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Multiple Mechanisms Contribute to Centriole Separation in C. elegans

Summary Centrosome function in cell division requires their duplication, once, and only once, per cell cycle. Underlying centrosome duplication are alternating cycles of centriole assembly and separation [1]. Work in vertebrates has implicated the cysteine protease separase in anaphase-coupled centriole separation (or disengagement) and identified this as a key step in licensing another round of assembly [2]. Current models have separase cleaving a physical link between centrioles, potentially cohesin [3, 4], that prevents reinitiation of centriole assembly unless disengaged. Here, we examine separase function in the C. elegans early embryo. We find that depletion impairs separation and consequently duplication of sperm-derived centrioles at the meiosis-mitosis transition. However, subsequent cycles proceed normally. Whereas mitotic centrioles separate in the context of cortical forces acting on a disassembling pericentriolar material, sperm centrioles are not associated with significant pericentriolar material or subject to strong forces. Increasing centrosomal microtubule nucleation restores sperm centriole separation and duplication in separase-depleted embryos, while forced pericentriolar material disassembly drives premature separation in mitosis. These results emphasize the critical role of cytoskeletal forces and the pericentriolar material in centriole separation. Separase contributes to separation where forces are limited, offering a potential explanation for results obtained in different experimental models [5–7].

. Localization of separase in C. elegans embryos, Related to the Results and Discussion Immunofluorescence micrographs of wild-type (A) and sep-1(RNAi) (B) embryos stained for DNA, microtubules and SEP-1. Insets show meiotic spindle, sperm pronucleus and later mitotic spindle. Separase localizes to meiotic and mitotic chromosomes, the spindle as well as mitotic centrosomes. No change in separase signal is seen at centrosomes from prophase to anaphase. At chromosomes, separase appears to spread from the holocentric centromere/kinetochore in prometaphase and metaphase to throughout the chromatin in anaphase. No residual signal was seen in sep-1(RNAi) embryos, indicating that staining is specific and depletion efficient. All embryos were exposed and processed identically for separase, except for control meiosis embryos, where exposure conditions were reduced 3x to avoid saturating signal. (C) Stills from widefield timelapse sequence of control embryo expressing GFP:SEP-1, showing redistribution of separase at anaphase onset. Scalebars are 10µm. Insets are magnified 2x (A, B), 1.5x (C).

Figure S2. Further analysis of the separase RNAi phenotype, Related to the Results and Discussion
(A) Quantitation of centrosome duplication phenotype in sep-1 single depletions, double depletions with dsRNAs targeting hyls-1 (control), zk430.5 (a non-essential gene related to sep-1) and single depletions using a second, non-overlapping dsRNA targeting sep-1, based on timelapse sequences as in Figure 1A. Centrosome duplication outcome is not significantly different between any of these conditions (Chi-square test, p>0.05). (B) Schematic representation of centrosome and centriole dynamics in wild-type and sep-1(RNAi) embryos. In wild-type embryos, a pair of centrioles is introduced into the oocyte with the sperm at fertilization. Sperm-derived centrioles separate and a new daughter centriole assembles alongside each parent such that by metaphase each centrosome contains two centrioles, one sperm-derived and one newly assembled in the embryo cytoplasm (in schematics parental centrioles are shown in grey, daughters in green). At the end of mitosis these centrioles separate and a new round of assembly occurs. In sep-1(RNAi) embryos, since spermatogenesis is complete prior to application of dsRNA, a normal pair of centrioles still enters the oocyte at fertilization. However, with the oocyte cytoplasm depleted of separase, centrioles do not separate sufficiently in the first mitosis. Consequently, monopolar spindles form and centriole duplication is blocked. At the end of mitosis I as well as in subsequent mitotic cycles, centrioles move apart and inhibition of centriole duplication is relieved. Since cytokinesis frequently fails, successive mitotic cycles occur in the same one-cell embryo, with two centrosomes in mitosis II (now comprised of a pair of centrioles) and four in mitosis III. (C) -(E) The pericentriolar material does not contribute to sperm centriole cohesion at the meiosis/mitosis transition. (C) Mating-based assay for SAS-6 recruitment. Similar to centriole duplication assay outlined in Figure 2A, except using SAS-6 in place of SAS-4. SAS-6 recruitment occurs at an earlier step in the centriole assembly pathway unaffected by depletion of γ-tubulin and therefore likely also by depletion of SPD-5[1]. Note that recruitment of SAS-6 (unlike that of SAS-4) does not indicate completion of centriole assembly. (D) Results of experiments performed as outlined in (C) on control, sep-1(RNAi), spd-5(RNAi), and sep-1;spd-5(RNAi) embryos, using spinning disk confocal microscopy. As expected, depletion of SPD-5 did not affect SAS-6 recruitment and initiation of centriole assembly. However, sep-1(RNAi) did perturb initiation of centriole assembly, to an extent not significantly altered by co-depletion of SPD-5 (Chi-square test, p>0.05). (E) Quantitation of centriole duplication assay shown in (D). Scalebars in (D) are 10µm, insets are magnified 4x.  Figure 2A, except using rec-8(ok978) males. Feminized hermaphrodites were used to eliminate the possibility of self-fertilization. Centriole duplication outcome is not significantly different compared to sep-1(RNAi) alone (Chi-square test, p>0.05). (C) Stills from spinning disk confocal timelapse sequences of embryos resulting from the indicated crosses, showing an overlay of DIC and SPD-2 images. (D) Cohesin depletion results in chromosome segregation defects in mitosis. Stills of timelapse spinning disk confocal movies of control and smc-1(RNAi) embryos expressing mCherry:Histone, aligned relative to nuclear envelope breakdown (t=0 seconds). Chromosomes fail to form a metaphase plate and display severe defects in anaphase segregation (n=12 embryos). (E) Cohesion depletion results in failure of sister chromatin cohesion during meiosis. Immunofluorescence micrographs of nuclei from diakinesis-stage wild-type and smc-1(RNAi) oocytes stained for DNA and HTP-3. (F) Cohesin mutants do not display premature centriole separation in mitosis. Immunofluorescence micrographs of wild-type and smc-3(t2553) mutant embryos stained for DNA, microtubules and SPD-5, as well as SAS-4 (insets). Scalebars are 10µm. Insets in (A) and (F) are magnified 4x. In sep-1(RNAi) embryos, anaphase onset was scored as onset of attempted chromosome segregation. Beginning and end of S-phase were assessed by recruitment and loss of GFP:CDC-45, measured as described in [2]. Error bars represent the 95% confidence interval. Asterisks indicate statistically significant differences from control (t-test, p<0.05; ns not significant). Note that sep-1(RNAi) embryos experience significant delays in entry into S-phase after exit from meiosis. However, duration of S-phase and subsequent entry into mitosis (nuclear envelope breakdown, NEBD) are largely normal. SMC-1 co-depletion rescues the sep-1(RNAi) centrosome duplication phenotype ( Figure 4A

RNA-mediated interference
RNAi experiments were performed by injection[1] or soaking [13] using the dsRNAs listed in table below. For double depletions, RNAs were mixed at equal concentrations of ≥1 mg/ml for each RNA, with HYLS-1 used as a control in corresponding single depletions. Standard RNAi conditions of 48h at 16C (72h for SMC-1/SMC-3) were used to ensure full depletion. For RNAi experiments involving anucleated sperm, injected worms were mated to emb-27(g48) mutant males for 24h at 25C to avoid reversion of the emb-27 phenotype, before removing males and returning hermaphrodites to 16C for the remaining 24h.

Live imaging
Embryos were filmed without compression [17] under attenuated fluorescence illumination conditions known not to perturb cell cycle progression. Widefield microscopy was performed using a 60x 1.42 NA Plan Apochromat lens on the DeltaVision microscope set-up described above. Spinning disk confocal microscopy was performed on a Yokogawa CSU X1-A1 spinning disk confocal mounted on a Zeiss Axio Observer Z1 inverted microscope equipped with a 63x 1.4NA Plan Apochromat lens, 100mW 488nm and 561nm solid-state lasers and CoolSNAP-HQ2 cooled CCD camera and controlled by VisiView software (Visitron Systems). For embryos coexpressing GFP:SPD-5 and mCherry:Histone, 6x2µm GFP/mCherry z-series as well as single plane DIC images were acquired every 30s from meiosis until the end of the second or third mitotic division. For embryos co-expressing GFP:CDC-45 and mCherry:Histone, 6x2µm GFP/mCherry z-series as well as single plane DIC images were acquired every 30s from meiosis until NEBD, with 2x2 camera binning used to enhance signal detection. For SAS-4 and SAS-6 recruitment analysis, 11x1µm GFP/mCherry z-series as well as single plane DIC images were acquired every 60s from early prophase until the end of the second mitotic division, with 2x2 binning to enhance signal detection. Image stacks were imported into MetaMorph for postacquisition processing. Panels are single-plane or maximum intensity projections of multiple planes depending on centrosome positioning within the embryo. Images are auto-scaled for best presentation unless otherwise noted.

Temperature shift experiments
spd-5(or213) temperature shift experiments were performed on a Zeiss Axioplan2 widefield microscope with a temperature-controlled 40x 1.3 NA Plan-Neofluar oil lens. Lens temperature was achieved by a custom 'cooling' collar (Bioptechs) that was heated by circulating antifreeze; antifreeze temperature was controlled by a circulating water bath. Images were acquired with a CoolSnap HQ (Photometrics) camera, using 488nm LED illumination (CoolLED, Andover). Image acquisition was controlled by VisiView (Visitron Systems). Worms were maintained at 16C prior to sample preparation. One-cell embryos were located at 10x magnification and ambient temperature (~20C) then shifted within one minute to the 40x lens at 25C. The temperature of the sample equilibrated to the lens temperature in less than two minutes, as judged by a temperature probe mounted as for embryos. 25x1µm GFP z-series and single plane DIC images were acquired at irregular intervals from immediately after the shift to 25C until the end of mitosis I.

Centriole separation measurements and statistical analyses
Centriole separation was measured on projected images in MetaMorph, disregarding any separation in the z-dimension. Each embryo was scored once per 100s interval. For the analysis in Table S1, distances were further averaged across the entire S phase interval such that each embryo is represented once in the overall average. Statistical comparisons were performed in Excel (Student's t-test, two-tailed), without disregarding any datapoints. Comparisons of centriole duplication outcome were performed in GraphPad Prism (Chi-square test) with data grouped into three categories (no duplication, partial duplication, normal duplication). Results were reported as significant if p value was <0.05.