hannan, S.B., Kern, J.V., Stanchev,
D.T., Koç, B., Jahn, T.R. and Rasse, T.M. (2017). The
KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic
density maturation and perisynaptic scaffold organization. Sci
Rep 7: 38172. PubMed ID: 28344334
The kinesin-3 family member KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104bris, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. This study shows that the unc-104bris mutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, there is a marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which can be rescued by pre- but not postsynaptic expression of unc-104. These results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation.
|Li, J., Zhang, Y. V., Asghari Adib, E., Stanchev, D. T., Xiong, X., Klinedinst, S., Soppina, P., Jahn, T. R., Hume, R. I., Rasse, T. M. and Collins, C. A. (2017). Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104. Elife 6. PubMed ID: 28925357
The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104's transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104's function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.
|Lim, A., Rechtsteiner, A. and Saxton, W. M. (2017). Two kinesins drive anterograde neuropeptide transport. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28904207
Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, the functional relationships were studied of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. This study showed that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). The results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNAi had no influence on anterograde DCV runs, and detailed co-localization analysis by super resolution microscopy revealed that Unc-104 and Khc co-associate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. A mechanism is suggested in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.
|Zong, W., Wang, Y., Tang, Q., Zhang, H. and Yu, F. (2018). Prd1 associates with the clathrin adaptor alpha-Adaptin and the kinesin-3 Imac/Unc-104 to govern dendrite pruning in Drosophila. PLoS Biol 16(8): e2004506. PubMed ID: 30142146
Refinement of the nervous system depends on selective removal of excessive axons/dendrites, a process known as pruning. Drosophila ddaC sensory neurons prune their larval dendrites via endo-lysosomal degradation of the L1-type cell adhesion molecule (L1-CAM), Neuroglian (Nrg). This study identified a novel gene, pruning defect 1 (prd1), which governs dendrite pruning of ddaC neurons. Prd1 colocalizes with the clathrin adaptor protein alpha-Adaptin (alpha-Ada) and the kinesin-3 immaculate connections (Imac)/Uncoordinated-104 (Unc-104) in dendrites. Moreover, Prd1 physically associates with alpha-Ada and Imac, which are both critical for dendrite pruning. Prd1, alpha-Ada, and Imac promote dendrite pruning via the regulation of endo-lysosomal degradation of Nrg. Importantly, genetic interactions among prd1, alpha-adaptin, and imac indicate that they act in the same pathway to promote dendrite pruning. These findings indicate that Prd1, alpha-Ada, and Imac act together to regulate discrete distribution of alpha-Ada/clathrin puncta, facilitate endo-lysosomal degradation, and thereby promote dendrite pruning in sensory neurons.
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).
A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, Kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism (Barkus, 2008).
To gain insight into mechanisms of axonal transport, the consequences of inhibition of a kinesin-3 were studied in Drosophila. The founder of the kinesin-3 subfamily, UNC-104, was discovered as a C. elegans protein required for coordinated crawling behavior. Subsequent studies of C. elegans UNC-104, mammalian Kif1A, B, and other kinesin-3 family members have revealed that different kinesin-3 motors, which move relatively fast toward microtubule plus-ends, can transport a variety of different cargoes, including endosomes, mitochondria, and various vesicles. The results indicate that Drosophila Unc-104 can carry at least two anterograde vesicle types in motor axons, the cardiac-specific atrial natriuretic factor (ANF) neuropeptide-carrying dense-core vesicles (DCVs), and synaptotagmin-bearing small transport vesicles (STVs). Unc-104 may also transport other types of organelles, but no evidence was found for transport of axonal mitochondria (Barkus, 2008).
It is known that axonal transport involves the energetic motion of individual organelles, each pulled along cytoskeletal filaments by motor proteins. The time-lapse analysis reported in this study emphasizes how distinct the transport behaviors of different organelles can be, and it raises questions about the mechanistic underpinnings of those differences. One possibility is that velocity varies inversely with organelle size, implying that cytoplasmic resistance to movement (viscous drag) is a key determinant of transport behavior and thus of cargo distribution dynamics. Mitochondria in Drosophila larval axons range widely in length, up to several micrometers, and they have an average diameter of 150 nm. DCVs are mostly spherical with diameters of about 100 nm. Mean DCV run velocity and length were, respectively, 4-fold and 20-fold greater than those of mitochondria, consistent with an inverse size-velocity relationship. However, although DCV diameter is two- to three-fold greater than that of STVs (~30 nm), means for DCV run velocity and length were, respectively, 1.5- and 4-fold greater than those of STVs. Furthermore, it was previously reported that run velocities for mitochondria in larval motor axons were independent of mitochondria lengths. These observations argue that transport behavior is determined mainly by organelle identity and organelle-specific differences in transport mechanisms, rather than by differences in size-dependent viscous drag (Barkus, 2008).
One likely source of transport mechanism differences is the intrinsic mechanochemical capabilities of different motors. The results presented in this study indicate that many anterograde DCVs in Drosophila motor axons use Unc-104 (kinesin-3). Previous work in the same system showed that anterograde mitochondria use Khc (kinesin-1). DCV runs have higher velocity and longer anterograde runs than mitochondria, consistent with in vitro tests showing that dimeric Unc-104 constructs move with higher velocity and processivity than dimeric Khc constructs. This sort of straightforward mechanochemical difference, however, fails to explain why synaptotagmin-tagged STVs, which also use Unc-104, have slower, shorter runs than DCVs. Furthermore, retrograde run velocities and lengths that were measured for the three organelle types were quite different, despite the fact that cytoplasmic dynein heavy chain (Dhc64C) is the only known fast retrograde microtubule motor available in Drosophila. Thus, although differences in the mechanochemical properties of motors are important to differential organelle transport behavior, it seems clear that motor performance can be influenced by cargo identity (Barkus, 2008).
Cargo-specific factors that might alter the output of a motor include posttranslational motor modification, motor-cargo linkage proteins, and the presence of other motors on the same organelle. Kinesin-3s are reported to be monomeric in vitro, and individual monomers move slowly on microtubules. However, artificially induced dimerization allows faster more processive motion, supporting the hypothesis that clustering of motors on an organelle may be an important determinant of transport behavior. Because Unc-104 may link directly to vesicle membranes via an FH lipid anchor domain, a variation in clustering controlled by lipid raft dynamics could produce variation in velocity and processivity. In addition, some cargoes are known to use multiple types of anterograde motors. Recent studies have shown that two different kinesins with distinct velocities, when active on the same dendritic cargo, generate motion at an intermediate velocity. Thus, the slower velocities of the STVs reported in this study might reflect mixed use of fast Unc-104 and slower Khc, whereas faster DCV velocities could reflect clusters of Unc-104 alone (Barkus, 2008).
Organelle tracking results suggest a specific positive influence of anterograde Unc-104 on retrograde DCV run velocity and length. A previous study of mitochondrial transport in Drosophila axons showed that kinesin-1 is critical for the dynein-driven retrograde flux of mitochondria. Although that sort of positive influence of an opposing motor might reflect a direct physical interaction between kinesin-1 and the dynein complex, it could also reflect simple logistical dependence for two reasons. (1) For normal numbers of mitochondria to move retrograde, normal numbers must be transported anterograde. Because kineisn-1 is the anterograde motor, Khc mutations result in low numbers of mitochondria in distal axons. (2) Dynein itself must be transported to the distal axon, before it can function in retrograde transport, and kinesin-1 is likely responsible for some of that anterograde dynein movement. In contrast, the retrograde DCV run velocity and length decreases observed in unc-104 mutant axons were not general, i.e., for STVs or mitochondria, statistically significant decreases in retrograde run velocity or length were not seen. This suggests that Unc-104 has an organelle-specific positive influence on the function of DCV-bound dynein (Barkus, 2008).
How could Unc-104 contribute to DCV retrograde transport? Possibly it might be responsible for delivering DCV-specific dynein regulatory factors into the axon that enhance retrograde run velocity and length. This would require no specific association of Unc-104 with retrograde organelles. However, the fact that retrograde movement of Unc-104::GFP has been observed in axons of C. elegans and Drosophila, along with a report that C. elegans UNC-104 is a retrograde cargo of dynein suggest more direct possibilities. (1) DCV-specific motor docking complexes might juxtapose anterograde and retrograde motors such that Unc-104 itself acts as an allosteric activator for dynein. (2) Unc-104 on DCVs might facilitate their retrograde transport biophysically, for example, intermittently generating reverse strain and motion that helps dynein-DCV complexes get past steric barriers in the axon (Barkus, 2008).
It is apparent that neurons use a diverse array of microtubule-based transport mechanisms to support long axons. Each type of organelle, RNP, and protein complex should have an ideal distribution and replacement rate for maintaining proper axon physiology and function. Thus, although it seems that only a few basic force-generating motors are used, diversity in their transport output via cargo-specific motor-motor influences and other regulatory schemes is likely important for optimizing nervous system function. Because motor proteins have complex effects on multiple processes in neurons and other cells, identifying cargo-specific motor control factors will be important, both for understanding the basic mechanisms of cytoplasmic organization and for providing new potential targets for drugs that can slow the progress of axonal transport-related neurodegenerative diseases (Barkus, 2008).
The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. This study shows that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. Kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant were identified. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity (Chang, 2011).
Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 (RhoGAP100F) as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. This study shows that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. The earlier defect coincides with a failure to localize synaptic vesicles (SVs), suggesting that it reflects a failure in presynaptic assembly. The relationship between syd-1 and Liprin-α in R7s was analyzed. It was found that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: Liprin-α was shown to promote Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. It was shown that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. It is therefore concluded that the two defects are caused by distinct molecular mechanisms. Trio overexpression was found to rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity (Holbrook, 2012).
GFP-fused SV proteins, such as Syt-GFP, are classic tools for studying presynaptic development but have not been used previously to analyze R7s. This study found that, as expected, Syt-GFP within R7s is enriched at sites known by electron microscopy to contain active zones. Loss of LAR, Liprin-α, or syd-1 causes R7 terminals to fail to contact their normal, M6, target layer. This study demonstrated that this morphological defect correlates temporally with a failure to localize SVs to presynaptic sites and is therefore likely to reflect a defect in R7 presynaptic development rather than simply in target layer selection (Holbrook, 2012).
Liprin-α is not only a scaffold for the assembly and retention of presynaptic components, including SVs, at presynaptic sites but also a positive regulator of Kinesin-3/Unc-104/Imac-dependent axon transport of those components. This study shows that, unlike Liprin-α, Syd-1 is not required for normal Kinesin-3/Unc-104/Imac-mediated transport. However, SVs are similarly mislocalized in Liprin-α and syd-1 mutant R7 axons that contact M6. A simple interpretation is that this mislocalization reflects a requirement for Liprin-α and syd-1 in retaining SVs within R7 terminals; in support of this, it was found that SVs are localized normally to syd-1 mutant R7 axon terminals at 24 h APF, before synaptogenesis. It was hypothesized that the additional disruption of axon transport in Liprin-α mutant R7s is reflected in their greater inability to maintain contact with M6; in support of this, it was found that imac mutant R7 axons also lose contact with M6 (Holbrook, 2012).
Although both Liprin-α and syd-1 are required for the clustering of SVs at en passant synapses in worm, syd-1 is not required for the localization of SVs to NMJ terminals in fly. The molecular mechanisms underlying presynaptic development at NMJ and in R7s have been shown previously to differ in several respects. The current finding further highlights the importance of analyzing synapse development using multiple neuron types (Holbrook, 2012).
Although mitochondria are often enriched at synapses, it remains unclear what proportion of them might be stably associated with presynaptic sites rather than transported there in response to acute energy needs. Within at least some axons, most clusters of stationary mitochondria reside at nonsynaptic sites. In R7s, Mito-GFP was found to be enriched at presynaptic sites. Because arthropod photoreceptor neurons continuously release neurotransmitter in response to light, this enrichment might simply be caused by continuous energy needs. However, this study found that mitochondria remained enriched at R7 terminals even in the absence of light-evoked activity, indicating that either spontaneous release is sufficient for their recruitment or an activity-independent mechanism is responsible. It is speculated that the permanently high energy demands at photoreceptor synapses may have selected for the activity-independent association of mitochondria with R7 synapses and that this localization requires syd-1 and Liprin-α. Mito-GFP is mislocalized in imac mutant R7s, despite previous work indicating that Kinesin-3/Unc-104/Imac is not required for transport of mitochondria. It is therefore thought that mitochondria are normally tethered at R7 presynaptic sites and that loss of imac indirectly causes their mislocalization by disrupting transport of the components required for tethering to occur (Holbrook, 2012).
Previous work identified two different phenotypes associated with loss of the LAR/Liprin/trio pathway: loss of LAR or Liprin-α caused R7 axons to terminate before their M6 target layer, whereas loss of Liprin-β or trio caused R7 axons to project extensions beyond M6. One possibility is that these two defects are simply different manifestations of the same cellular defect: a decrease in the stability of the synaptic contact between R7s and their targets. However, this study has shown that loss of a single gene, syd-1, causes both defects and that the defects occur at distinct developmental time points, suggesting that they occur by distinct mechanisms. In support of this, Liprin-α overexpression can rescue the early but not the late syd-1 defect (Holbrook, 2012).
The earlier defect, failure to contact M6, correlates with the failure to localize SVs, suggesting, as mentioned above, that this represents a failure to assemble synapses. However, the cause of the later morphological defect and the precise nature of the extensions remain unclear. It is noted that the extensions often terminate in small varicosities that can contain Syt-GFP, and Mito-GFP, indicating that they are not simply filopodia but may instead represent sites of ectopic presynaptic assembly. One possibility is that, as at NMJ, loss of syd-1 causes ectopic accumulations of Liprin-α, Brp, Nrx-1, or other presynaptic proteins and that these might then promote ectopic, abnormal presynaptic assembly. A second possibility is that the extensions may instead be an indirect consequence of the role of syd-1 in postsynaptic development: perhaps the extensions are the response of the syd-1 mutant R7 terminal to defects in its postsynaptic target. Loss of Liprin-α causes no such postsynaptic effect, providing an explanation for why Liprin-α mutant R7s do not form extensions. A third possibility is that R7s form distinct types of synapses at different time points. Failure to assemble one type of synapse, which R7s assemble first, causes decreased contact with M6, whereas failure to assemble a second type, which occur later, results in extensions. Consistent with this model, R7s form synapses with more than one neuron type (Holbrook, 2012).
Loss of syd-1 has a significantly weaker effect on fly NMJ development than does loss of Liprin-&alpha. Likewise, this study shows that the early phase of R7 terminal development, during which presynaptic components are localized, is less affected by loss of syd-1 than by loss of Liprin-α. A possible explanation for this difference is identified: loss of Liprin-α, but not of syd-1, significantly decreases Kinesin-3/Unc-104/Imac-mediated axon transport, and Kinesin-3/Unc-104/Imac is required for R7s to form boutons in M6 (Holbrook, 2012).
In both worm and fly, Syd-1 is required for the normal localization of Liprin-α and Brp/ELKS to presynaptic sites. In worm, loss of syd-1 can be rescued either by overexpressing full-length wild-type Liprin-α, or by overexpressing a domain of Liprin-α that promotes oligomerization of Liprin-α proteins, or by a mutation that enhances the ability of Liprin-α to bind Brp/ELKS. These results suggest that the primary function of Syd-1 is to potentiate Liprin-α activities. However, this sutyd found that Liprin-α overexpression only partially rescues the early defect that syd-1 mutant R7s have in assembling synapses. This suggests that, as in worm, Liprin-α can act partly independently of Syd-1 during presynaptic assembly but that, unlike in worm, Syd-1 also has some Liprin-α-independent function. In contrast, Liprin-α overexpression does not at all rescue the late extensions caused by loss of syd-1. As it speculated above, one possibility is that these extensions might be caused by mislocalized Liprin-α, Brp, or Nrx-1 (Holbrook, 2012).
Unlike Liprin-α, Trio overexpression fully rescues the early and partly rescues the late defect caused by loss of syd-1, suggesting that Syd-1 promotes R7 synaptic terminal development primarily by potentiating Trio activity. Consistent with this model, loss of trio phenocopies loss of syd-1 from R7s, and overexpressing Syd-1 or Trio bypasses the need for LAR to similar degrees. At fly NMJ, Trio promotes presynaptic development by acting as a GEF for Rac1. Syd-1 has a RhoGAP domain, albeit one that has not been shown to interact with GTPases. Syd-1 may act distantly upstream of Trio. However, it is also possible that Syd-1 might instead regulate one or more small GTPases in parallel with Trio. GAPs and GEFs have opposite effects on GTPases, but loss of trio or syd-1 causes similar defects at both NMJ and in R7s. One possibility, therefore, is that Syd-1 acts as a GAP not for Rac1 but for Rho, which often functions in opposition to Rac. Alternatively, Syd-1 might act as an atypical GAP for Rac1 -- perhaps lacking GAP activity but able to bind and protect Rac1-GTP from conventional GAPs -- or Syd-1 might yet act as a conventional GAP for Rac1 if it is the rate of cycling between GDP- and GTP-bound states of Rac1 (rather than simply the amount of the GTPase that is in the 'active,' GTP-bound, state) that promotes presynaptic development (Holbrook, 2012).
Autophagy is a cellular degradation process important for neuronal development and survival. Neurons are highly polarized cells in which autophagosome biogenesis is spatially compartmentalized. The mechanisms and physiological importance of this spatial compartmentalization of autophagy in the neuronal development of living animals are not well understood. This study determines that, in Caenorhabditis elegans neurons, autophagosomes form near synapses and are required for neurodevelopment. It was shown through unbiased genetic screens and systematic genetic analyses that autophagy is required cell autonomously for presynaptic assembly and for axon outgrowth dynamics in specific neurons. Autophagosome biogenesis in the axon near synapses was observed, and this localization was found to depend on the synaptic vesicle kinesin, KIF1A/UNC-104 (see Drosophila Unc-104). KIF1A/UNC-104 coordinates localized autophagosome formation by regulating the transport of the integral membrane autophagy protein ATG-9 (see Drosophila Atg9). These findings indicate that autophagy is spatially regulated in neurons through the transport of ATG-9 by KIF1A/UNC-104 to regulate neurodevelopment (Stavoe, 2016).
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date revised: 12 November 2017
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