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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 physically interacts with UNC-76, as shown by in vitro GST pull-down assays. (a) Full-length UNC-76 (UNC-76 FL) specifically binds to full-length GST-UNC-69 but not GST-CBP. The E1A-CBP interaction was used as a positive control. (b) Serial deletions of UNC-76: a portion of the carboxy-terminal region (deleted in UNC-76 Δγ but contained within UNC-76 B3 and A3) is necessary for interaction with GST-UNC-69. (c) Point mutation L287P or a small 19-amino-acid deletion (UNC-76 Δ19), which deletes amino acids 281–299, totally abolishes the ability of UNC-76 to bind GST-UNC-69. (d) Summary of the deletion analysis, as well as the results of rescuing experiments. Gray shading indicates conserved regions. Note that UNC-76 Δ19 not only loses its binding ability but also its rescuing activity for the unc-76(e911) mutants. The 19-amino-acid region (green) lies within a conserved region and overlaps with a region we predicted to form a coiled-coil domain (purple). A previously described axonal targeting sequence [14] is in red. The positions of different unc-76 alleles are indicated.
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Figure 7: UNC-69 physically interacts with UNC-76, as shown by in vitro GST pull-down assays. (a) Full-length UNC-76 (UNC-76 FL) specifically binds to full-length GST-UNC-69 but not GST-CBP. The E1A-CBP interaction was used as a positive control. (b) Serial deletions of UNC-76: a portion of the carboxy-terminal region (deleted in UNC-76 Δγ but contained within UNC-76 B3 and A3) is necessary for interaction with GST-UNC-69. (c) Point mutation L287P or a small 19-amino-acid deletion (UNC-76 Δ19), which deletes amino acids 281–299, totally abolishes the ability of UNC-76 to bind GST-UNC-69. (d) Summary of the deletion analysis, as well as the results of rescuing experiments. Gray shading indicates conserved regions. Note that UNC-76 Δ19 not only loses its binding ability but also its rescuing activity for the unc-76(e911) mutants. The 19-amino-acid region (green) lies within a conserved region and overlaps with a region we predicted to form a coiled-coil domain (purple). A previously described axonal targeting sequence [14] is in red. The positions of different unc-76 alleles are indicated.

Mentions: To identify potential UNC-69 interactors, we screened three C. elegans yeast two-hybrid libraries using full-length UNC-69 as bait. From these screens, we isolated at least 34 independent clones of UNC-76, a 385-amino-acid protein that was previously shown to be involved in axonal outgrowth and fasciculation in C. elegans [12-14]. The Drosophila homolog of UNC-76 was identified as a KHC-binding protein and shown to be a regulator of axonal transport [15]. A mammalian homolog of UNC-76, FEZ1, is a substrate for PKCζ [16]. Worm, fly and mammalian UNC-76 proteins are not only conserved in amino-acid sequence but also have several conserved regions (Figure 7d) predicted to be capable of forming coiled-coil domains [14,15]. UNC-76 localizes to axons, and worms harboring mutations in unc-76 have a severe Unc phenotype and coil ventrally, phenotypes very similar to those observed in unc-69 mutants [14].


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 physically interacts with UNC-76, as shown by in vitro GST pull-down assays. (a) Full-length UNC-76 (UNC-76 FL) specifically binds to full-length GST-UNC-69 but not GST-CBP. The E1A-CBP interaction was used as a positive control. (b) Serial deletions of UNC-76: a portion of the carboxy-terminal region (deleted in UNC-76 Δγ but contained within UNC-76 B3 and A3) is necessary for interaction with GST-UNC-69. (c) Point mutation L287P or a small 19-amino-acid deletion (UNC-76 Δ19), which deletes amino acids 281–299, totally abolishes the ability of UNC-76 to bind GST-UNC-69. (d) Summary of the deletion analysis, as well as the results of rescuing experiments. Gray shading indicates conserved regions. Note that UNC-76 Δ19 not only loses its binding ability but also its rescuing activity for the unc-76(e911) mutants. The 19-amino-acid region (green) lies within a conserved region and overlaps with a region we predicted to form a coiled-coil domain (purple). A previously described axonal targeting sequence [14] is in red. The positions of different unc-76 alleles are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 7: UNC-69 physically interacts with UNC-76, as shown by in vitro GST pull-down assays. (a) Full-length UNC-76 (UNC-76 FL) specifically binds to full-length GST-UNC-69 but not GST-CBP. The E1A-CBP interaction was used as a positive control. (b) Serial deletions of UNC-76: a portion of the carboxy-terminal region (deleted in UNC-76 Δγ but contained within UNC-76 B3 and A3) is necessary for interaction with GST-UNC-69. (c) Point mutation L287P or a small 19-amino-acid deletion (UNC-76 Δ19), which deletes amino acids 281–299, totally abolishes the ability of UNC-76 to bind GST-UNC-69. (d) Summary of the deletion analysis, as well as the results of rescuing experiments. Gray shading indicates conserved regions. Note that UNC-76 Δ19 not only loses its binding ability but also its rescuing activity for the unc-76(e911) mutants. The 19-amino-acid region (green) lies within a conserved region and overlaps with a region we predicted to form a coiled-coil domain (purple). A previously described axonal targeting sequence [14] is in red. The positions of different unc-76 alleles are indicated.
Mentions: To identify potential UNC-69 interactors, we screened three C. elegans yeast two-hybrid libraries using full-length UNC-69 as bait. From these screens, we isolated at least 34 independent clones of UNC-76, a 385-amino-acid protein that was previously shown to be involved in axonal outgrowth and fasciculation in C. elegans [12-14]. The Drosophila homolog of UNC-76 was identified as a KHC-binding protein and shown to be a regulator of axonal transport [15]. A mammalian homolog of UNC-76, FEZ1, is a substrate for PKCζ [16]. Worm, fly and mammalian UNC-76 proteins are not only conserved in amino-acid sequence but also have several conserved regions (Figure 7d) predicted to be capable of forming coiled-coil domains [14,15]. UNC-76 localizes to axons, and worms harboring mutations in unc-76 have a severe Unc phenotype and coil ventrally, phenotypes very similar to those observed in unc-69 mutants [14].

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