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First published online 12 September 2006
doi: 10.1242/jcs.03187

Journal of Cell Science 119, 4088-4100 (2006)
Published by The Company of Biologists 2006
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IFTA-2 is a conserved cilia protein involved in pathways regulating longevity and dauer formation in Caenorhabditis elegans

Jenny C. Schafer1, Marlene E. Winkelbauer1, Corey L. Williams1, Courtney J. Haycraft1, Renee A. Desmond2 and Bradley K. Yoder1,*

1 Department of Cell Biology, University of Alabama at Birmingham Medical Center, Birmingham, AL 35294, USA
2 Division of Biostatistics and Bioinformatics, University of Alabama at Birmingham Medical Center, Birmingham, AL 35294, USA


Figure 1
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  		Fig. 1. Using BLAST, the closest homologs of IFTA-2 are human and mouse RabL5 proteins. IFTA-2, human RabL5, and mouse RabL5 share homology with human Rab15 and human Rab5A. Asterisks indicate identical residues in all species. Colons indicate conserved substitutions according to small and/or hydrophobic (AVFPMILW), acidic (DE), basic, (RHK) and hydroxyl/amine/basic/Q (STYHCNGQ) amino acids. Full stops indicate semi-conserved substitutions. G1, G2 G3 and G4 indicate conserved nucleotide-binding domains. Arrows indicate point mutations created in IFTA-2::GFP (T42N and D123L).

 

Figure 2
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  		Fig. 2. (A) The ifta-2 promoter fusion to GFP shows expression specifically in ciliated sensory neurons. Data for amphid and labial neurons in the head are shown. Expression is also present in the ciliated phasmid neurons in the tail (data not shown). In panels A-D, the head of the worm is towards the left and in B and D, arrows indicate cilia localization. (B) IFTA-2::GFP translational fusion protein localizes to the base of cilia and within the cilia axoneme in the head and tail (not shown) of the adult hermaphrodite. (C) A single point mutation (T42N) in the GTP binding domain of IFTA-2::GFP causes diffuse localization of the fusion protein throughout the ciliated sensory neurons with no cilia localization. (D) A single point mutation (D123L) in the G3 nucleotide binding domain of IFTA-2::GFP does not disrupt cilia localization of the fusion protein. (E) Immunofluorescence localization of the RabL5 protein in inner medullary collecting duct cells derived from the mouse kidney, detected with an antibody against mammalian RabL5 (green) and acetylated tubulin (red). Both proteins colocalize to the primary cilia. RabL5 is also concentrated at the basal body at the base of cilia. Inset shows RabL5 localization alone.

 

Figure 3
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  		Fig. 3. (A) OSM-5::GFP, CHE-11::GFP, and XBX-1::YFP localize to cilia in both wild type and ifta-2(tm1724) mutants. (B) IFTA-2::GFP localizes properly to cilia in wild-type, osm-5(m184), che-11(e1810), and che-3(e1124) worms. In A and B, upward arrowhead indicates base of cilia and arrow indicates cilia axonemes. In B, downward arrowheads indicate malformed cilia.

 

Figure 4
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  		Fig. 4. (A) Survival analysis on wild-type, osm-5(m184) and ifta-2(tm1724) strains show that ifta-2(tm1724) worms have a greatly extended lifespan compared with wild type. (B) Survival analysis shows that rescue of IFTA-2::GFP in ifta-2(tm1724) mutants results in greatly reduced lifespan compared with the original ifta-2(tm1724) mutant and is not different from wild type. IFTA-2::GFP in wild type is also similar to wild type, indicating that the transgene has no effect on lifespan. (C) Survival analysis shows that ifta-2(tm1724);daf-2(e1370) double mutants have a lifespan similar to that of daf-2(e1370) alone or ifta-2(tm1724) alone, indicating that there is no additive effect of the ifta-2 mutation on the daf-2 longevity phenotype. (D) Survival analysis shows daf-16 is epistatic to ifta-2 because ifta-2(tm1724);daf-16(mu86) double mutants have a lifespan similar to that of daf-16(mu86) alone and is reduced compared with the ifta-2(tm1724) alone. (E) Survival analysis shows osm-5(m184);ifta-2(tm1724) double mutants have a lifespan similar to that of ifta-2(tm1724) alone.

 

Figure 5
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  		Fig. 5. (A) Immunofluorescence of a wild-type adult hermaphrodite shows IFTA-2::GFP (green) and DAF-2 (red) colocalized in cilia in the head. Immunofluorescence of an ifta-2(tm1724) adult hermaphrodite shows that DAF-2 is properly localized to cilia. (B) Immunofluorescence of a wild-type adult hermaphrodite shows IFTA-2::GFP (green) and AGE-1 (red) colocalized in cilia in the head. Immunofluorescence of an ifta-2(tm1724) adult hermaphrodite shows that AGE-1 is properly localized to cilia. DNA is shown in blue, cilia are indicated by an arrowhead, the head of the worm is directed to the left.

 

Figure 6
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  		Fig. 6. (A) Representative image of cytoplasmic DAF-16::GFP localization in an L1 worm. (B) Representative image of intermediate DAF-16::GFP localization in an L1 worm showing both cytoplasmic and nuclear localization. (C) Representative image of nuclear DAF-16::GFP localization in an L1 worm. (D) DAF-16::GFP in daf-16(mu86) (labeled WT) and DAF-16::GFP in ifta-2(tm1724);daf-16(mu86) [labeled ifta-2(tm1724)] were compared for nuclear vs cytoplasmic DAF-16::GFP localization. Individual worms were classified as cytoplasmic, intermediate or nuclear by three researchers. Numbers are the average (to the nearest whole number) of the scores of three researchers. The proportion of nuclear, intermediate and cytoplasmic DAF-16::GFP localization for each strain was compared by the Chi2 test. *P<0.001, Chi2=9.2475.

 

Figure 7
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  		Fig. 7. (A) In the wild-type state, signaling through DAF-2, AGE-1 and IFTA-2 leads to phosphorylated DAF-16, which remains cytoplasmic and results in wild-type lifespan and wild-type dauer formation. (B) In ifta-2 mutants, disruption of the pathway between DAF-2 and DAF-16 leads to an accumulation of DAF-16 in the nucleus, extended lifespan and altered dauer formation.

 

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© The Company of Biologists Ltd 2006