The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis

Background: The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however. Results: We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. BioMed Central Journal of Biology Journal of Biology 2006, 5:9 Open Access Published: 25 May 2006 Journal of Biology 2006, 5:9 The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/5/4/9 Received: 16 March 2005 Revised: 23 December 2005 Accepted: 5 April 2006 © 2006 Su and Tharin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background At its simplest, a neuron is composed of three major structures, a central cell body and two networks of extensively branched membrane structures, the dendrite and the axon. Growing axons respond to a wide variety of extracellular attractive and repulsive signals that direct migration to a fated location. Although many guidance receptors have been identified on extending growth cones, little is known about how activation of receptors mediates coordinated neurite extension. In addition to signaling cues in the extracellular matrix, neurite elongation and growth-cone extension depend on a concerted effort of vesicular transport and regulated membrane addition. For growth cones to extend, vesicles derived from the Golgi apparatus fuse with the plasma membrane by a process of regulated exocytosis [1]. Likewise, synapse formation also requires transport of preand post-synaptic components supplied in membranous organelles [2,3]. These vesicles are not only transported but are also differentially sorted into dendrites or axons [4,5]. To fulfill these tasks, intrinsic cytosolic factors are required to regulate transport of the vesicles [6] and to differentially control dendritic versus axonal growth and morphogenesis. The nematode Caenorhabditis elegans has been extensively used to study vesicular transport in neuronal development. For example, monomeric kinesin UNC-104/KIF1A, UNC-116/kinesin heavy chain (KHC), kinesin light chain KLC-2, and various cytoplasmic dynein complex components regulate various vesicle trafficking events [7-9]. KLC-2 might regulate the transport of various axonal and synaptic cargos by recruiting adaptor and regulatory proteins such as UNC-16, UNC-14 and UNC-51 [9,10]. In the absence of UNC-16 (a JNK-scaffolding protein), a glutamate receptor and synaptic vesicles containing the synaptobrevin homolog SNB-1 dislodge from the postand pre-synaptic terminals [7]. UNC-16 binds directly to the tetratricopeptide repeat (TPR) domain of KLC-2, whereas the RUNdomain-containing protein UNC-14 associates with UNC-16 in the presence of KLC-2 [9]. UNC-14 interacts physically with the serine/threonine kinase UNC-51, and both proteins are required for axonal outgrowth [10,11]. Noticeably, although membranous structures with variable size accumulate within axons in unc-51 [12,13] and unc-14 [13] mutants, suggesting that both genes are involved in axonal transport, synaptic vesicles are normally clustered in presynaptic terminals in these mutants [13]. C. elegans UNC-76 and its homologs have been implicated in both axonal outgrowth and synaptic transport via association with the heavy chain of Kinesin-1. In worms mutant for unc-76, the nervous system is disorganized: the axons fail to extend and axonal bundles are defasciculated [13,14]. In Drosophila, Unc-76 interacts with the tail of KHC and is important for transporting synaptic cargos in the axons [15]. The mechanism of UNC-76-mediated transport remains elusive, although there is some evidence that secondary modification by protein kinase C (PKC ) or polyubiquitination of the fasciculation and elongation protein zygin/zeta 1 (FEZ1), one of the mammalian UNC-76 homologs, contributes to its neurite outgrowth activity [16,17]. In this study we report the cloning and characterization of UNC-69, a small, evolutionarily conserved coiled-coil domain-containing protein that acts as a novel binding partner of UNC-76 in C. elegans. Whereas a weak reductionof-function allele of unc-69 results in a selective defect in mislocalization of a synaptic vesicle marker, strong unc-69 mutants show extensive defects in axonal outgrowth, fasciculation and guidance. Mutations in UNC-69 preferentially disrupt membrane traffic within axons. We show that UNC-69 and UNC-76 participate in a common genetic pathway necessary for axon extension and cooperate to regulate the size and position of synaptic vesicles in axons. Moreover, both proteins colocalize as puncta in neuronal processes. We propose that UNC-69 and UNC-76 form a conserved protein complex in vivo to regulate axonal transport of vesicles. 9.2 Journal of Biology 2006, Volume 5, Article 9 Su and Tharin et al. http://jbiol.com/content/5/4/9 Journal of Biology 2006, 5:9 In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects. Conclusions: We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly. Results unc-69 encodes a conserved short coiled-coil domain-containing protein unc-69 was identified in a large-scale behavioral screen for uncoordinated (Unc) mutants [18]. unc-69 loss-of-function (lf) mutants move poorly, coil ventrally and are phenotypically similar to other coiler Unc mutants, many of which are defective in axonal outgrowth and guidance. Additionally, unc-69 mutant hermaphrodites lay more eggs in the absence of food than wild-type worms do (see Additional data file 1, available with the online version of this article), suggesting a defect in the hermaphrodite-specific neurons (HSNs), which control egg-laying behavior. Previous genetic data placed unc-69 between lin-12 and tra-1 on chromosome III, 0.12 map units to the left of ced-9 [19]. Using cosmid rescue, we were able to identify the predicted gene T07A5.6a (previously named T07C4.10b) as unc-69 (Figure 1a). The unc-69 gene encodes a 108-amino-acid protein and contains a short coiled-coil domain in its carboxyl terminus (Figure 1b). Although UNC-69 could possibly form a homodimer via its coiled-coil domain, we failed to detect any homophilic interactions of UNC-69 (see Additional data file 1). The original alleles of unc-69, unc-69(e587) and unc-69(e602), are both nonsense mutations in the carboxyterminal half of the protein (see Figure 1b). The unc-69(e602) mutation causes a T-to-A transversion and replaces a leucine with an amber stop codon at position 77; unc-69(e587) results in a C-to-T transition, changing a glutamine to an amber stop codon at position 86; both of these mutations lie within the well conserved coiled-coil domain. Both unc-69(e602) and unc-69(e587) are candidate genetic null alleles, as the axon extension and branching defects of the neurons named ALM and AVM were not enhanced significantly when either of these two alleles was placed in trans to the deficiency nDf40 (Table 1, Figure 2). We also isolated a hypomorphic allele, ju69, which results in a G-to-A transition at the start codon and changes the initiator methionine to an isoleucine. Theoretically, the M-to-I substitution (M1I) should abolish translation initiation and hence synthesis of the UNC-69 protein. As the phenotype of unc-69(ju69) mutants is much weaker than that of the other two alleles, however, we suspect that a small amount of UNC-69 functional protein is still being produced, either by leaky translation initiation at the original site, or through initiation at the internal, in-frame ATG site at residue 49, which would leave the coiled-coil domain intact. Indeed, overexpression of a mutant fusion protein of UNC-69 with green fluorescent protein (UNC-69(M1I)::GFP) or a carboxyterminal fragment of UNC-69 (residues 41-108) could partially suppress the locomotion defect of the unc-69(e587) mutants (data not shown, and see Additional data file 1). Finally, we analyzed a small deletion, ok339, which completely eliminates the unc-69 locus. Unfortunately, this deletion also removes the essential neighboring gene T07A5.5 and was therefore not studied further (see Additional data file 1). Expressed sequence tag (EST) analysis suggested that the unc-69 locus encodes two splice variants (see Figure 1a and see Additional data file 1). Northern blot analysis of poly(A)+ RNA from mixed-stage worms as well as from embryos revealed a 0.65 kb major transcript (Figure 1c), consistent with the predicted size of the T07A5.6a transcript. UNC-69 is conserved from single-celled eukaryotes to complex metazoans We found that UNC-69 is highly conserved through evolution and encodes the C. elegans homolog of mammalian SCOCO (short coiled-coil protein), a protein recently found to interact with dominant-negative ARF-like 1 (ARL1) protein in a yeast two-hybrid screen [20]. The Saccharomyces cerevisiae UNC-69 homolog, Slo1p (SCOCO-like open reading frame protein), has been shown to interact with Arl3p, a homolog of mammalian ARFRP1, another ARF-like protein, which is involved in endoplasmic reticulum-Golgi and post-Golgi transport [21,22]. Uncharacterized UNC-69/SCOCO homologs can also be found in many other animal species (Figure 3a and Additional data file 1). All of the UNC-69 homologs are predicted to form a coiledcoil structure near their carboxyl termini (the underlined region in Figure 3a). In an alignment of the S. cerevisiae, C. elegans, C. briggsae, mosquito, fly, Fugu, zebrafish, Xenopus, mouse and human protein sequences, identity over the coiled-coil regions is 32.6% (Figure 3a). The identity in the coiled-coil region jumps to 73.9% if the yeast sequence is excluded. Except for yeast, an acidic region immediately upstream of the coiled-coil domain as well as a serine/ threonine-rich region and a basic region downstream appear also to be highly conserved. In contrast, the amino terminus of UNC-69 and its homologs is highly divergent, both in length and in amino-acid sequence. The function of UNC-69 proteins seems to be conserved, since expression of human SCOCO as a transgene under the unc-69 promoter restored locomotion to unc-69 mutants (Figure 3c). We assessed the tissue distribution of human SCOCO transcripts by probing a human fetal tissue northern blot. This probe detected a single transcript of approximately 2.1 kb in all tissues examined (brain, lung, liver and kidney; Figure 3b). Human SCOCO mRNA appeared to be enriched in fetal brain, possibly hinting at a role for SCOCO in mammalian nervous system development. http://jbiol.com/content/5/4/9 Journal of Biology 2006, Volume 5, Article 9 Su and Tharin et al. 9.3 Journal of Biology 2006, 5:9 UNC-69 is expressed in the nervous system and other tissues from early embryogenesis to adulthood We generated transgenic animals expressing either aminoor carboxy-terminally gfp-tagged unc-69 fusion constructs under the control of the endogenous unc-69 promoter. Both translational fusion constructs rescued the Unc phenotype of unc-69 mutants, suggesting that the fusion proteins were correctly expressed and biologically functional. UNC-69::GFP expression was first detectable 9.4 Journal of Biology 2006, Volume 5, Article 9 Su and Tharin et al. http://jbiol.com/content/5/4/9 Journal of Biology 2006, 5:9 Figure 1 The unc-69 locus encodes a 108-amino-acid protein with a short coiled-coil domain. (a) Genetic and physical maps of chromosome III in the vicinity of the unc-69 locus. unc-69 is close to and left of ced-9. Cosmids and subclones able to rescue the locomotion defect of unc-69(e587) mutants are shown in bold. B: BamHI; H: HindIII; M: MluI; P: PstI; R: EcoRI; S: Sac