A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans

Abstract

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

Fig. 1.. Induced degradation of LMN-1 in the C. elegans germ line causes apoptosis-independent nuclear collapse in maturing oocytes.

(A) Domain organization of C. elegans LMN-1, indicating where we inserted a degron (AID) and V5 epitope. (B) LMN-1::V5::AID immunofluorescence in gonads dissected from adult hermaphrodites. Meiosis progresses from left to right. 4′,6-Diamidino-2-phenylindole (DAPI), magenta; anti-V5, green. Grayscale images show LMN-1::V5::AID alone. Following auxin treatment, LMN-1::V5::AID is retained in the somatic distal tip and sheath cells, which do not express TIR1. Images are maximum-intensity projections and were scaled identically. Scale bar, 10 μm. (C) Differential interference contrast (DIC) images of embryos laid by hermaphrodites following auxin treatment for 48 hours and (−)auxin controls. Scale bars, 20 μm. Some embryos are typically overlooked when counting, resulting in viability counts exceeding 100%. (D) Immunofluorescence of CED-1::GFP. Following depletion of LMN-1 depletion, nuclei in the proximal gonad have hypercondensed chromatin, and some are surrounded by CED-1::GFP, which marks engulfing cells. Dashed lines indicate the direction of meiotic progression. Scale bar, 10 μm. (E) Germ lines in living animals showing gonad morphology (DIC) and apoptotic nuclei stained with acridine orange. Red dashed lines and arrows indicate the contour of gonad and the direction of meiotic progression. Scale bar, 10 μm. (F) Quantification of apoptotic nuclei based on acridine orange staining. All worms were homozygous for P_sun-1::TIR1. Each dot represents one animal. Medians (black crossbars) and means (black boxes) are shown. (G) Nuclear morphology during late meiotic prophase. All worms were homozygous for P_sun-1::TIR1. Meiotic prophase progresses from top to bottom; stages are annotated on the basis of their anatomical positions within control gonads. Scale bar, 10 μm. (H) Quantification of nuclear size. Medians (black crossbars) and means (black boxes) are shown. Colors correspond to the regions shown in (G). All statistics are in Materials and Methods and data S1.

Fig. 2.. Dynamics of nuclear collapse.

(A to E) Prolonged LINC complex clustering and mobility following LMN-1 depletion. (A) Maximum-intensity projection images of SUN-1::mRuby fluorescence at different stages of meiotic prophase. Images are scaled identically. Scale bar, 5 μm. (B) Mean SUN-1::mRuby intensity per nucleus, normalized against transition zone nuclei. The periphery of each nucleus was manually segmented and quantified from additive projection after background subtraction. TZ, transition zone; MP, mid-pachytene; LP, late pachytene; Dip, diplotene. (C) SUN-1::mRuby clustering, defined as the ratio of the standard deviation (SD) to the mean fluorescence intensity at the periphery of each nucleus, measured as in (B) (also see fig. S8A). (D) Grayscale images of SUN-1::mRuby overlaid with color-coded trajectories of SUN-1::mRuby patches. Scale bar, 5 μm. (E) Mean speed of individual SUN-1::mRuby patches at the NE. (F) Representative time-lapse images showing a collapsing diplotene nucleus after LMN-1 depletion. Maximum-intensity projections were scaled identically. The frame showing initial contact between NE and SC was shown. Note the asymmetric distribution of ZYG-12::GFP at the NE before and during collapse. Time stamps, min:s. Scale bar, 5 μm. (G) Another example of a collapsing late pachytene/early diplotene nucleus, displayed as temporal projections of three successive time points to highlight the shrinking of NE without concomitant contraction of SC from 40 to 50 s. Time stamps are min:s. Scale bar, 2 μm. (H) Quantification of NE and SC sizes measured from time-lapse images. The point of contact between NE and SC was used to align data, and the final frame was used to for normalization. Means ± SD are plotted. (I) Quantification of the collapse rate before (“pre-”) and after (“post-”) contact between NE and SC. Medians (black crossbars) and means (black boxes) are shown (B, C, E, and I). All statistics are in Materials and Methods and data S1.

Fig. 3.. Nuclear collapse is rescued by disrupting dynein or LINC complexes, but not the connections between LINC complexes and pairing centers.

(A) Composite images showing mRuby::SYP-3 (magenta) and ZYG-12::GFP (green) in diplotene nuclei of indicated genotypes or treatments. All images are maximum-intensity projections and were scaled identically. Scale bar, 5 μm. (B) Polarization of ZYG-12::GFP fluorescence intensities in diplotene nuclei, mapped to angular coordinates. The brightest point was designated as 180° and the intensity at 0° was normalized to 1 (see fig. S8B). For detailed statistics, see data S1. (C) Nuclear morphology at later stages of meiotic prophase. All worms were homozygous for P_sun-1::TIR1. Colored dashed lines mark meiotic stages on the basis of the anatomical positions of gonads from control animals. Scale bars, 10 μm. (D) Nuclear morphology at later stages of meiotic prophase, from gonads dissected from animals of indicated genotypes or treatments. Pairing center proteins HIM-8, ZIM-1, ZIM-2, and ZIM-3 are expressed in ieDf2/+ heterozygotes but absent in homozygotes. Colored dashed lines indicate meiotic stages on the basis of their distributions in controls. Scale bar, 10 μm. (E) Normalized nuclear size in worms of indicated genotypes or treatments. Medians (black crossbars) and means (black boxes) are shown. The colors correspond to dashed lines in (C) and (D). All statistics are in Materials and Methods and data S1.

Fig. 4.. Nuclear collapse due to lack of LMN-1 is exacerbated by co-depletion of SAMP-1 or EMR-1/LEM-2.

(A and C) Nuclear morphology throughout meiosis. All worms were homozygous for P_sun-1::TIR1 or P_gld-1::TIR1 (omitted in the figure). Scale bars, 10 μm. Insets for early pachytene are 3× magnified. (B and D) Fraction of collapsed nuclei as a function of meiotic progression. Schematic at the top shows distal gonad being divided into six zones of equal lengths, so that the percent of nuclei with collapsed/condensed chromatin in each zone can be quantified. Three worms were measured per condition. Means ± SD are plotted. Pairwise comparisons for proportions were used to compute the P values (adjusted by the Benjamini-Hochberg method; see data S1).

Fig. 5.. NE stiffness is reduced following LMN-1 depletion.

(A) Meiotic nuclei isolated from adult hermaphrodites expressing ZYG-12::GFP. Green arrowheads indicate nuclei, and white arrowheads indicate debris. Scale bars, 5 μm. (B) The mechano-NPS platform consists of a microfluidic channel segmented by a node. A pair of electrodes at either end of the channel enables a four-terminal measurement of the current. Nuclear diameter is measured within the sizing segment, and nuclei are compressed under a constant applied strain in the contraction segment. The expected current pulse caused by a nucleus transiting the channel is shown. ΔI_S corresponds to the initial current drop caused by a nucleus transiting the sizing segment. t_s and t_c correspond to the transit time of a nucleus passing through the sizing and contraction segments, respectively. Nuclear size is determined by the magnitude of ΔI_S (eq. S1). Nuclear stiffness is determined by the transit time of a nucleus passing through the contraction segment (t_c): stiffer nuclei take longer than softer ones. To normalize with respect to nuclear size, the whole-cell deformability index, wCDI, which is inversely related to the Young’s modulus, is used (eqs. S2 and S3). (C) A mechano-NPS device. The microfluidic channel corresponding to that shown in the schematic in (B) is located between the inlet and the outlet. Scale bar, 6 mm. (D) A nucleus about to enter the contraction segment. The pink shaded area was used to make the kymograph aligned below the snapshot. Scale bars, 5 μm and 71.6 ms. (E) Representative current pulses of a control (pink), LMN-1–depleted (green), and LMN-1 and SAMP-1 co-depleted (blue) nucleus transiting the sizing and the contraction segments. Scale bars, 100 ms and 0.2 nA. (F) Quantification of the wCDI. Black cross bars indicate means. All statistics are in Materials and Methods and data S1.

Fig. 6.. A cooperative network stabilizes oocyte nucleus against mechanical forces.

(A) Illustration of components influencing nuclear stability during oogenesis. LMN-1 (magenta) and INM proteins (EMR-1, LEM-2, and SAMP-1) likely contribute to the mechanical stability of the nucleus either by providing rigidity at the INM that withstand forces generated by microtubule/dynein and transmitted by the LINC complex or by spreading LINC-mediated forces over a larger area of the NE. Depletion of LMN-1 leads to polarization of LINC complexes that transmits unbalanced forces, leading to nuclear collapse in diplotene. Co-depletion of components/regulators of the dynein motor, SUN-1 or ZYG-12, rescues nuclear collapse. Simultaneous depletion of EMR-1 and LEM-2 or of SAMP-1 leads to exacerbated, earlier nuclear collapse. (B) Force balance at the nuclear envelope. External pushing forces are depicted by arrows pointing inward to the nucleus, and resisting forces are depicted by arrows pointing outward from inside the nuclear envelope. Schematics show balanced forces across the NE in a wild-type nucleus and unbalanced forces due to LMN-1 depletion leading to diplotene collapse. Upon LMN-1 depletion, force balance can be restored by reducing LINC-mediated mechanotransduction. However, the imbalance can be exacerbated by co-depleting SAMP-1 (or EMR-1 and LEM-2), which likely causes increased forces applied on the NE from outside because of more polarized LINC complex distribution. (C) Regulation of nuclear stability during C. elegans oogenesis. Factors inhibiting collapse are depicted with blunt arrows, while those that promote collapse are depicted with pointed arrows.