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Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrin's receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.
Neurons in the developing nervous system must extend their axons to precise targets. Axon guidance molecules, located on cell membranes or in the extracellular milieu, provide positional information (Tessier-Lavigne & Goodman 1996; Yu & Bargmann 2001; Dickson 2002; Chilton 2006; Killeen & Sybingco 2008; Lai Wing Sun et al. 2011) by binding to receptors on the growth cone, a specialized structure at the tip of the axon, and inducing cytoskeletal changes.
Netrin, an evolutionarily conserved axon guidance molecule, both attracts and repels axons to guide their growth (Fig. 1A) (Serafini et al. 1994; Colamarino & Tessier-Lavigne 1995). UNC-6, the single Caenorhabditis elegans Netrin homologue, is required for dorsal-ventral axon guidance (Hedgecock et al. 1990; Ishii et al. 1992; McIntire et al. 1992; Hao et al. 2001). UNC-6 is expressed by ventral cells, including epidermoblasts, glia, neurons, muscle cells, and vulval precursor cells (Wadsworth et al. 1996; Asakura et al. 2007). As UNC-6 is thought to provide positional information by establishing a concentration gradient (Fig. 1B) (Wadsworth 2002), UNC-6 must itself be properly localized to provide accurate guidance.
The C. elegans UNC-6/Netrin receptors UNC-5 and UNC-40/DCC belong to the immunoglobulin superfamily (Fig. 1A) (Leung-Hagesteijn et al. 1992; Chan et al. 1996). Each has a single transmembrane domain, and both are required for ventral UNC-6 to repulse axons fated to extend dorsally (Fig. 1B) (Wadsworth 2002). Axons extending ventrally, however, are attracted to UNC-6, and only the UNC-40 receptor is required for this response (Fig. 1B). UNC-5 and UNC-40 function at growth cones, which are generally a long distance from the neural cell body; therefore, the localization (trafficking) of UNC-5 and UNC-40 from the neural cell body to the growth cone is critical for their function.
Since UNC-6 and its receptors provide positional information in C. elegans, their proper localization is an essential aspect of their function. This review discusses mechanisms governing the localization of UNC-6 and its receptors, UNC-5 and UNC-40, in C. elegans, focusing largely on the results of our recent reports. The precise axon guidance mechanisms and detailed functions of Netirn and its receptors are covered in other excellent reviews (Tessier-Lavigne & Goodman 1996; Yu & Bargmann 2001; Dickson 2002; Chilton 2006; Killeen & Sybingco 2008; Lai Wing Sun et al. 2011).
Localization mechanisms of the axon guidance molecule UNC-6
UNC-6 is mainly expressed in ventral cells, including epidermoblasts, glia, neurons, muscle cells, and vulval precursor cells (Wadsworth et al. 1996; Asakura et al. 2007), and is also expressed in dorsal muscle cells in the tail and in the ray of male worms. Mutant analysis has shown that although many cell types express UNC-6, each cell type has a specific mechanism for regulating UNC-6's localization (Asakura et al. 2010). We will discuss the localization mechanism for UNC-6 in muscle cells and neurons (Asakura et al. 2010).
Neurons non-cell-autonomously regulate UNC-6′s localization in muscle cells
In wild-type muscle cells, UNC-6 is excluded from the nucleus and is distributed throughout the cytoplasm (Fig. 2A) (Asakura et al. 2007). In the unc-18 mutant, the unc-68 mutant, and the syd-1; rpm-1 double mutant, UNC-6 accumulates aberrantly in the muscle cells (Fig. 2B); therefore, these genes regulate UNC-6′s localization in or secretion from muscle cells.
UNC-18 belongs to the SM (Sec1/Munc18-like) protein family (Gengyo-Ando et al. 1993; Malsam et al. 2008; Südhof & Rothman 2009), which regulates vesicle exocytosis by interacting with syntaxin, a SNARE protein found in neurons (Sassa et al. 1999; Weimer et al. 2003; Malsam et al. 2008; Südhof & Rothman 2009). Mutations in other genes required for vesicle exocytosis do not cause UNC-6 to accumulate abnormally, indicating that the traditional exocytosis machinery is not involved in UNC-18′s effect on UNC-6 localization or secretion. How, then, does UNC-18 regulate UNC-6′s localization in muscle cells?
Although the unc-18 mutant accumulates UNC-6 abnormally in muscle cells (Fig. 2B), UNC-18 is expressed in neurons, not in muscle cells (Gengyo-Ando et al. 1993). In addition, expressing unc-18 specifically in muscles does not rescue the UNC-6 accumulation in unc-18 mutants, whereas neuron-specific UNC-18 expression does. This suggests that UNC-18 in neurons regulates the UNC-6′s localization or secretion in muscle cells.
Because UNC-18 is an important regulator of neurotransmitter release, a neurotransmitter may be involved in regulating UCN-6′s localization or secretion in muscles. Oddly enough, UNC-6′s localization is not defective in muscle in unc-13, unc-25, or unc-31 mutant. UNC-13/Munc13, UNC-25/glutamate decarboxylase 1, and UNC-31/CAPS are required for acetylcholine (ACh) secretion (Maruyama & Brenner 1991), GABA synthesis (Jin et al. 1999), and neuropeptide secretion (Berwin et al. 1998; Speese et al. 2007), respectively, suggesting that traditional neurotransmitters such as ACh, GABA, or neuropeptides are not involved in UNC-18′s effect on UNC-6′s localization or secretion in muscle cells.
It is unlikely that defective muscle contraction causes the abnormal UNC-6 localization, since UNC-6 localizes normally in the unc-13 mutant, which has a paralysis phenotype. GABA-regulated muscle contractility (Garcia et al. 2007) is also unlikely to cause UNC-6 to accumulate abnormally, since UNC-6′s localization is normal in the unc-25 mutant.
We propose that UNC-6′s localization in and secretion from muscle is regulated by an unknown signal sent from neurons and mediated by UNC-18 (Fig. 2C). The signaling molecule(s) are probably secreted into synapses, since UNC-6 accumulates in the muscle cells of syd-1; rpm-1 double mutants, in which presynaptic components are reduced and disrupted (Nakata et al. 2005). The secretion machinery associated with this unknown signal is probably different from that of traditional neurotransmitters such as acetylcholine, GABA, and neuropeptides.
UNC-6 also accumulates in muscle cells in the unc-68 mutant. UNC-68 is homologous to ryanodine receptors (RyRs), a class of Ca2+ channels (Maryon et al. 1996; Sakube et al. 1997; Zalk et al. 2007). UNC-68 is important in muscle contraction, since it mediates Ca2+-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER). In addition, CICR regulates both evoked and spontaneous neurotransmitter releases (Liu et al. 2005). Therefore, the simplest explanation for UNC-68′s function is that in muscle cells, neurons, or both, UNC-68 mediates CICR from the ER, and this CICR is required for UNC-6 to be properly localized in or secreted from muscle cells (Fig. 2C).
Although UNC-6′s localization is defective in unc-18 and unc-68 mutant muscle cells, unc-18 and unc-68 do not appear to participate in known UNC-6 functions, including AVM axon guidance, DA9 synaptic development, or distal tip cell migration. UNC-6 may have unknown functions when expressed by muscle cells, or the functions of the UNC-6 expressed by muscle cells may be masked by the UNC-6 expressed by neurons.
Localization mechanisms of UNC-6 in neurons
UNC-6 is expressed in ventral neurons (Wadsworth et al. 1996; Asakura et al. 2007). In wild-type neurons, UNC-6 is excluded from the nucleus and has a punctate distribution pattern throughout the cytoplasm and axons (Fig. 3A). The genes required for UNC-6′s localization in neurons appear to include genes required for membrane trafficking. In unc-51, unc-14, and unc-104 mutants, UNC-6 accumulates in neuronal cell bodies, but little is detected in the axons (Fig. 3B), suggesting that these genes regulate UNC-6′s transport from the neuronal cell body to the axon. The UNC-6/Netrin-mediated dorso-ventral axonal guidance is also defective in these mutants (McIntire et al. 1992). In addition, unc-51, unc-14, and unc-104 interact genetically with unc-6 (Ogura & Goshima 2006; Asakura et al. 2010). These findings suggest that the defective UNC-6 transport in these mutants disrupts normal UNC-6 secretion from the neurons.
In unc-51 and unc-14 mutants, UNC-6 accumulates unevenly in the cell body. UNC-51 is a serine/threonine kinase that binds UNC-14, a RUN domain protein (Ogura et al. 1994, 1997). With regard to neural function, UNC-51, UNC-14, and UNC-51′s homologues in other species are predicted to be important in vesicle trafficking, including early endosome functions (McIntire et al. 1992; Tomoda et al. 2004; Sakamoto et al. 2005; Ogura & Goshima 2006; Toda et al. 2008). Therefore, UNC-51 and UNC-14 may regulate the processing involved in UNC-6′s localization, including UNC-6′s maturation, selection, or transport (Fig. 3C). UNC-51 is also required for autophagy in C. elegans (Meléndez et al. 2003). However, it is unlikely that the traditional autophagy pathway participates in UNC-6 localization, since the RNAi of other genes required for autophagy (bec-1/atg-6, atg-7, lgg-1/atg-8, and atg-18) does not disrupt the normal UNC-6 localization.
UNC-104 is a homologue of the kinesin motor protein KIF1A, which transports precursors of synaptic vesicles (SVs) and dense core vesicles (DCVs) from neuronal cell bodies to synapses (Hall & Hedgecock 1991; Otsuka et al. 1991; Yonekawa et al. 1998; Zahn et al. 2004; Hirokawa & Noda 2008). In the unc-104 mutant, UNC-6 accumulates throughout the neuronal cell body, and the SVs and DCVs show a very similar pattern (Fig. 3B) (Hall & Hedgecock 1991; Nonet 1999; Zahn et al. 2004). These reports support our hypothesis that UNC-104 transports vesicles containing UNC-6 from the neuronal cell body to the axon (Fig. 3C). Our hypothesis is also supported by a report that UNC-6 is required for synaptic partner recognition of the PHB and AVA neurons (Park et al. 2011). In the AVA neuron, UNC-6 should localize to the synaptic region far from the cell body, and UNC-104 is required for this localization. In addition, UNC-6 may be secreted by the synapse, since UNC-104/KIF1A transports synaptic vesicle precursors (Hirokawa & Noda 2008).
A model for Netrin's patterning mechanism has been proposed in Drosophila melanogaster (Hiramoto et al. 2000). In this model, Netrin's localization is regulated by interaction with its receptor, Frazzled/UNC-40. In C. elegans, UNC-6′s localization is not altered in unc-40 mutants. Since the C. elegans nervous system is extremely simple compared with that of D. melanogaster, a similar interaction between UNC-6 and UNC-40 might not be required for UNC-6 to function. It will be important for future studies to address whether a similar Netrin interaction is conserved in mammalian species.
Localization mechanisms of the UNC-6/Netrin receptor UNC-5
UNC-5′s localization is regulated by UNC-51 and UNC-14 (Ogura & Goshima 2006), which also regulate UNC-6′s localization (Asakura et al. 2010). Thus, UNC-51 and UNC-14 regulate the localization of both the UNC-6 ligand and its receptor, UNC-5. UNC-5 is a single trans-membrane protein with immunoglobulin domains at its extra-cellular region (Fig. 1A) (Leung-Hagesteijn et al. 1992). In unc-51 and unc-14 mutants, UNC-5 accumulates at neural cell bodies (Fig. 4B), and little UNC-5 is observed at axons (Fig. 4B). UNC-51 and UNC-14 may regulate the processing involved in UNC-5′s localization in neuronal cell bodies, including UNC-5′s maturation, selection, or transport (Fig. 4C). Like many other proteins (although not SNB-1/VAMP2), another UNC-6 receptor, UNC-40, localizes normally in unc-51 and unc-14 mutants, suggesting that UNC-51 and UNC-14 specifically regulate the localization of UNC-6 and its receptor UNC-5.
UNC-51 cooperates with PP2A (protein phosphatase 2A) to guide axon growth (Ogura et al. 2010). Therefore, PP2A may cooperate with UNC-51 for the neural localization of UNC-6 and UNC-5 (Fig. 4C). UNC-51 and UNC-14 are predicted to be important in vesicle trafficking, including early endosome functions (McIntire et al. 1992; Tomoda et al. 2004; Sakamoto et al. 2005; Toda et al. 2008).
Interestingly, in unc-51 and unc-14 mutants, UNC-5′s localization in non-neural cells (distal tip and excretory cells) is normal, as is UNC-6′s localization (Asakura et al. 2010). This suggests that UNC-51 and UNC-14 specifically regulate the localization of UNC-6 and UNC-5 in neurons only, and may regulate axonal UNC-6 and UNC-5 trafficking in neurons. UNC-51 is also required for autophagy in C. elegans (Meléndez et al. 2003). As with UNC-6, UNC-5′s localization is not affected by loss-of-function mutations of the autophagy genes bec-1/atg-6, atg-7, lgg-1/atg-8, or atg-18, indicating that the traditional autophagy pathway probably does not participate in UNC-5′s localization. With regard to neuronal trafficking, the localization mechanisms of UNC-5 and UNC-6 differ. The UNC-6 transporter UNC-104/KIF1A is not involved in UNC-5′s localization, since its mutant does not have a mislocated UNC-5 phenotype. No candidate motor proteins responsible for UNC-5′s localization have been determined. Future studies should address the question of how UNC-51, UNC-14, and motor proteins coordinately regulate the trafficking and localization of UNC-6 and UNC-5.
Localization mechanisms of the UNC-6/Netrin receptor UNC-40/DCC
The UNC-6 receptor UNC-40 is a single transmembrane protein with immunoglobulin domains at its extra-cellular regions (Fig. 1A) (Chan et al. 1996). UNC-6 induces UNC-40 to localize ventrally in the hermaphrodite specific motor neurons (HSN) neuron growth cones (Fig. 5) (Adler et al. 2006), which causes MIG-10, a downstream actin regulator, to also localize ventrally. A ventral UNC-40 localization induced by UNC-6 is also reported in anchor cells (Ziel et al. 2009). In these cases, UNC-6 is secreted from the ventral regions of the HSN neurons and the anchor cells.
Several studies have found that levels of the receptors for axon guidance molecules affect how axons are guided. The levels of UNC-40 and of SAX-3/Robo, a receptor for the axon guidance molecule Slit, regulate the direction of axon extension from neural cell bodies, by a mechanism independent of their ligands (Fig. 6) (Levy-Strumpf & Culotti 2007; Watari-Goshima et al. 2007). The kinesin-like protein VAB-8 promotes the proper localization of the receptors for axon guidance molecules UNC-6 and SLT-1. The VAB-8-binding protein UNC-73, a guanine nucleotide exchange factor, and a Rac-like small GTPase participate in this function. Both VAB-8 and UNC-73 are binding partners of UNC-51 (Lai & Garriga 2004; our unpubl.data, 2000), and UNC-51 regulates localization of an UNC-6 receptor UNC-5 that also participates in the function (Levy-Strumpf & Culotti 2007; Watari-Goshima et al. 2007). VAB-8 and UNC-73 may cooperate with UNC-51 to regulate the localization or trafficking of these receptors (Fig. 6). These cytoplasmic proteins were previously thought to act downstream of the receptors. However, at least in this case, they act upstream of the receptors and influence their expression. RPM-1 and CLEC-38 are also axon guidance regulators that regulate the expression of SAX-3/Robo, UNC-5, and UNC-40 (Kulkarni et al. 2008; Li et al. 2008).
In this review, we discuss possible localization mechanisms for UNC-6/Netrin and its receptors, UNC-5 and UNC-40/DCC, in C. elegans. However, their precise molecular localization mechanisms are still unclear and will require detailed analysis. As many of the gene products identified as being involved in their localization are evolutionarily conserved, it will be interesting to analyze the functional relationships of their homologues in other species.
In addition to guiding axons, UNC-6 and its receptors also provide positional information for cell migration (Hedgecock et al. 1990), synapse formation (Colón-Ramos et al. 2007; Poon et al. 2008), cell polarity (Adler et al. 2006; Ziel et al. 2009), axonal branching (Hao et al. 2010), synaptic partner recognition (Park et al. 2011), and muscle arm extension (Seetharaman et al. 2011), as well as promoting dendritic growth (Teichmann & Shen 2011). Thus, determining the precise localization mechanisms of UNC-6 and its receptors, UNC-5 and UNC-40, will provide novel insight into their functions as well.
We thank The Company of Biologists and the Genetics Society of America for permitting us to reuse the contents of our published papers (Ogura & Goshima 2006; Asakura et al. 2010). We regret that many valuable findings could not be included in this review due to space restrictions. Support for the work discussed in this review was provided by CREST (Core Research for Evolutional Science and Technology) of JST (Japan Science and Technology Agency) (Y.G.), Grants-in-aid for Scientific Research in a Priority Area (Y.G. and K.O.) from the Ministry of Education, Science, Sports and Culture, and the Yokohama Foundation for Advancement of Medical Science (T.A. and K.O.).