CRL2^LRR-1 E3-Ligase controls HIM-3 loading on chromosomes.

<p>A- Schematic drawing showing that mitotic germ cells proliferate throughout the germline with no sign of meiotic entry in the <i>gld-3 nos-3</i> double mutant germlines (upper panel). B- A simplified pathway with key regulators of the mitosis/meiosis decision is shown in the lower panel. C- HTP-3 accumulates in <i>gld-3 nos-3</i> tumorous germlines upon <i>cul-2(RNAi)</i>. Total <i>gld-3 nos-3</i> worm extracts from <i>control</i> or <i>cul-2(RNAi)</i> were separated by SDS-PAGE and immunoblotted with HTP-3 (upper panel) and actin (lower panel) antibodies (loading control). The value at the bottom is the ratio between HTP-3 and actin signal intensities. The wild-type value was arbitrary defined as 1. D- Representative images of dissected gonads, of the indicated genotypes stained with HIM-3 (red) and SUN-1 Ser8-Pi (P-SUN-1, green) antibodies and with DAPI (blue). RNAi treatments were performed from the L1 (control, <i>cul-2</i>, <i>lrr-1</i>, <i>cye-1</i>) or L3 (<i>pbs-5</i>) stage at 20°C. Germlines in each panel were treated identically and fluorescent images taken at the same settings. Scale bars: 50 µM. The boxed regions, encompassing representative nuclei, are shown at higher magnification on the right panels. Scale bar: 5 µm. E- Graph showing the percentage of tumorous <i>gld-3 nos-3</i> germlines HIM-3 and SUN-1 Ser8-Pi negative (white bars), HIM-3 positive and P-SUN-1 negative (light grey), and both, HIM-3 and P-SUN-1 positive (dark grey), as scored by counting the number of nuclei rows that contain at least 5 gcd with cells expressing HIM-3 and P-SUN-1 positive.</p

CRL2^LRR-1 regulates entry into meiosis.

<p>The axial element HTP-3 (red) promotes the assembly of the synaptonemal complex by triggering the recruitment of the other axial element components HIM-3, HTP-1, HTP-2 (bleu) and the transverse filaments (SYP-1/2/3/4) (green). CRL2^LRR-1 inhibits the accumulation of HTP-3 whereas FBF-1/2 prevent the expression of structural components of meiotic chromosomes in the mitotic region of the germline. CRL2^LRR-1 also acts independently of HTP-3 to prevent meiotic entry presumably by controlling the stability of unknown meiotic promoting factor(s).</p

Figure 3

<p> A- Representative images of dissected gonads of animals of the indicated genotypes, maintained at 20°C from L1 and taken 24 hours after L4, stained with HIM-3 (red), SUN-1 Ser8-Pi (green) antibodies and with DAPI (blue) (left panel). Arrowhead, distal end of the germline. Yellow arrow, germ cells in G2-M phase of the cell cycle and white arrow, nuclei with crescent-shape morphology typical of meiotic prophase. Quantification of the size of the mitotic zone in germ cell diameters from the distal end is shown on the right. B- Representative images of dissected gonads of animals of the indicated genotypes maintained at 20°C from L1 and taken 24 hours after the L4 stage stained with HIM-3 antibodies (green) and counterstained with DAPI (red) (left panel). Arrowhead, distal end of the germline. Arrow, nuclei with crescent-shape morphology typical of meiotic prophase. Quantification of the size of the mitotic zone in germ cell diameters from the distal end is shown on the right. Note that although <i>fbf-1</i> and <i>fbf-2</i> genes are largely redundant for promoting mitosis, they have opposite roles in regulating the size of the mitotic region. The mitotic region is smaller than normal in <i>fbf-1(0)</i> mutants but larger than normal in <i>fbf-2(0)</i> mutants. This opposite role is largely due to different spatial regulation and reciprocal 3′UTR repression; <i>fbf-2</i> expression is limited to the distal germline whereas <i>fbf-1</i> expression is much broader <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003375#pgen.1003375-Lamont1" target="_blank">[58]</a>.</p

<i>or209</i> is a conditional <i>cul-2</i> temperature-sensitive allele.

<p>A- Schematic representation of the CRL2^LRR-1 E3-ligase. This complex is composed of two modules nucleated around the CUL-2 subunit (arc-shaped in blue): the substrate recognition module and the catalytic site. The substrate recognition module comprises the adaptor protein ELC-1, ELB-1 (blue) and the LRR-1 substrate recognition subunit (purple), whereas the catalytic module contains the RING finger protein RBX-1 (grey). B- Schematic drawing of the RNAi-based screen used to search for temperature-sensitive (ts) alleles affecting the function of the CRL2^LRR-1 complex. <i>lrr-1(tm3543)</i> mutant animals <i>(lrr-1(0))</i> are sterile but recover fertility upon <i>atl-1</i> depletion by RNAi (upper panel). Therefore, we searched for ts mutants that are sterile at 25°C but recover fertility upon inactivation of <i>atl-1</i>. The <i>or209</i>ts mutant fulfilled these criteria (lower panel). C- The <i>or209</i>ts mutation phenocopies inactivation of the CRL2^LRR-1 complex. Graph showing the percentage of fertile <i>or209</i> animals (25°C) after control (orange bars) and RNAi-mediated depletion of <i>atl-1</i> (green bars). An average of eight different experiments is presented with 30 animals analysed in each experiment. D- Structure of the <i>cul-2</i> gene (upper panel); red asterisk depicts the location of the <i>or209</i> mutation. Chromatograms showing the T to A transversion found in the <i>cul-2</i>(<i>or209</i>ts) mutant (lower panels). E- Embryonic extracts of the indicated gentotype were separated by SDS-PAGE and immunoblotted with CUL-2 (upper panel) and tubulin (lower panel) antibodies (loading control). The asterisk marks the position of truncated forms of CUL-2 that probably lack the C-terminal part of the protein. The value at the bottom is the ratio between CUL-2 and tubulin signal intensities. The wild-type value was arbitrary defined as 1. F- The sex determination factor TRA-1 accumulates in <i>cul-2</i>(<i>or209</i>ts) mutant animals, presumably causing the feminisation of the germline. Micrographs of adult worms of the indicated genotypes analysed by DIC microscopy (left panel). The germline is outlined. Note the accumulation of packed oocytes (black arrows) in <i>cul-2</i>(<i>or209</i>ts) animals, which is characteristic of a feminisation (fem) phenotype. This phenotype is observed in 13% (n = 551) of animals maintained at 15°C and in 27% (n = 228) of animals shifted during 20 hours at 25°C from the L3 stage, but is never observed in wild-type (n>200). Right panel: worm extracts of synchronised <i>cul-2</i>(<i>or209</i>ts) animals cultivated at 15°C or shifted 20 hours at 25°C from the L3/L4 stage were separated by SDS-PAGE and blotted with TRA-1 (upper panel) antibodies. The asterisk marks the position of a non-specific band. The value at the bottom is the ratio between TRA-1 and actin signal intensities. The wild-type value was arbitrary defined as 1.</p

CRL2^LRR-1 E3-ligase promotes germ cell proliferation.

<p>A- Schematic drawing of an adult distal germline. The distal tip cell (DTC) niche is located at the distal end. The DTC expressed the Notch ligand LAG-2, which is shown in grey. The mitotic zone contains germ cells in the mitotic cell cycle (red), including a germline stem cell (GSC) pool distally and possibly transit-amplifying germ cells proximally; at its proximal edge, some germ cells have entered pre-meiotic S-phase. The transition zone contains germ cells in meiotic S-phase and in meiotic prophase (nuclear crescents) that start to accumulate HIM-3 (green), a marker of meiotic prophase. The dashed line marks the boundary between the mitotic and transition zones (left panel). A simplified pathway with key regulators of the balance between GSC renewal and meiotic differentiation is shown in the right panel. Positive regulation (arrows) and negative regulation (bars). B- Representative images of dissected gonads of animals of the indicated genotypes taken 24 hours after the mid-L4 stage stained with HIM-3 antibodies (green) and DAPI (red). The distal end of the germline (arrowhead) is oriented toward the left, and the proximal end is oriented toward the right in this and other figures. Arrow, nuclei with crescent shape that is typical of nuclei in meiotic prophase. Germlines in each panel were treated identically and fluorescent images taken at the same settings. Scale bars: 50 µM. C- Graphs showing the mean of the total number of germ cells in the mitotic zone of the germline and D- the quantification of the size of the mitotic zone, in germ cell diameters (gcd) from the distal end in animals of the indicated genotypes. At least ten germlines of each genotype were scored.</p

CRL2^LRR-1 E3-ligase acts through HTP-3 in the <i>C. elegans</i> germline.

<p>A- Flow-chart of the <i>cul-2</i>(ts) RNAi-based suppressor screen. At 15°C, synchronised <i>cul-2</i>(ts) L1 larvae were fed with bacteria expressing dsRNA, then were shifted to a semi-permissive temperature (23°C) at the L4 stage. After 11 hours, young adults were removed, and embryonic viability was determined and plotted. B- Graphs showing the percent viability of <i>cul-2</i>(<i>or209</i>ts) animals after RNAi depletion of the indicated genes. C- Representative images of dissected gonads of the indicated genotypes stained with HIM-3 antibodies (red) and DAPI (green) are shown. Scale bars: 50 µM. Arrow marks the distal end of the germline D- Reduction of <i>htp-3</i> function suppresses <i>lrr-1(0)</i> mutant animal sterility. Micrographs of adult worms of the indicated genotypes were analysed by DIC microscopy. Thirty-four percent of <i>lrr-1(0)</i>; <i>htp-3(vc75)</i> double mutants are fertile (n = 112). The germline is outlined, and the presence of oocytes and embryos is indicated. The asterisk marks the distal region of the germline. E- The DNA replication checkpoint is functional in <i>htp-3(vc75)</i> mutant animals. Animals of indicated genotypes were exposed to 10 mM Hydroxyurea (HU) and germlines were dissected 24 h post-L4 and stained with DAPI (upper panel). The number of germ nuclei in a given volume was determined and plotted (bottom panel). Note that HU treatment of wild-type (N2) animals leads to S-phase arrest that manifests as enlarged nuclei with an overall reduction in the number of nuclei in the mitotic zone of the germline. HU treatment similarly arrested <i>htp-3(vc75)</i> mutant germ cells, whereas checkpoint deficient <i>atl-1(tm853)</i> mutant germ cells fail to arrest cell cycle progression, indicating that the DNA replication checkpoint is functional in the <i>htp-3(vc75)</i> mutant. F- Pathway diagram summarizing the observed genetic interactions. CUL-2 and LRR-1 counteracts the DNA replication checkpoint pathway, which is composed of MUS-101 and ATL-1, and inhibits HTP-3, which promotes the recruitment of HIM-3, SYP-1 and the assembly of the synaptonemal complex (SC). Positive regulation (arrows) and negative regulation (bars).</p

CRL2^LRR-1 E3-ligase regulates HTP-3 levels in the mitotic zone of the germline.

<p>A- HTP-3 accumulates to high levels in <i>cul-2</i>(<i>or209</i>ts), <i>lrr-1(0)</i> and <i>pbs-5(RNAi)</i> germlines. Representative images of dissected gonads of the indicated genotypes stained with HTP-3 (green) antibodies and DAPI (red) (left panel). Insets show germ cells located in the most distal region of the germline (right panel) B- HTP-3 physically interacts with LRR-1. A flow chart of the experiment is presented in the left panel. Briefly, total worm extracts were incubated with the recombinant 6xHis-LRR-1/ELC-1/T7-ELB-1 trimeric complex immobilised on T7-agarose beads (green oval). As a control, we used a trimeric complex in which the substrate-binding interface LRR-1 (purple rectangle) was deleted. The middle panel shows a Coomassie-stained gel of the recombinant trimeric complexes containing either truncated or full-length LRR-1 protein (lane 1 and 2 respectively). Right panel: the trimeric complexes and associated proteins were eluted with loading buffer and analysed by western blot using MUS-101- and HTP-3-specific antibodies. T, total; FT, Flow-Through; E, elution. Lower right: total extracts of N2 and <i>htp-3(0)</i> worms were separated by SDS-PAGE and immunoblotted with HTP-3 antibodies.</p