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An SCF-like ubiquitin ligase complex that controls presynaptic differentiation

Abstract

During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-β family members between presynaptic and postsynaptic termini are proposed to govern synapse formation1,2,3. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. Here we report the identification of FSN-1, a novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes4,5,6,7. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains8,9. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. We provide evidence that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase10), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation.

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Figure 1: fsn-1(hp1) mutants have similar synaptic defects to rpm-1 animals.
Figure 2: FSN-1 is present at some periactive zones and non-synaptic regions, and forms a complex with RPM-1.
Figure 3: FSN-1 antagonizes the activity of T10H9.2/ALK.
Figure 4: Ectopic and elevated expression of ALK–GFP in fsn-1 and rpm-1 mutants.

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Acknowledgements

We thank the Caenorhabditis Genetics Centre, Y. Jin, M. Nonet, B. Bamber, J. Kaplan, I. Greenwald, K.-L. Chow, J. Miwa and D. Pilgrim for C. elegans strains; The Gene Knockout Consortium for the T10H9.2(ok565) deletion strain; Y. Kohara for EST cDNAs; A. Fire for GFP vectors; the Sanger Institute for cosmids; M. Nonet, M. Tyers and Y. Xiong for antibodies; M. Ailion, Y. Jin, B. Meyer, D. Reiner and J. Thomas for communicating unpublished results; Y. Wang for technical support; members of the Zhen laboratory for discussions; and S. Cordes, J. Culotti, Y. Jin, A. Pawson, J. Rossant, M. Tyers and E. Yeh for comments on the manuscript. M.Z. isolated the fsn-1(hp1) mutant when she was a postdoctoral fellow in the laboratory of Y. Jin at UC, Santa Cruz. B.A. is supported by a National Institute of Health grant to Y. Jin. This work was funded by a grant to M.Z. from the Canadian Institute of Health Research, Canada.

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Correspondence to Mei Zhen.

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Supplementary information

Supplementary Figure 1

Cloning of fsn-1. This figure describes the cloning of fsn-1, providing details on constructs used, protein structures and comparisons with FSN-1 homologues from Drosophila and mouse. It also shows the specificity of the anti-FSN-1 antibody. (PPT 718 kb)

Supplementary Figure 2

fsn-1 is necessary and sufficient in presynaptic neurons. This figure shows details of the mosaic analysis of fsn-1. It also shows the results of the rescue experiment with exclusive FSN-1 expression in GABAergic neurons or body wall muscles. (PPT 621 kb)

Supplementary Figure 3

Specificity of anti-Skp1 and anti-cullin antibodies against C. elegans homologues proteins. This figure shows the specificities of anti-Skp1 and anti-cullin antibodies on C. elegans lysates. (PPT 473 kb)

Supplementary Figure 4

C. elegans RBX-1 is unlikely a component of the RPM-1/FSN-1 protein complex. Panel A of this figure shows the specificity of the anti-rbx1 antibody used on C. elegans lysates. Panel B shows that RBX-1 fails to co-precipitate with RPM-1::GFP whereas FSN-1 is part of a complex with RPM-1. (PPT 327 kb)

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Liao, E., Hung, W., Abrams, B. et al. An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430, 345–350 (2004). https://doi.org/10.1038/nature02647

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