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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.

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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 affects axonal but not dendritic trafficking. (a,c) SNB-1::GFP is seen as evenly spaced puncta along the (a) VNC and (c) DNC in wild-type animals. (b,d,e) In unc-69(ju69) mutants, SNB-1::GFP puncta are on average bigger and often are absent from the VNC (arrowhead in (b)) and the DNC (arrowheads in (d,e)). In addition, SNB-1::GFP sometimes diffuses into the commissure (arrow in (d)). (a,b,e) Lateral views; (c,d) dorsal views of adult hermaphrodites. (f-i) As in (f,h) wild-type animals , neuronal morphology is grossly normal in (g,i) unc-69(ju69) mutants, and commissures still routinely reach the DNC. D-type GABAergic neuron morphology is visualized with the Punc-25::gfp transgene juIs76. (f,g) Lateral views; (h,i) dorsal views. (j) Distribution of SNB-1::GFP puncta in a stretch of axon labeled with Punc-25::DsRed monomer in the DNC in a unc-69(ju69) mutant hermaphrodite. SNB-1::GFP puncta are unevenly distributed, even though the DNC anatomy is grossly normal. (k) SNB-1::GFP is not significantly mislocalized into DD dendrites in unc-69(ju69) mutants. Animals carrying an snb-1::gfp transgene were scored at the L1 larval stage. Whereas 90% of unc-16(ju146) L1 larvae (n = 32) show dorsal GFP, 0% of wild-type L1s (n = 47) and 3% unc-69(ju69) L1s (n = 59) show dorsal GFP. Error bars represent the standard error of the mean. (l-n) The diacetyl odorant receptor ODR-10::GFP is targeted efficiently into AWB cilia both in (l) wild-type worms and in (m) unc-69(ju69) mutants. (n) In contrast, ODR-10::GFP becomes diffused in the dendritic targeting mutant unc-101. The arrow indicates the cilia; arrowheads indicate packets of ODR-10::GFP that shuttle in the dendrites. Anterior is to the left and dorsal is up.
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Figure 6: unc-69 affects axonal but not dendritic trafficking. (a,c) SNB-1::GFP is seen as evenly spaced puncta along the (a) VNC and (c) DNC in wild-type animals. (b,d,e) In unc-69(ju69) mutants, SNB-1::GFP puncta are on average bigger and often are absent from the VNC (arrowhead in (b)) and the DNC (arrowheads in (d,e)). In addition, SNB-1::GFP sometimes diffuses into the commissure (arrow in (d)). (a,b,e) Lateral views; (c,d) dorsal views of adult hermaphrodites. (f-i) As in (f,h) wild-type animals , neuronal morphology is grossly normal in (g,i) unc-69(ju69) mutants, and commissures still routinely reach the DNC. D-type GABAergic neuron morphology is visualized with the Punc-25::gfp transgene juIs76. (f,g) Lateral views; (h,i) dorsal views. (j) Distribution of SNB-1::GFP puncta in a stretch of axon labeled with Punc-25::DsRed monomer in the DNC in a unc-69(ju69) mutant hermaphrodite. SNB-1::GFP puncta are unevenly distributed, even though the DNC anatomy is grossly normal. (k) SNB-1::GFP is not significantly mislocalized into DD dendrites in unc-69(ju69) mutants. Animals carrying an snb-1::gfp transgene were scored at the L1 larval stage. Whereas 90% of unc-16(ju146) L1 larvae (n = 32) show dorsal GFP, 0% of wild-type L1s (n = 47) and 3% unc-69(ju69) L1s (n = 59) show dorsal GFP. Error bars represent the standard error of the mean. (l-n) The diacetyl odorant receptor ODR-10::GFP is targeted efficiently into AWB cilia both in (l) wild-type worms and in (m) unc-69(ju69) mutants. (n) In contrast, ODR-10::GFP becomes diffused in the dendritic targeting mutant unc-101. The arrow indicates the cilia; arrowheads indicate packets of ODR-10::GFP that shuttle in the dendrites. Anterior is to the left and dorsal is up.

Mentions: The C. elegans synaptobrevin/vesicle-associated membrane protein (VAMP) homolog SNB-1 is a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) on synaptic vesicles (SVs). Tagged SNB-1 can be used to follow SVs as they are transported to presynaptic regions [26]. We isolated an allele of unc-69, ju69, in a visual genetic screen for mislocalization of a SNB-1::GFP reporter in D-type GABAergic motor neurons. In wild-type worms, SNB-1::GFP expressed in the D neurons can be localized to discrete puncta along the VNC and DNC, at sites of neuromuscular junctions (Figure 6a,c). In unc-69(ju69) mutant nerve cords, SNB-1::GFP puncta were irregular in size and position, on average larger than in wild type, and often completely missing for extended stretches (Figure 6b,d,e). In addition, we occasionally observed puncta that abnormally diffused from the nerve cords into the commissures (Figure 6d). Despite the abnormal shape and distribution of presynaptic regions, the overall morphology of DD and VD neurons was grossly normal (Figure 6f–i) and only occasionally (<10%; n = 50) did one commissure fail to exit the VNC. We made similar observations in touch neurons using worms carrying the Pmec-4::gfptransgene zdIs5 (data not shown), a strain chosen for reconfirming findings made on D-type GABAergic motor neurons.


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 affects axonal but not dendritic trafficking. (a,c) SNB-1::GFP is seen as evenly spaced puncta along the (a) VNC and (c) DNC in wild-type animals. (b,d,e) In unc-69(ju69) mutants, SNB-1::GFP puncta are on average bigger and often are absent from the VNC (arrowhead in (b)) and the DNC (arrowheads in (d,e)). In addition, SNB-1::GFP sometimes diffuses into the commissure (arrow in (d)). (a,b,e) Lateral views; (c,d) dorsal views of adult hermaphrodites. (f-i) As in (f,h) wild-type animals , neuronal morphology is grossly normal in (g,i) unc-69(ju69) mutants, and commissures still routinely reach the DNC. D-type GABAergic neuron morphology is visualized with the Punc-25::gfp transgene juIs76. (f,g) Lateral views; (h,i) dorsal views. (j) Distribution of SNB-1::GFP puncta in a stretch of axon labeled with Punc-25::DsRed monomer in the DNC in a unc-69(ju69) mutant hermaphrodite. SNB-1::GFP puncta are unevenly distributed, even though the DNC anatomy is grossly normal. (k) SNB-1::GFP is not significantly mislocalized into DD dendrites in unc-69(ju69) mutants. Animals carrying an snb-1::gfp transgene were scored at the L1 larval stage. Whereas 90% of unc-16(ju146) L1 larvae (n = 32) show dorsal GFP, 0% of wild-type L1s (n = 47) and 3% unc-69(ju69) L1s (n = 59) show dorsal GFP. Error bars represent the standard error of the mean. (l-n) The diacetyl odorant receptor ODR-10::GFP is targeted efficiently into AWB cilia both in (l) wild-type worms and in (m) unc-69(ju69) mutants. (n) In contrast, ODR-10::GFP becomes diffused in the dendritic targeting mutant unc-101. The arrow indicates the cilia; arrowheads indicate packets of ODR-10::GFP that shuttle in the dendrites. Anterior is to the left and dorsal is up.
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Figure 6: unc-69 affects axonal but not dendritic trafficking. (a,c) SNB-1::GFP is seen as evenly spaced puncta along the (a) VNC and (c) DNC in wild-type animals. (b,d,e) In unc-69(ju69) mutants, SNB-1::GFP puncta are on average bigger and often are absent from the VNC (arrowhead in (b)) and the DNC (arrowheads in (d,e)). In addition, SNB-1::GFP sometimes diffuses into the commissure (arrow in (d)). (a,b,e) Lateral views; (c,d) dorsal views of adult hermaphrodites. (f-i) As in (f,h) wild-type animals , neuronal morphology is grossly normal in (g,i) unc-69(ju69) mutants, and commissures still routinely reach the DNC. D-type GABAergic neuron morphology is visualized with the Punc-25::gfp transgene juIs76. (f,g) Lateral views; (h,i) dorsal views. (j) Distribution of SNB-1::GFP puncta in a stretch of axon labeled with Punc-25::DsRed monomer in the DNC in a unc-69(ju69) mutant hermaphrodite. SNB-1::GFP puncta are unevenly distributed, even though the DNC anatomy is grossly normal. (k) SNB-1::GFP is not significantly mislocalized into DD dendrites in unc-69(ju69) mutants. Animals carrying an snb-1::gfp transgene were scored at the L1 larval stage. Whereas 90% of unc-16(ju146) L1 larvae (n = 32) show dorsal GFP, 0% of wild-type L1s (n = 47) and 3% unc-69(ju69) L1s (n = 59) show dorsal GFP. Error bars represent the standard error of the mean. (l-n) The diacetyl odorant receptor ODR-10::GFP is targeted efficiently into AWB cilia both in (l) wild-type worms and in (m) unc-69(ju69) mutants. (n) In contrast, ODR-10::GFP becomes diffused in the dendritic targeting mutant unc-101. The arrow indicates the cilia; arrowheads indicate packets of ODR-10::GFP that shuttle in the dendrites. Anterior is to the left and dorsal is up.
Mentions: The C. elegans synaptobrevin/vesicle-associated membrane protein (VAMP) homolog SNB-1 is a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) on synaptic vesicles (SVs). Tagged SNB-1 can be used to follow SVs as they are transported to presynaptic regions [26]. We isolated an allele of unc-69, ju69, in a visual genetic screen for mislocalization of a SNB-1::GFP reporter in D-type GABAergic motor neurons. In wild-type worms, SNB-1::GFP expressed in the D neurons can be localized to discrete puncta along the VNC and DNC, at sites of neuromuscular junctions (Figure 6a,c). In unc-69(ju69) mutant nerve cords, SNB-1::GFP puncta were irregular in size and position, on average larger than in wild type, and often completely missing for extended stretches (Figure 6b,d,e). In addition, we occasionally observed puncta that abnormally diffused from the nerve cords into the commissures (Figure 6d). Despite the abnormal shape and distribution of presynaptic regions, the overall morphology of DD and VD neurons was grossly normal (Figure 6f–i) and only occasionally (<10%; n = 50) did one commissure fail to exit the VNC. We made similar observations in touch neurons using worms carrying the Pmec-4::gfptransgene zdIs5 (data not shown), a strain chosen for reconfirming findings made on D-type GABAergic motor neurons.

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