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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 does not interact with ARL-1, ARL-3 or ARFRP. (a) Plasmids containing LexA-unc-69 or LexA-human SCOCO were cotransformed into yeast cells with vector alone or vectors containing GAD-unc-76γ, GAD-arl-1, GAD-arl-3, or GAD-arfrp. Protein-protein interactions were measured as β-galactosidase activity by using ONPG liquid assays. UNC-69 did not interaction with any of the three ARL proteins. SCOCO did not interact with any of the three ARL proteins either (data not shown). (b) Auxotrophic growth assays for interactions between LexA-UNC-119 and GAD-ARL-3 or GAD-UNC-76γ. Cells (3 × 104) were plated onto +His or -His plates, and serial tenfold dilutions of cells were then subsequently plated. Cells were grown at 30°C for 48 h before images were taken. Note that the -His plate did not contain 3-amino triazol. The strength of interaction between UNC-119 and ARL-3Δ17 was only a fifth of that between UNC-119 and ARL-3 FL, as assayed by β-galactosidase activity (data not shown).
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Figure 10: UNC-69 does not interact with ARL-1, ARL-3 or ARFRP. (a) Plasmids containing LexA-unc-69 or LexA-human SCOCO were cotransformed into yeast cells with vector alone or vectors containing GAD-unc-76γ, GAD-arl-1, GAD-arl-3, or GAD-arfrp. Protein-protein interactions were measured as β-galactosidase activity by using ONPG liquid assays. UNC-69 did not interaction with any of the three ARL proteins. SCOCO did not interact with any of the three ARL proteins either (data not shown). (b) Auxotrophic growth assays for interactions between LexA-UNC-119 and GAD-ARL-3 or GAD-UNC-76γ. Cells (3 × 104) were plated onto +His or -His plates, and serial tenfold dilutions of cells were then subsequently plated. Cells were grown at 30°C for 48 h before images were taken. Note that the -His plate did not contain 3-amino triazol. The strength of interaction between UNC-119 and ARL-3Δ17 was only a fifth of that between UNC-119 and ARL-3 FL, as assayed by β-galactosidase activity (data not shown).

Mentions: UNC-69 homologs in S. cerevisiae and mammals have been reported to interact physically with members of the family of ARF-like small GTPases. To investigate whether a similar interaction occurs in C. elegans, we first used yeast two-hybrid assays to study protein-protein interactions between UNC-69 and three closely related but distinct ARF-like small GTPases, ARL-1 (F54C9.10), ARL-3 (F19H8.3), and ARFRP (Y54E10BR.2) [38]. Whereas UNC-69 readily interacted with the carboxyl terminus of UNC-76 (UNC-76γ), it did not interact with any of the three ARF-like proteins (Figure 10a). As human SCOCO was isolated as an effector for GTP-bound ARL1 [20], we also tested the ability of UNC-69 to interact with GTPase-defective forms of ARL-1 and ARFRP. UNC-69 did not interact with either ARL-1(Q70L) or ARFRP(Q79L) (Figure 10a). Deletion of the amino-terminal myristoylation site [39] also had no effect: UNC-69 did not interact with the amino-terminal deletion ARL mutants, ARL-1Δ16 (with or without the GTPase-defective mutation) or or ARL-3Δ17 (data not shown). In contrast, we readily detected the previously reported interaction between ARL-3 and UNC-119 [40], a homolog of human retinal gene 4 (HRG4) [41-43] (Figure 10b). Thus, the failure to detect any interaction between UNC-69 and the three ARF-like proteins might not have been due to inappropriate protein folding or subcellular compartmentalization in yeast.


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 does not interact with ARL-1, ARL-3 or ARFRP. (a) Plasmids containing LexA-unc-69 or LexA-human SCOCO were cotransformed into yeast cells with vector alone or vectors containing GAD-unc-76γ, GAD-arl-1, GAD-arl-3, or GAD-arfrp. Protein-protein interactions were measured as β-galactosidase activity by using ONPG liquid assays. UNC-69 did not interaction with any of the three ARL proteins. SCOCO did not interact with any of the three ARL proteins either (data not shown). (b) Auxotrophic growth assays for interactions between LexA-UNC-119 and GAD-ARL-3 or GAD-UNC-76γ. Cells (3 × 104) were plated onto +His or -His plates, and serial tenfold dilutions of cells were then subsequently plated. Cells were grown at 30°C for 48 h before images were taken. Note that the -His plate did not contain 3-amino triazol. The strength of interaction between UNC-119 and ARL-3Δ17 was only a fifth of that between UNC-119 and ARL-3 FL, as assayed by β-galactosidase activity (data not shown).
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Figure 10: UNC-69 does not interact with ARL-1, ARL-3 or ARFRP. (a) Plasmids containing LexA-unc-69 or LexA-human SCOCO were cotransformed into yeast cells with vector alone or vectors containing GAD-unc-76γ, GAD-arl-1, GAD-arl-3, or GAD-arfrp. Protein-protein interactions were measured as β-galactosidase activity by using ONPG liquid assays. UNC-69 did not interaction with any of the three ARL proteins. SCOCO did not interact with any of the three ARL proteins either (data not shown). (b) Auxotrophic growth assays for interactions between LexA-UNC-119 and GAD-ARL-3 or GAD-UNC-76γ. Cells (3 × 104) were plated onto +His or -His plates, and serial tenfold dilutions of cells were then subsequently plated. Cells were grown at 30°C for 48 h before images were taken. Note that the -His plate did not contain 3-amino triazol. The strength of interaction between UNC-119 and ARL-3Δ17 was only a fifth of that between UNC-119 and ARL-3 FL, as assayed by β-galactosidase activity (data not shown).
Mentions: UNC-69 homologs in S. cerevisiae and mammals have been reported to interact physically with members of the family of ARF-like small GTPases. To investigate whether a similar interaction occurs in C. elegans, we first used yeast two-hybrid assays to study protein-protein interactions between UNC-69 and three closely related but distinct ARF-like small GTPases, ARL-1 (F54C9.10), ARL-3 (F19H8.3), and ARFRP (Y54E10BR.2) [38]. Whereas UNC-69 readily interacted with the carboxyl terminus of UNC-76 (UNC-76γ), it did not interact with any of the three ARF-like proteins (Figure 10a). As human SCOCO was isolated as an effector for GTP-bound ARL1 [20], we also tested the ability of UNC-69 to interact with GTPase-defective forms of ARL-1 and ARFRP. UNC-69 did not interact with either ARL-1(Q70L) or ARFRP(Q79L) (Figure 10a). Deletion of the amino-terminal myristoylation site [39] also had no effect: UNC-69 did not interact with the amino-terminal deletion ARL mutants, ARL-1Δ16 (with or without the GTPase-defective mutation) or or ARL-3Δ17 (data not shown). In contrast, we readily detected the previously reported interaction between ARL-3 and UNC-119 [40], a homolog of human retinal gene 4 (HRG4) [41-43] (Figure 10b). Thus, the failure to detect any interaction between UNC-69 and the three ARF-like proteins might not have been due to inappropriate protein folding or subcellular compartmentalization in yeast.

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