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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.015883

Journal of Cell Science 120, 3179-3187 (2007)
Published by The Company of Biologists 2007
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The AAA-ATPase FIGL-1 controls mitotic progression, and its levels are regulated by the CUL-3MEL-26 E3 ligase in the C. elegans germ line

Sarah Luke-Glaser1, Lionel Pintard2,*,{ddagger}, Mike Tyers2 and Matthias Peter1,{ddagger}

1 Institute of Biochemistry, HPM G8, ETH Hönggerberg, 8093 Zürich, Switzerland
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G1X5, Canada


Figure 1
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  		Fig. 1. MEL-26 interacts with FIGL-1 by means of the substrate-binding MATH domain. (A) The domain structure of MEL-26 and the interaction with MEI-1, POD-1 and CUL-3 are schematically indicated. The C94Y mutation lies in the MATH domain. The numbers mark the amino acids starting from the amino-terminus (N) to the carboxy-terminus (C). (B) Bacterially expressed GST-MEL-26 and, as a control, GST alone were incubated with S. cerevisiae extracts containing HA–FIGL-1. Bound proteins were eluted and immunoblotted with antibodies against HA. 5% of the supernatant (input) was loaded for comparison with 5% of the beads. (C) The interaction between wild-type and the C94Y mutant of MEL-26 fused to the Gal4 DNA-binding domain (BD) was characterized by two-hybrid assay with FIGL-1 fused to the Gal4 activation domain (AD). The expression of the β-galactosidase reporter was analyzed by filter assay. An empty plasmid (AD-empty) was included as a control.

 

Figure 2
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  		Fig. 2. FIGL-1 and MEI-1 belong to subgroup seven of the AAA-ATPases. (A) Members of subgroup seven of the AAA-ATPases are depicted in a phylogenetic tree. The closer two proteins cluster, the more recently they diverged during evolution. Whereas human and mouse contain fidgetin and fidgetin-like 1, the C. elegans genome only contains one gene F32D1.1, which was named figl-1 and encodes Fidgetin-like 1 protein (Yakushiji et al., 2006Go). Species abbreviations are as follows: Hs, Homo sapiens; Sc, S. cerevisiae; Ce, C. elegans. (B) Alignment of MEI-1 and FIGL-1. Identical residues are shown in black and conserved substitutions on a gray background. The catalytic domain of AAA-ATPases (predicted by SMART) is underlined. Note that MEI-1 and FIGL-1 show some sequence homology outside the ATPase domain. (C) The indicated concentration (µM) of purified tubulin was polymerized in vitro, incubated with GST–FIGL-1 or yeast coronin (Crn1) as a positive control, and separated by high-speed centrifugation over a glycerol cushion. The entire pellet (P) fraction was analyzed by SDS-PAGE, whereas only 10% of the top supernatant (S1) and the bottom supernatant (S2) were loaded. The co-sedimentation of FIGL-1 with polymerized tubulin was quantified at 0 µM, 2 µM and 4 µM tubulin (n=1 for 2 µM and 4 µM tubulin, n=2 for 0 µM), and shown as a percentage (%) of the total input recovered in the pellet (bound) fraction (lower panel). In the human Cul3 negative control, only 7% (for 2 µM, n=2) and 6% (for 4 µM, n=2) of in vitro-translated Cul3 was recovered under these conditions (data not shown).

 

Figure 3
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  		Fig. 3. FIGL-1 is required for timely mitotic progression in the germ line. (A) L3 larvae were injected with figl-1 dsRNA and the resulting embryonic lethality and sterility of their F1 progeny was quantified and expressed as a percentage (%) of the total number (n) of progeny analyzed. Although the embryonic lethality is low, the percentage of sterile progeny reaches 89%. (B) The gonadal arms at the tail end of wild-type (left image), figl-1(RNAi)-depleted progeny and figl-1(tm808)-homozygous animals (right image) were visualized within the worm by Hoechst staining. The stars mark the start of the `mitotic gonad' in the figl-1(RNAi) progeny, and the arrows indicate the mitotic and meiotic progression in the U-shaped gonad. The few nuclei that were left in the figl-1(RNAi) and figl-1(tm808) progeny all display abnormal sizes and structures (insets). Bar, 10 µm. (C) figl-1(RNAi) animals express the distal-tip cell marker Lag2::GFP. GFP expressed by a Lag-2::GFP transgene (arrows) is visualized with a fluorescent microscope in control (bottom left) or figl-1(RNAi) animals (bottom right). Upper panels show animals visualized by bright-field microscopy. Bar, 100 µm. (D,E) The number of mitotic nuclei was determined in gonads of wild-type (WT) and figl-1(RNAi) animals by immunofluorescence with antibodies against phosphorylated histone H3 (D, left images). The DNA was visualized by Hoechst staining (D, right images). The result was quantified and plotted as a percentage (%) of mitotic cells per gonad (E). Note that the number of nuclei with a positive phospho-H3 signal is higher in figl-1(RNAi) animals (P=1.17x10–7).

 

Figure 4
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  		Fig. 4. FIGL-1 activity is vital for C. elegans embryos in the absence of a functional spindle-assembly checkpoint. (A) The localization of FIGL-1 and MEL-26 was determined by immunofluorescence with affinity-purified antibodies in wild-type and figl-1(RNAi) early embryos. The DNA was visualized by Hoechst staining (right panels). Bar, 10 µm. (B,C) L3 larvae were injected with double-stranded RNA against figl-1 or the mitotic checkpoint components san-1 (S. cerevisiae MAD3), mdf-1 (S. cerevisiae MAD1) or Y54G9A.6 (S. cerevisiae BUB3) either alone or in combination. Embryonic lethality was scored after the times indicated and plotted (24-48 hours). The values obtained after 24 and 48 hours of inactivation are listed in panel C, and schematically presented in panel B.

 

Figure 5
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  		Fig. 5. FIGL-1 is expressed in the mitotic zone of the germ line and accumulates in mel-26 mutants or cul-3-depleted animals. (A) The localization of FIGL-1 and MEL-26 was determined by immunofluorescence in the gonads of wild-type animals by using affinity-purified antibodies against FIGL-1 (top left) and MEL-26 (top right). The DNA was visualized by Hoechst staining (bottom row). The beginning of meiosis (transition zone) is marked by arrowheads and is defined as the region where 60% of the nuclei are in meiotic prophase, as indicated by the crescent-shaped nuclei (depicted in yellow). The white box delineates the area shown at higher magnification in the inset. Stars mark nuclei before meiotic prophase; the short arrows in insets point to crescent-shaped nuclei in meiotic prophase. Note that the staining of FIGL-1 is significantly reduced (P<0.05) in cells entering into meiosis compared with cells in mitosis. Bars, 10 µm. (B) The expression of FIGL-1 (top panel) was investigated by immunofluorescence as described for panel B in gonads of mel-26(ct61sb4) animals. All stainings were performed under the same conditions to allow comparison. The DNA was visualized by Hoechst staining (bottom panel). The inset shows a higher magnification of the boxed area. (C) The fluorescence intensity of FIGL-1 staining in a set area in the mitotic (blue) and meiotic (red) zone of multiple wild-type, mel-26(sb45), mel-26(ct61sb4) and cul-3(RNAi) gonads was quantified as described above (see Materials and Methods) and shown in a bar diagram along with standard deviations. Note that FIGL-1 accumulates in gonads of worms that are defective for CUL-3MEL-26 function, and its expression extends into the meiotic zone.

 

Figure 6
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  		Fig. 6. The CUL-3MEL-26 ubiquitin ligase might target two distinct AAA-ATPases for degradation at different developmental stages. The CUL-3MEL-26 complex degrades FIGL-1 during meiotic stages in the gonad. By contrast, FIGL-1 is largely protected from degradation in the mitotic zone, possibly because of spatial restriction of MEL-26. At the meiosis-to-mitosis transition, the same E3 ligase targets MEI-1 for degradation (Pintard et al., 2003bGo). The different timing of degradation of FIGL-1 and MEI-1 might be regulated by phosphorylation by means of kinases present at these different developmental stages.

 

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© The Company of Biologists Ltd 2007