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First published online 3 August 2006
doi: 10.1242/dev.02503

Development 133, 3441-3450 (2006)
Published by The Company of Biologists 2006
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The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

Ken-ichi Ogura1,* and Yoshio Goshima1,2

1 Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, 3-9 Fukura, Kanazawa-ku, Yokohama, 236-0004, Japan.
2 CREST, JST, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.


Figure 1
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  		Fig. 1. Morphology of DD/VD motoneurons and the phenotypes of the unc-51, unc-14, unc-6 and unc-5 mutants. (A) Wild type. (B) Schematic diagram of a single DD/VD neuron. (C) unc-51(e369). (D) unc-14(e57). (E) unc-6(ev400). (F) unc-5(e53). DD/VD neurons were visualized by using oxIs12 (unc-47p::gfp). Anterior is upper left. Dorsal is upper right. Arrows show abnormally positioned axons. Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Accumulation of functional UNC-5::GFP in the cell bodies of DD/VD neurons in unc-51 and unc-14 mutants. (A-E) Localization of UNC-5::GFP (A,B,D) and of mRFP (C,E), which marks cell bodies and axons of DD/VD neurons. (A) Wild type. (B,C) The same region in unc-51(e369). (D,E) The same region in unc-14(e57). Arrowheads point to DD/VD cell bodies. Open triangles point to the abnormal accumulation of UNC-5::GFP. Scale bar: 10 µm. UNC-5::GFP was localized to small vesicles throughout the axons and cell bodies in wild-type animals (A). UNC-5::GFP accumulated in the cell bodies of DD/VD neurons in unc-51 (B) and unc-14 (D) mutants. A small amount of UNC-5::GFP was present in axons (B). (F,G) Quantification of GFP fluorescence at cell bodies (F) and axons (G). In each case, 20 cell bodies and axons were examined and the results were averaged. The numbers at the bottom are in arbitrary units that were measured by using an LSM510 confocal microscope (Zeiss). Error bars show the standard error. * P<0.01 (Bonferroni correction). NS, not significant. We determined their cell bodies and axons by using the fluorescence of mRFP. In unc-51(e369) mutants, large amounts of UNC-5::GFP accumulated in cell bodies. In addition, the amount of UNC-5::GFP in axons reduced. In unc-14(e57) mutants, although certain accumulation of UNC-5::GFP was observed in the cell bodies (D), the fluorescence amounts of the cell bodies and the axons were statistically not different from that of wild type.

 

Figure 3
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  		Fig. 3. Localization of UNC-5::GFP in cell bodies. (A) Wild type. (B) unc-51(e369). (C) unc-14(e57). Arrows point to the abnormal accumulation of UNC-5::GFP. The accumulation was never observed in wild type but commonly observed in the unc-51 and unc-14 mutants. Scale bar: 1 µm.

 

Figure 4
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  		Fig. 4. UNC-5::GFP expression in non-neural cells and UNC-40::GFP expression are normal in unc-51 and unc-14 mutants. (A-J) Wild type (A,D,H); unc-51(e369) (B,E,I); unc-14(e57) (C,F,J); unc-40(n324) (G). (A-C) Dotted lines show the approximate outline of a distal tip cell (DTC). Anterior is left. Dorsal is up. (D-F) Excretory cells. Anterior is upper left. Dorsal is upper right. (G) Morphology of DD/VD motoneurons of unc-40(n324) mutants. Anterior is upper left. Dorsal is upper right. Arrows show abnormally positioned axons. (H-J) UNC-40::GFP expression. UNC-40::GFP was expressed at the cell surface of DD/VD cells (H) and its localization was normal in unc-51 (I) and unc-14 mutants (J). Scale bars: 10 µm in A-F,H-J; 100 µm in G.

 

Figure 5
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  		Fig. 5. Genetic interactions among unc-5, unc-6, unc-40, unc-51 and unc-14. (A-C) To analyze the axon-guidance defects of the mutants, we counted the number of DD/VD axons that could reach the dorsal nerve cord in L4 larvae or young adults. At least 20 worms were examined and the results were averaged for each strain. unc-5(e53)/+ and unc-40(n324)/+ represent the heterogygote of each of mutants. Error bars show the standard error. *1, P<0.01; NS, not significant (Bonferroni correction). (A) Genetic interactions among unc-5, unc-51 and unc-14. The low dose of unc-5 strongly enhanced the defects of both unc-51 and unc-14 (*1). *2, unc-14(e57) did not enhance the phenotype of unc-51(e369), indicating that these genes were in the same genetic pathway for axon guidance. (B) Genetic interactions among unc-6, unc-51 and unc-14. *3, strong enhancement of mutant phenotypes by unc-6(ju152). (C) Genetic interactions between unc-40 and unc-51. The low dose of unc-40 did not enhance the defects of unc-51. In these analyses, we used hT2 (I, III) balancer for a making the heterozygote strain of unc-40(n324). As the balancer strain includes qIs48 that expresses GFP at the pharynx, we could not analyze one axon that passed near the pharynx. We excluded the axon from our counting.

 

Figure 6
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  		Fig. 6. The combination of the transmembrane and cytoplasmic regions of UNC-5 were necessary and sufficient for the regulation by UNC-51. (A-R) UNC-5 has two Ig domains, two TSP domains, a transmembrane domain (TM), a ZU-5 domain and a DD domain. SS represents the UNC-40 signal sequence. (A,H,I) Full-length UNC-5. (B,J,K) Deletion of the extracellular region. (C,L,M) Deletion of the cytoplasmic region. (D,N) Only the cytoplasmic region. (E,O) Extracellular region alone. (F,P) Transmembrane domain alone. (G,Q,R) Full-length UNC-5, including the UNC-40 signal sequence at the N-terminus. All the proteins were expressed as GFP-fusion proteins in DD/VD neurons using the unc-25 promoter. `Wild type' indicates that the fusion proteins were expressed in wild type. `unc-51(e369)' indicates that the fusion proteins were expressed in unc-51(e369). (H,J,L,M,Q) Localization to small vesicles. In the cases of L and M, the vesicles were larger than those of wild type. (I,K,O,R) Accumulation in the cell bodies. (N) Localization to the nucleus. (P) Localization to the cytoplasm.

 

Figure 7
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  		Fig. 7. UNC-5 and UNC-51 or UNC-5 and UNC-14 were partly co-localized in DD/VD neurons. (A) UNC-51::GFP. (B,E) UNC-5::mRFP. (C) Merged image of A and B. (D) UNC-14::GFP. (F) Merged image of D and E. (A-C) The same cell bodies. (D-F) The same axons. Arrowheads represent the co-localization of UNC-5 and UNC-51 or UNC-5 and UNC-14. Although their co-localization was observed in DD/VD neurons, their co-localization levels were very different in each cell. In this paper, we have shown the good co-localization cases. Scale bars: 1 µm.

 

Figure 8
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  		Fig. 8. Models. (A) Possible function of UNC-51 and UNC-14 in the regulation of UNC-5 localization. UNC-51 and UNC-14 may affect the formation, selection or transport of the vesicles and their associated UNC-5. (B) An UNC-5 silencing model. The gray ovals represent Netin/UNC-6. The green `Y's represent UNC-5. UNC-5 may be packaged in small vesicles to inhibit its surface expression in DD/VD neurons. Some unknown `UNC-5 surfacing signal' may relocate UNC-5 to the cell membrane where the growth cones respond to Netrin/UNC-6, resulting in dorsal extension. In this model, we show that UNC-5 is localized at the cell surface after the `UNC-5 surfacing signal'; however, we did not observe the surface localization of UNC-5::GFP by using a fluorescent microscope at the L4 stage. We think that part of UNC-5::GFP localizes at the cell surface, and that this is enough for the function. Alternatively, the surface localization may be temporary.

 

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© The Company of Biologists Ltd 2006