RMH-1 and SMC-5 cooperate to prevent accumulation of aberrant interhomolog connections.

<p>(A) In <i>smc-5(ok2421)</i>, RMH-1 foci increase in mid pachytene nuclei (MP) (green square), while in late pachytene (LP) (yellow square), a zone with fewer foci is still present, as in WT. (B) Quantification of RMH-1 foci in MP nuclei in WT (<i>n</i> = 205) and <i>smc-5(ok2421)</i> (<i>n</i> = 203). In the mutant, we frequently observe nuclei with more than 25 foci, never seen in the WT. Distribution of mid pachytene GFP::RMH-1 foci is significantly different between WT and <i>smc-5</i> mutant (Mann Whitney test, **** <i>p</i> < 0.0001). (C) Quantification of RMH-1 foci in LP nuclei; data are represented as mean +/- SD with ns (not significant). Quantification of hatch rate (D), larval arrest (E), and DAPI bodies in diakinesis oocytes (F) for WT, <i>rmh-1(jf54)</i>, <i>smc-5(ok2421)</i>, and <i>rmh-1(jf54); smc-5</i> (<i>n</i> = 35–45 hermaphrodites per genotype). Data are represented as mean +/- SD with ns (not significant) and * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. (G–J) Images of individual diakinesis bivalents stained for long arm and short arm markers. Both <i>rmh-1(jf54)</i> and <i>smc-5</i> single mutants exhibit abnormally structured bivalents at low frequency (H,I). In <i>rmh-1; smc-5</i>, all diakinesis nuclei contain bivalents with abnormal structures; typical of these abnormalities is a side-by-side organization of the long arms of the bivalents (J′), presumably reflecting the presence of persistent interhomolog associations at NCO sites. (K) Quantification of the frequencies of diakinesis nuclei (-2 and -1) containing at least one abnormal bivalent (<i>n</i> = 13–25 nuclei per genotype). Data are represented as percentage with ns (not significant) and ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, and **** <i>p</i> < 0.0001 (Chi² test) (L–N) Images of chromosomes in diakinesis nuclei from <i>zhp-3; smc-5</i> and <i>rmh-1(jf54) zhp-3; smc-5</i> worms. Despite the absence of the canonical meiotic CO machinery component ZHP-3, fewer than 12 DAPI structures are observed in some <i>zhp-3; smc-5</i>–1 oocytes, indicating the presence of ectopic connections (L,M). Such ectopic connections occur at high frequency in the <i>rmh-1(jf54) zhp-3; smc-5</i> triple mutant (N–N′). The quantification is presented in (O) with <i>n</i> = 13–36 oocytes per genotype. Data are represented as mean +/- SD with ns (not significant), * <i>p</i> < 0.05 and **** <i>p</i> < 0.0001.</p

Dose-dependent uptake of fluid phase FITC-dextran by erythrocytes treated with amphiphilic drugs.

<p>Erythrocytes were suspended in FITC-labeled dextran and incubated with the indicated amphiphilic drugs to induce endovesiculation. Upon washing, the uptake of fluorescent label was quantified by flow cytometry. Representative histograms are shown. The concentrations were 1.5, 0.4 and 0.375 mM (blue) and 3.0, 0.6 and 0.75 mM (green) for primaquine, chlorpromazine and imipramine, respectively (red is a control incubation without drug).</p

RMH-1 promotes the bias for CO formation on chromosome arms.

<p>(A) Schematics of crosses to obtain the progeny of singled F2 individuals subjected to Next Generation Sequencing (NGS) for SNP analysis. White insert indicates the WT (Bristol) background, and black insert indicates the Hawaiian background. (B) Quantification of the overall recombination frequencies for assayed chromosomes; stacked bar graph indicates the fraction of meiotic products with zero, one, or two COs. For WT (<i>n</i> = 36 chromatids), for <i>rmh-1(jf54)</i> (<i>n</i> = 40 chromatids), and for <i>rmh-1(tn309)</i> (<i>n</i> = 45 chromatids). The frequency of COs was not found to be different between WT and both mutants (Chi² test). (C) Scheme of the different chromosomes used during the recombination assay. The chromosome domains (left arm in blue, center in yellow, and right arm in purple) are correlated with the physical map of each chromosome. (D) Locations of the recombination events (assayed for chromosomes X, IV, and V) in WT (<i>n</i> = 17 COs: three events on X, four on II, four on IV, and six on V), for <i>rmh-1(jf54)</i> (<i>n</i> = 20 COs: 11 events on II and 9 on V), and <i>rmh-1(tn309)</i> (<i>n</i> = 21 COs: nine events on X, nine on IV, and three on V); also see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#sec015" target="_blank">Experimental Procedures</a> section. The relative distribution of COs in the center versus arm domains differed from the WT for <i>tn309</i> (<i>p</i> = 0.046, Chi² test) and for <i>jf54</i>, (p = 0.062, Chi² test).</p

Impaired drug-induced endovesiculation in erythrocytes of patients with acanthocytosis.

<p>Erythrocytes from patients (ChAc, PKAN+, PKAN-) and control donors were subjected to drug-induced endovesiculation using 3 mM primaquine. The amount of FITC-dextran positive cells (in %) was assessed by flow cytometry as described. Respective pairs of patient and control donors are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p

Altered LPA-induced PS exposure and calcium uptake in erythrocytes of patients with acanthocytes.

<p>Erythrocytes from patients and control donors were treated with LPA as described in Materials and Methods and either PS exposure (A) or calcium uptake (B) was monitored by flow cytometry. The percentage of FITC-annexin V-positive cells (A) and of Fluo-3-positive cells (B) of patient and control samples are shown and respective pairs are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p

Correlations between drug-induced endovesiculation, LPA-induced PS exposure and calcium uptake.

<p>The data of ChAc patients (circles) and respective control samples (triangles) for % of cells with endovesicles upon incubation with primaquine, for % annexin V-positive cells and % Fluo-3-positive cells upon LPA treatment are blotted against each other as indicated (data derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g007" target="_blank">7</a>). Linear regression lines are shown with R2 (shown as inserts). In each combination a positive correlation is observed with Pearson’s r and the 2-tailed significance (given as inserts).</p

RMH-1 contributes to successful bivalent formation at several levels during prophase I.

<p>Top: scheme of <i>C</i>. <i>elegans</i> gonad showing prophase I divided in zones: transition zone (leptotene/zygotene), pachytene (early, mid, and late), diplotene, and diakinesis. Bottom: summary of localization and functions of RMH-1. Bold: key words referring to localization or function; italics: used when mechanistic insight is proposed. First row: localization of RMH-1: numerous foci in mid pachytene, six bright foci in late pachytene: RMH-1 is absent in diakinesis. Second row: genetic requirements and characteristics of RMH-1 localization. Third row: anti CO function: RMH-1 prevents accumulation of joint molecules and discourages CO formation in chromosome centers. Last row: pro CO functions: role of RMH-1 in CO designation, in assurance of chiasma formation and, finally, its potential function in supporting the geometry of recombination intermediates. We propose timing for the different functions based on our data and previous publications. However, the <i>C</i>. <i>elegans</i> gonad is a continuous production line of meiocytes, and we do not intend to imply sharper transitions between meiotic stages than exist.</p

RMH-1 (but not RMH-2) contributes to reliable chiasma formation and chromosome segregation in meiosis.

<p>(A) Schematics of RMH-1 and RMH-2. (A′) Location of the three <i>rmh-1</i> mutations. In the <i>jf92</i> allele, the coding sequence was disrupted after the START codon by the insertion of the <i>unc-119</i> gene by the CRISPR technology. In the <i>jf54</i> allele, the G-to-A transition affects the first nucleotide of intron 1 and therefore, destroys the splice donor site of the preceding exon 1. qRT-PCR revealed the presence of different splicing variants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#pbio.1002412.s001" target="_blank">S1 Fig</a>). In the <i>tn309</i> allele, the G-to-A transition introduces a premature STOP codon at position aa 640, leading to the deletion of the OB2 domain (for more details, see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#sec015" target="_blank">Experimental Procedures</a> section). (B–E) Oocyte nuclei at the diakinesis stage of meiotic prophase. Each image shows the complete set of DAPI-stained chromosomes from a single nucleus. The wild-type (WT) and <i>rmh-2(jf94)</i> nuclei contain six bivalents (homolog pairs connected by chiasmata), whereas the <i>rmh-1</i> mutant nuclei contain a mixture of bivalents and univalents. (F) Quantification of the average number of DAPI-positive structures in diakinesis oocytes in the -1 position. WT <i>n</i> = 25, <i>rmh-1(jf92) n</i> = 21, <i>rmh-1(jf54) n</i> = 36, <i>rmh-1(tn309) n</i> = 30, and <i>rmh-2(jf94) n</i> = 18. (G–I) Quantification of embryonic hatch rates (G). Frequencies of male offspring (H) and larval arrest (I) among the progeny of WT and <i>rmh-1</i> and <i>rmh-2</i> mutant worms (<i>n</i> = 35–45 hermaphrodites per genotype). Data for F–I are represented as mean +/- SD; ns stands for not significant, differences are highlighted with stars (* <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. and **** <i>p</i> < 0.0001). Scale bars, 2 μm.</p

Microscopic comparison of patient’s and control erythrocytes in drug-induced endovesiculation.

<p>Erythrocytes of a PKAN+ patient (B and D) and a control donor (A and C) were treated with 3 mM primaquine (A and B) or 0.8 mM chlorpromazine (C and D) in the presence of FITC-dextran to monitor the formation of endovesicles by confocal microscopy. Representative phase contrast (left panels), fluorescence (middle panels) and overlay (right panels) images are shown.</p

Foci of RMH-1 and BLM are resolved as doublets or elongated structures during the pachytene stage.

<p>(A–G) Single pachytene nuclei imaged by SIM. (A–B) In the WT, HIM-6 and RMH-1 colocalize in MP. Foci appear elongated or as a doublet (see insets). (C–E) In LP, RMH-1 and HIM-6 are concentrated at CO sites contained in a structure resolvable into a doublet (see insets). (F) Colocalization of HIM-6 and COSA-1 at CO sites in WT. (G) In <i>rmh-1(jf54)</i>, HIM-6 does not colocalize with COSA-1 at CO sites (white arrow) but can be found in close proximity (white arrows). Scale bar 2μm.</p