Matefin/SUN-1 phosphorylation is part of a surveillance mechanism to coordinate chromosome synapsis and recombination with meiotic progression and chromosome movement

Abstract

Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In Caenorhabditis elegans, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a chk-2- and plk-2-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation-dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end-led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.

Figure 1. Prolonged SUN-1 phosphorylation correlates with meiotic failure.

(A) Wild-type (WT) hermaphrodite gonad stained with DAPI (top and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). Arrow highlights a nucleus in mid/late pachytene zone with clustered chromatin and phosphorylated SUN-1. Magnifications at the bottom of representative TZ or early pachytene nuclei to highlight differences in SUN-1 phosphorylation patterns. Schematics on top (in A, C, D, and E) delineate quantifications of nuclei with and without phosphorylation of SUN-1 (S8, S12, S24, and S43) in the meiotic part of the gonad (quantified from meiotic prophase entry to beginning of cellularization/diplotene in WT, marked with dotted lines in DAPI channels). Orange represents cell rows with ≥50% of nuclei with SUN-1 phosphorylation. n, number of gonads scored for each genotype. (B) Wild-type hermaphrodite gonad stained with DAPI (left and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-RAD-51 (red in merge). Box, upper right: magnification of representative nuclei in late pachytene with clustered chromatin, phosphorylated SUN-1, and numerous RAD-51signals. Red channel boosted in merge picture to better visualize RAD-51 foci in all nuclei. (C and D) syp-2(ok307) (C) and rad-51(ok2218) (D) mutant hermaphrodite gonad stained with DAPI (top and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). (E) Wild-type hermaphrodite gonad dissected 90 min after 90 Gy gamma irradiation stained with DAPI (top and blue in merge), anti-RAD-51 (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). (F) Wild-type hermaphrodite gonad dissected 27 h after 70 Gy gamma irradiation; anti-SUN-1 S12Pi (red), anti-SUN-1 S43Pi (green), and DAPI (blue). Scale bars, 10 µm.

Figure 2. SUN-1 phosphorylation is prolonged in the absence of DSBs and can be decreased to wild-type length by low-dosage irradiation.

WT (left) and spo-11(ok79) (right) hermaphrodite gonad, not irradiated (top) or irradiated with 7.5 Gy (bottom) dissected 8 h post-irradiation and stained with DAPI (top, blue in merge) and anti-SUN-1 S8Pi (bottom, green in merge). Schematics on the bottom of each gonad represent nuclei with and without phosphorylation of SUN-1 (S8, S12, S24, and S43) in the meiotic part of the gonad (from meiotic prophase entry to beginning of cellularization/diplotene in WT, marked with dotted lines in DAPI channels). Orange represents cell rows with ≥50% of nuclei with SUN-1 phosphorylation.

Figure 3. Effect of sun-1 phosphosite mutations on the duration of meiotic stages, DSB turnover, SUN-1 aggregates, chromosome movement, and synapsis.

(A) TZ nuclei of sun-1(wt); sun-1(ok1282) (top), and sun-1(allA); sun-1(ok1282) (bottom) hermaphrodite gonads stained with DAPI (left; blue in merged picture) and anti-GFP (middle; green in merged picture). Scale bars, 10 µm. (B) First frame of in vivo time-lapse GFP-recorded TZ nuclei of sun-1(wt); sun-1(ok1282) (top, left) and sun-1(allA); sun-1(ok1282) (bottom, left) hermaphrodite gonads. Displacement tracks of SUN-1 aggregates represent 2D plotted chromosome end movements over 3 min (right). Average speed of SUN-1 aggregates in TZ (n = 9 nuclei, followed over 3 mins). Scale bar, 2 µm. (C) Schematic of a wild-type hermaphrodite gonad with nuclei in the corresponding zones, as used in the quantifications in Figure 3D: chromatin (blue) and SUN-1 morphology (green). Gonad is subdivided into ten zones, as used for the RAD-51 foci quantification in Figure 3E. Meiotic entry and beginning of cellularization are indicated. (D) Representation of relative time of residency in different meiotic stages, as assessed by presence of SUN-1 aggregates in different mutant backgrounds, normalized to the length of the gonad (from meiotic entry [0%] to start of meiocyte cellularization at diplotene [100%]). Nuclei were sorted into three categories: “TZ” (dark green, more than one SUN-1 aggregate), “early pachytene” (light green, one SUN-1 aggregate), and “late pachytene” (red, no SUN-1 aggregate). Categories were assigned once ≥50% of nuclei in a cell row fulfilled one of these criteria. At least eight gonads were counted per genotype. (E) Average numbers of RAD-51 foci per nucleus in the hermaphrodite gonad of different sun-1 phosphosite mutants. Gonads were divided into ten zones, as schematically indicated in (B). Three gonads per genotype were counted. (F) Nuclei from early and late pachytene zones of sun-1(wt); sun-1(ok1282) (top) and sun-1(allA); sun-1(ok1282) (bottom) hermaphrodite gonads stained with anti-HTP-3 (left, green in merged) and anti-SPY-1 (middle, red in merged). Scale bars, 10 µm.

Figure 4. Effect of nonphosphorylatable SUN-1 on aggregate persistence, PLK-2 localization, and chromosome mobility beyond TZ.

Hermaphrodite gonad of sun-1(wt); sun-1(ok1282), syp-2(ok307) (top) and sun-1(allA); sun-1(ok1282); syp-2(ok307) (bottom) stained with DAPI (blue), anti-GFP to highlight SUN-1 (green), and anti-PLK-2 (red). Dark green and light green frames highlight zones with distinct chromosome movement patterns: magnifications of nuclei in the corresponding zones below, showing displacement tracks of 2D plotted chromosome end movements over 3 min (left) and PLK-2 (red) and DAPI (blue). Bottom: average number of aggregates, aggregate velocity (in nm/sec), and fusion/split events (per nucleus/min) for nuclei from corresponding zones. Scale bar, 10 µm.

Figure 5. SUN-1 S12 phosphorylation in male meiosis.

(A) Representative TZ nuclei of a sun-1(wt) hermaphrodite gonad (upper panel) and a sun-1(wt) male gonad (lower panel) to highlight SUN-1 (anti-GFP, green), anti-Him-8 (red), and DAPI (blue). (B) Representative nuclei from wild-type hermaphrodite TZ (upper panel) and wild-type male TZ (lower panel) stained with anti-SUN-1 S12Pi (green), anti-HIM-8 (red), and DAPI (blue). (B′) Representative nuclei from him-3 (gk149) hermaphrodite TZ (upper panel) and him-3 (gk149) male TZ (lower panel) stained with anti-SUN-1 S12 Pi (green), anti-HIM-8 (red), and DAPI (blue). (B″) Representative nuclei from sun-1(jf18) hermaphrodite TZ (upper panel) and sun-1(jf18) male TZ (lower panel) stained with anti-SUN-1 S12 Pi (green), anti-HIM-8 (red), and DAPI (blue). (C) Representative wild-type TZ nuclei of a hermaphrodite gonad (upper panel) and a male gonad (lower panel) costained with anti-PLK-2 (green), anti-Him-8 (red), and DAPI (blue). (D) Relative time of residency in different meiotic stages in different sun-1 phosphorylation mutant backgrounds in males, assessed by presence of SUN-1 aggregates; zones were normalized to the individual length of the scored gonad (from meiotic entry to diplotene). Nuclei were sorted into two categories: “TZ” (dark green, more than one SUN-1 aggregate) and “pachytene” (red, no SUN-1 aggregates). (E) Pachytene zone nuclei of sun-1(wt) and sun-1(6E) stained with anti-HIM-8 (blue), anti-SYP-1 (yellow), and DAPI (red). Note that the single male X chromosome was unsynapsed in both WT and 6xE substitution lines.