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First published online November 11, 2004
doi: 10.1242/10.1242/dev.01457

Development 131, 5991-6000 (2004)
Published by The Company of Biologists 2004
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The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans

Tina Lai and Gian Garriga*

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA



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  		Fig. 1. VAB-8 and UNC-51 interactions. (A) Yeast two-hybrid interactions between full-length VAB-8 and various UNC-51 fragments. VAB-8L was fused to the GAL4 DNA binding domain (GAL4BD), and UNC-51 fragments were fused to the GAL4 activation domain (GAL4AD). Binding was determined by ß-galactosidase activity using filter lift assays (Durfee et al., 1993Go). +, most colonies had turned blue overnight; –, no colony had turned blue overnight. (B) GST and GST-UNC-51(750-856) fusion proteins were expressed and purified from E. coli, and bound to glutathione-conjugated beads. Various protein domains of VAB-8 were transcribed and translated in vitro using reticulocyte lysates (see Materials and methods). The + and – signs represent the strength of binding of each VAB-8 fragment to UNC-51(750-856) as compared to GST alone. (C) Domains of VAB-8 sufficient to bind UNC-51(750-856). (D) Schematic representation of binding interactions between UNC-51 and both VAB-8 and UNC-14. The hatched boxes represent the domains that are sufficient for binding. VAB-8 contains two domains that are sufficient to bind to UNC-51.

 


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  		Fig. 2. CAN axon morphology in wild-type, vab-8 and unc-51 animals. (A) Schematic representation of a CAN cell body and its axons. The centrally positioned CAN cell body extends an axon anteriorly to the nerve ring and an axon posteriorly to the tail near the PHA/B sensory neurons. (B-F) Fluorescence photomicrographs of larvae carrying a Pceh-23::gfp transgene, which expresses GFP in the CANs, as well as sensory neurons in the head and the tail. The CAN cell bodies (large arrowheads) and their axons can be visualized using this transgene. An arrow indicates the position of an axon termination. (B) Wild-type third larval stage hermaphrodite. (C) vab-8(ev411) first larval stage hermaphrodite. (D) vab-8(ev411) fourth larval stage hermaphrodite. The asterisk indicates the position where the posterior axon had reversed and extended anteriorly. The penetrance of this misrouting defect is 29% for ev411. (E,F) The small arrowheads point to large varicosities that often flank the CAN cell bodies in unc-51 mutants. (E) Anterior half of an unc-51(e369) third larval stage hermaphrodite. (F) Posterior half of a different unc-51(e369) larva. The open arrowhead points to an out-of-focus CAN cell body. Scale bars: 20 µm.

 


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  		Fig. 3. vab-8 and unc-51 act autonomously in the CAN cell. vab-8 or unc-51 full-length cDNAs was expressed from the ceh-23 promoter. At the top is a schematic representation of the posterior half of C. elegans, showing a CAN cell body and its posterior axon. Also shown are two sensory neurons (PHA/B) that express the Pceh-23::gfp transgene and mark the position where the CAN axon terminates. We scored the extent to which the posterior axons extended, with the position of the CAN cell body representing 0% extension and the position of the PHA/B sensory neurons representing 100% extension (see Materials and methods). The numbers in the boxes represent the percentages of axons that terminated or turned in that interval. Axons that completed 95-100% of the distance from the CAN cell body to the sensory neurons are considered wild type. n is the number of axons scored. To simplify the statistical analysis, only percentages of the wild-type axons (to the right of the vertical line) were compared. *The two-tailed Z test was used to compare the differences in the distributions of the two populations of axons in the wild-type position.

 


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  		Fig. 4. Expression of the binding domains of VAB-8 and UNC-51 in the CANs disrupted posteriorly directed axon outgrowth. (A) At the top is a schematic representation of the transgene that expresses the VAB-8 peptide in the CANs. A cDNA containing this UNC-51-binding domain of VAB-8 was fused in frame to a GFP cDNA and driven from the ceh-23 promoter. The fluorescence photomicrograph shows a wild-type larva that carries this transgene. The arrowhead indicates the position of the CAN cell body, and the arrow indicates the end of its truncated posterior axon. The scale bar represents 20 µm. (B) The distribution of axon termination positions in wild type, mutants and animals that expressed VAB-8 and/or UNC-51 peptides in the CANs. The vab-8 allele used was ev411; the unc-14 allele used was e866. See Fig. 3 for quantification of axon phenotypes and statistical analysis.

 


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  		Fig. 5. Overexpression of vab-8L partially suppressed the CAN posterior axon outgrowth defect of unc-51 mutants. A full-length vab-8 cDNA was expressed from the vab-8 promoter. See Fig. 3 for quantification of axon phenotypes and statistical analysis.

 


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  		Fig. 6. UNC-51 autophosphorylation. (A) Autoradiograph of samples from in vitro kinase assays. FLAG-tagged wild-type and mutant UNC-51 proteins were immunoprecipitated from COS cell extracts and incubated with [{gamma}-32P]ATP. UNC-51(K39R) had 9% and UNC-51({Delta}AIKAI) had 0.6% of the wild-type UNC-51 autophosphorylation activity. See Materials and methods for quantification of kinase activity. (B) Loading control. The western blot was probed with anti-FLAG antibodies to detect various forms of UNC-51 proteins. Similar amounts of immunoprecipitated UNC-51 proteins were used in the kinase assay.

 


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  		Fig. 7. UNC-51-dependent phosphorylation of UNC-14 and VAB-8. (A,B) Western blots were probed with anti-HA antibodies to detect UNC-14. (A) COS cell extracts expressing UNC-14 alone, or with wild-type or mutant UNC-51 proteins. UNC-14 was phosphorylated when coexpressed with wild-type UNC-51, partially phosphorylated when coexpressed with UNC-51(K39R) and not phosphorylated when coexpressed with UNC-51({Delta}AIKAI). (B) All lanes contained UNC-14 that was immunoprecipitated from COS cells expressing both wild-type UNC-51 and UNC-14. The sample from the second lane was treated with {lambda} phosphatase ({lambda} PPase) to yield a lower molecular mass band that ran at the same size as the product from cells expressing only UNC-14. Adding the phosphatase inhibitors Na3VO4 or EDTA inhibited the ability of {lambda} PPase to yield the lower molecular mass UNC-14 band. (C) GST and GST-UNC-14 proteins were expressed and purified from E. coli, and incubated with [{gamma}-32P]ATP and wild-type UNC-51 purified from COS cells. GST-UNC-14 was labeled, whereas no labeling was seen with the GST control. (D,E) Western blots were probed with anti-Myc antibodies to detect VAB-8. (D) COS cell extracts expressing VAB-8 alone, or with wild-type or mutant UNC-51 proteins. VAB-8 was phosphorylated when coexpressed with wild-type UNC-51 or with UNC-51(K39R), but not phosphorylated when coexpressed with UNC-51({Delta}AIKAI). (E) All lanes contained VAB-8 that was immunoprecipitated from COS cells that expressed both wild-type UNC-51 and VAB-8. {lambda} PPase treatment of the sample in the second lane converted multiple bands into one lower molecular mass band that ran at the same size as the product from cells expressing only VAB-8. Adding the phosphatase inhibitors Na3VO4 or EDTA inhibited the ability of {lambda} PPase to yield the lower molecular mass VAB-8 band.

 

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© The Company of Biologists Ltd 2004