QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Luke-Glaser, S. Articles by Peter, M. Search for Related Content PubMed PubMed Citation Articles by Luke-Glaser, S. Articles by Peter, M.

The AAA-ATPase FIGL-1 controls mitotic progression, and its levels are regulated by the CUL-3^MEL-26 E3 ligase in the C. elegans germ line

Sarah Luke-Glaser¹, Lionel Pintard^2,*,, Mike Tyers² and Matthias Peter^1,

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

View larger version (18K): [in this window] [in a new window]   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 -galactosidase reporter was analyzed by filter assay. An empty plasmid (AD-empty) was included as a control.

View larger version (32K): [in this window] [in a new window]   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., 2006). 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).

View larger version (36K): [in this window] [in a new window]   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).

View larger version (49K): [in this window] [in a new window]   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.

View larger version (55K): [in this window] [in a new window]   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.

View larger version (12K): [in this window] [in a new window]   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., 2003b). 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.