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

Su CW, Tharin S, Jin Y, Wightman B, Spector M, Meili D, Tsung N, Rhiner C, Bourikas D, Stoeckli E, Garriga G, Horvitz HR, Hengartner MO - J. Biol. (2006)

Bottom Line: UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. chengwensu@gmail.com

ABSTRACT

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. 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.

Conclusion: 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.

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UNC-69 and UNC-76 cooperate to regulate the size and position of synaptic vesicles. (a-d) Lateral view of adult hermaphrodites 52–54 h after hatching, single section. (e,f) Lateral view of the DNC of adult hermaphrodites 52–54 h after hatching, flattened images of confocal z-stack. Anterior is to the left and dorsal is up. (a,e) SNB-1::GFP is evenly distributed along the DNC in wild-type animals. (b) Removing one copy of unc-69 does not affect SNB-1::GFP distribution. (c,d,f) SNB-1::GFP becomes diffused and the puncta becomes larger (arrows) in unc-69(e587)/+; unc-76(e911)/+ and (unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes. Occasionally, SNB-1::GFP is missing altogether from a stretch of the DNC (bracket in (d)). The genotypes are as follows: (a,e) juIs1 [Punc-25::snb-1::gfp], (b) qC1/unc-69(e587); juIs1, (c) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(e911), (d,f) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(n2457). Scale bars represent 10 μm.
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Figure 8: UNC-69 and UNC-76 cooperate to regulate the size and position of synaptic vesicles. (a-d) Lateral view of adult hermaphrodites 52–54 h after hatching, single section. (e,f) Lateral view of the DNC of adult hermaphrodites 52–54 h after hatching, flattened images of confocal z-stack. Anterior is to the left and dorsal is up. (a,e) SNB-1::GFP is evenly distributed along the DNC in wild-type animals. (b) Removing one copy of unc-69 does not affect SNB-1::GFP distribution. (c,d,f) SNB-1::GFP becomes diffused and the puncta becomes larger (arrows) in unc-69(e587)/+; unc-76(e911)/+ and (unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes. Occasionally, SNB-1::GFP is missing altogether from a stretch of the DNC (bracket in (d)). The genotypes are as follows: (a,e) juIs1 [Punc-25::snb-1::gfp], (b) qC1/unc-69(e587); juIs1, (c) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(e911), (d,f) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(n2457). Scale bars represent 10 μm.

Mentions: We showed above that UNC-69 is required for localization of synaptic vesicles in axons. Does UNC-76 also have a role in this process, and if so, does UNC-76 control presynaptic organization together with UNC-69? Unfortunately, all existing unc-76 alleles have severe axonal outgrowth defects, making interpretations of defect in synaptic vesicle localization difficult. To bypass this problem and to reveal possible genetic interactions between unc-69 and unc-76, we looked at the localization of the synaptobrevin SNB-1::GFP puncta in unc-69(lf)/+; unc-76(lf)/+ double heterozygotes (Figure 8). In wild-type adult hermaphrodites, SNB-1::GFP can be seen as evenly distributed puncta along the DNC [7] (Figure 8a,e). The distribution pattern of GFP puncta in DNC was not significantly different in unc-69(e587)/+ heterozygotes (Figure 8b) as compared with wild-type animals. However, in both unc-69(e587)/+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygous hermaphrodites, SNB-1::GFP puncta were occasionally more diffused, larger, or completely absent within a stretch of DNC (Figure 8c,d,f); the absence of SNB-1::GFP puncta may be due to either transport or axon extension defects. In addition, unc-69(e587)/+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes occasionally had a slight Unc phenotype in locomotion, resembling weak synaptic transmission mutants. The weak locomotion defect could be a direct or indirect effect of the synaptic vesicle mislocalization defect.


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

Su CW, Tharin S, Jin Y, Wightman B, Spector M, Meili D, Tsung N, Rhiner C, Bourikas D, Stoeckli E, Garriga G, Horvitz HR, Hengartner MO - J. Biol. (2006)

UNC-69 and UNC-76 cooperate to regulate the size and position of synaptic vesicles. (a-d) Lateral view of adult hermaphrodites 52–54 h after hatching, single section. (e,f) Lateral view of the DNC of adult hermaphrodites 52–54 h after hatching, flattened images of confocal z-stack. Anterior is to the left and dorsal is up. (a,e) SNB-1::GFP is evenly distributed along the DNC in wild-type animals. (b) Removing one copy of unc-69 does not affect SNB-1::GFP distribution. (c,d,f) SNB-1::GFP becomes diffused and the puncta becomes larger (arrows) in unc-69(e587)/+; unc-76(e911)/+ and (unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes. Occasionally, SNB-1::GFP is missing altogether from a stretch of the DNC (bracket in (d)). The genotypes are as follows: (a,e) juIs1 [Punc-25::snb-1::gfp], (b) qC1/unc-69(e587); juIs1, (c) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(e911), (d,f) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(n2457). Scale bars represent 10 μm.
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Figure 8: UNC-69 and UNC-76 cooperate to regulate the size and position of synaptic vesicles. (a-d) Lateral view of adult hermaphrodites 52–54 h after hatching, single section. (e,f) Lateral view of the DNC of adult hermaphrodites 52–54 h after hatching, flattened images of confocal z-stack. Anterior is to the left and dorsal is up. (a,e) SNB-1::GFP is evenly distributed along the DNC in wild-type animals. (b) Removing one copy of unc-69 does not affect SNB-1::GFP distribution. (c,d,f) SNB-1::GFP becomes diffused and the puncta becomes larger (arrows) in unc-69(e587)/+; unc-76(e911)/+ and (unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes. Occasionally, SNB-1::GFP is missing altogether from a stretch of the DNC (bracket in (d)). The genotypes are as follows: (a,e) juIs1 [Punc-25::snb-1::gfp], (b) qC1/unc-69(e587); juIs1, (c) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(e911), (d,f) qC1/unc-69(e587); nT1[qIs51]/juIs1; nT1[qIs51]/unc-76(n2457). Scale bars represent 10 μm.
Mentions: We showed above that UNC-69 is required for localization of synaptic vesicles in axons. Does UNC-76 also have a role in this process, and if so, does UNC-76 control presynaptic organization together with UNC-69? Unfortunately, all existing unc-76 alleles have severe axonal outgrowth defects, making interpretations of defect in synaptic vesicle localization difficult. To bypass this problem and to reveal possible genetic interactions between unc-69 and unc-76, we looked at the localization of the synaptobrevin SNB-1::GFP puncta in unc-69(lf)/+; unc-76(lf)/+ double heterozygotes (Figure 8). In wild-type adult hermaphrodites, SNB-1::GFP can be seen as evenly distributed puncta along the DNC [7] (Figure 8a,e). The distribution pattern of GFP puncta in DNC was not significantly different in unc-69(e587)/+ heterozygotes (Figure 8b) as compared with wild-type animals. However, in both unc-69(e587)/+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygous hermaphrodites, SNB-1::GFP puncta were occasionally more diffused, larger, or completely absent within a stretch of DNC (Figure 8c,d,f); the absence of SNB-1::GFP puncta may be due to either transport or axon extension defects. In addition, unc-69(e587)/+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes occasionally had a slight Unc phenotype in locomotion, resembling weak synaptic transmission mutants. The weak locomotion defect could be a direct or indirect effect of the synaptic vesicle mislocalization defect.

Bottom Line: UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. chengwensu@gmail.com

ABSTRACT

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. 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus