Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

Abstract

Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid-Sad1p, UNC-84-domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid "twin" nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly.

Keywords: recombination/synapsis; sisters versus homologs.

Fig. 1.

Progression from crozier to meiotic pachytene in WT (Upper) and slp1 (Lower). Chromosome axes are visualized by Spo76/Pds5–GFP.

Fig. 2.

Pre- and postkaryogamy in slp1 compared with WT. (A–I) Antitubulin staining. (A and B) MTs in crozier before (A) and during mitosis (B). Spindles (B) are positioned to leave the two upper nuclei in the crook, which, after septum formation will become the ascus (see Fig. S4, Upper). (C–F) Prekaryogamy nuclei are surrounded by MTs in both slp1 (C) and WT (E); D and F, corresponding DAPI. (G–I) Cortical MTs of young asci in WT (G) and slp1 (H and I). Red arrows point to the WT diploid nucleus (G, DAPI in red) and the twin nuclei of slp1 (I). (J and K) slp1 nuclei have moved very close with their SPBs marked by Sun1–GFP (green arrows) facing each other; (K) corresponding DAPI. (L and M) NEs of the two slp1 nuclei are flattened and parallel to one another (red arrows) when observed by Mad2–GFP (L) or ER-Tracker (M). (Scale bars, 5 μm.)

Fig. 3.

Slp1–GFP localization in WT. (A–C) Colocalization of Slp1–GFP with ER-Tracker at the NE (red arrows) during early prophase; (A) corresponding DAPI. (D and E) As in WT (C), ER-Tracker makes a clear ring around slp1 twin nuclei (red arrows in D); (E) corresponding DAPI. (F–I) Colocalization of Slp1–GFP with ER-Tracker at late prophase. They show a similar cytoplasmic reticular pattern and net-like structures (arrow) around the NE (G–I); (F) corresponding DAPI. (Scale bars, 5 μm.)

Fig. 4.

Axis formation and Spo76/Pds5 requirement. (A) slp1 nuclei exhibit two chromatids (arrows), clear indication of correct premeiotic S phase. (B and C) Spo76–GFP staining in WT (B) and slp1 twin nuclei (C). (D and E) Rec8–GFP staining in slp1 (C) and WT (E). (F and G) Diffuse chromatin in spo76-1 (F) and spo76-1 slp1 (G) prophase nuclei. (H and I) Premature loss of sister cohesion in spo76-1 slp1 (H), whereas centromeres remain attached in slp1 simple mutant (I). (J) Degenerated spo76-1 slp1 nuclei after their anaphase I arrest. (K and L) Anaphase I onset in slp1 twin nuclei. Spindles form normally (K) despite random segregation of chromosomes (L). (Scale bars, 1 μm.)

Fig. 5.

SC formation in slp1. (A–F) slp1: synchronous loading of Zip4–GFP (A) and Sme4–GFP (D) along all chromosomes stained by DAPI (B and E) and merge (C and F). The Upper nucleus in D–F is perpendicular to the Lower nucleus and thus less clear in the merged Z sections. (G–I) Similar loading of Sme4–GFP (G) along all WT homologs (H, DAPI) and (I) merge. (J–M) slp1: Sme4–GFP (J) forms continuous lines along all seven chromosomes. Loading occurs between sister chromatids (arrows in J and K); note that, contrary to WT (H) sister chromatids are separated (arrows in K). (L) Merge and corresponding cartoon (M). (N and O) Spread nuclei of slp1 showing clear individualization of the seven chromosomes (stained by Spo76–GFP) in both nuclei; (O) corresponding cartoon. (P) Late pachytene slp1 nucleus with nonhomologous synapsis (arrows). (Scale bars, 2 μm.)

Fig. 6.

Synchronous loading of recombination proteins in slp1. Numbers of foci are indicated on the twin slp1 nuclei in red and green and in red for WT. (A–D) Rad51 in the twin nuclei of slp1 (A and B) compared with WT (C and D). (E–H) Mer3–GFP in WT (E) and slp1 (F–H). Arrows in H point to evenly spaced foci. (I and J) Msh4 foci in slp1. (K–Q) Hei10 foci in slp1 (K–P) and WT (Q). Each chromosome exhibits at least one focus at early–mid (K and L) and late pachytene (M) like in WT (Q). Foci form also, but rarely, between two nonhomologous chromatids (arrow in K and L). Foci remain through the diffuse stage attached to small SC segments (arrows in N). The number of Hei10 foci (O) is about 0.5 of WT for all chromosomes. Their distribution along chromosomes (P) is almost WT-like for the two longer chromosomes (Upper) but much flatter for the smaller chromosomes (Lower). nu, nucleolus. (Scale bars, 5 μm.)