Absence of SUN-Domain Protein Slp1 Blocks Karyogamy and Switches

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

Christelle Vasniera, Arnaud de Muyta,b, Liangran Zhangc, Sophie Tesséa, Nancy E. Klecknerc,1, Denise Zicklera,1, and Eric Espagnea,1

aInstitut de Génétique et Microbiologie, Unité Mixte de Recherche 8621, Université Paris-Sud, 91405 Orsay, France; bInstitut Curie, 75248 Paris Cedex 05, France; and cDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Contributed by Nancy E. Kleckner, August 18, 2014 (sent for review June 5, 2014) Karyogamy, the process of nuclear fusion is required for two haploid (7). Blast search of the basidiomycete Cryptococcus neoformans gamete nuclei to form a zygote. Also, in haplobiontic organisms, genome identified four of the known KAR genes; interestingly karyogamy is required to produce the diploid nucleus/cell that then only the kar7 mutant showed a defect in vegetative nuclear enters meiosis. We identify sun like protein 1 (Slp1), member of the movement and hyphae mating but not in premeiotic karyogamy mid–Sad1p, UNC-84–domain ubiquitous family, as essential for kary- (8), likely reflecting the different evolution of ascomycetes and ogamy in the filamentous fungus Sordaria macrospora, thus uncov- basidiomycetes. ering a new function for this protein family. Slp1 is required at the Whereas karyogamy studies of fungi have provided significant last step, nuclear fusion, not for earlier events including nuclear insight into the steps and molecules involved, less is known con- movements, recognition, and juxtaposition. Correspondingly, like cerning the outcome of the remaining nonfused haploid nuclei. other family members, Slp1 localizes to the endoplasmic reticulum Such “twin” meiosis has been analyzed in fission yeast in three and also to its extensions comprising the nuclear envelope. Remark- cases: in the presence of the mating-type allele h90 (9) or in ably, despite the absence of nuclear fusion in the slp1 null mutant, absence of either the microtubule plus-end tracking protein “ ” meiosis proceeds efficiently in the two haploid twin nuclei, by the Eb1/Mal3 (7, 10) or the type 1 membrane protein Tht1 (11), but same program and timing as in diploid nuclei with a single dramatic without detailed analysis of the meiotic process per se. exception: the normal prophase program of recombination and syn- To further investigate these issues, we cloned and characterized apsis between homologous chromosomes, including loading of re- the two Sad1p, UNC-84 (SUN)-domain genes of the fungus Sor- combination and synaptonemal complex proteins, occurs instead daria macrospora. In most organisms investigated thus far, SUN- between sister chromatids. Moreover, the numbers of recombination- domain proteins are NE associated and are good candidates to initiating double-strand breaks (DSBs) and ensuing recombinational play roles in karyogamy because they (i) transmit forces between interactions, including foci of the essential crossover factor Homo the nucleus and cytoskeletal complexes; (ii) have conserved roles sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in meiotic chromosome movements, and (iii) in yeasts, are asso- in each haploid nucleus, implying per-chromosome specification of ciated with the SPB (reviewed in ref. 12). One Sordaria gene is DSB formation. Further, the distribution of Hei10 foci shows interfer- a member of the canonical SUN1/SUN2/MPS3/SAD1 family. Be- ence like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. ing essential for cell viability, it could not be analyzed for roles in the sexual cycle. The other gene is not essential for viability and is These observations imply that the prophase program senses absence SLP1 OPT/SUCO/SUN3/4/5/SUNB – of karyogamy and/or absence of a homolog partner and adjusts the a member of the / mid SUN- domain family (13–15). Like the other proteins of this family,

interchromosomal interaction program accordingly. GENETICS

sisters versus homologs | recombination/synapsis Significance

aryogamy is the process by which two nuclei fuse to produce Meiosis is the specialized cellular program that generates gametes Ka single nucleus. This process is critical in diploid organisms for sexual reproduction. In the fungus Sordaria macrospora kar- (e.g., mammals and plants) when haploid egg and sperm nuclei yogamy is required to produce the diploid cell that enters the fuse to produce a diploid nucleus and zygote. In organisms with meiotic program. In absence of the mid-Sad1p, UNC-84–domain a haploid vegetative cycle (e.g., fungi and most algae), karyogamy sun like protein 1, karyogamy does not occur. Meiosis none- is required to produce the diploid nucleus/cell, which will then theless proceeds efficiently in the two haploid nuclei, but with enter meiosis. In both situations, karyogamy involves two steps: the entire program of interhomolog events now occurring in- nuclear movement leading to nuclear juxtaposition (congression) stead between sister chromatids, including spatially patterned and final fusion of the nuclear membranes. Cooperation of the recombination and synaptonemal complex formation. As a re- cytoskeleton components is required for nuclear movement and sult, significant levels of gametes are still formed. In contrast, correct positioning of the nuclei (e.g., ref. 1). other cases of meiosis in haploid genome complements exhibit In budding yeast, premeiotic karyogamy requires several genes inefficient or aberrant chromosomal programs. We thus pro- (named Kar1 to Kar9) with their mutant defects corresponding pose that Sordaria can sense the absence of karyogamy so as to to two steps: nuclear congression, which involves cytoskeleton trigger an appropriately regular response. components, motor proteins and the spindle pole body (SPB, Author contributions: C.V., A.d.M., D.Z., and E.E. designed research; C.V., A.d.M., S.T., mammal centrosome equivalent) and fusion of the two haploid D.Z., and E.E. performed research; L.Z. and N.E.K. contributed new reagents/analytic tools; nuclear envelopes (NEs), which involves either the endoplasmic L.Z., N.E.K., D.Z., and E.E. analyzed data; and N.E.K., D.Z., and E.E. wrote the paper. reticulum (ER) per se or protein translocation into the peri- The authors declare no conflict of interest. – nuclear space (reviewed in refs. 2 4 and references therein). Kar 1To whom correspondence may be addressed. Email: denise.zickler@igmors.u-psud.fr, homologs are present in fission yeast and Candida albicans (5, 6) [email protected], or [email protected]. and two fission-yeast proteins required for proper microtubule This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (MT) integrity, Mal3 and Mto1, are also required for karyogamy 1073/pnas.1415758111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1415758111 PNAS | Published online September 10, 2014 | E4015–E4023 Downloaded by guest on September 24, 2021 Sordaria sun like protein 1 (Slp1) is localized in both the ER and because karyogamy occurs just after formation of the dikaryotic NE. The precise role of those proteins remains unknown. Budding cells, with the resulting diploid nucleus immediately entering yeast Slp1 is implicated in folding of integral membrane proteins meiosis (Fig. 1). and is involved directly or indirectly in Sun1/Mps3 localization to the NE (15, 16). Dictyostellum discoideum SunB protein plays roles Slp1 Is Not Required for Nuclear Congression or Microtubule or SPB in both cell proliferation and differentiation (14). The mouse Dynamics. To further characterize the karyogamy defect seen in ab- ortholog osteopotentia (Opt) protein is a key regulator of bone sence of Slp1, we analyzed the slp1Δ mutant with respect to the two formation (17). The two splicing variants (CH1 and C1orf9) of the steps identified in budding yeast karyogamy mutants (Introduction). human SUCO gene were respectively found overexpressed in First, we analyzed in detail, in both WT and slp1Δ, nuclear breast cancer cells or linked to the hereditary prostate cancer lo- migration and formation of the MT network by immunostaining cus, HPC1 (18, 19). using alpha-tubulin antibodies from the crozier stage onward, i.e., Interestingly, the Sordaria SLP1 gene is absolutely crucial for before, at, and after the karyogamy stage. The mutant shows karyogamy but not for the overall meiotic program. In an slp1Δ null WT-like nuclear migration (Fig. 1). The two mutant haploid nuclei mutant, the two juxtaposed haploid nuclei progress synchronously of the hook-shaped crozier (Fig. S4 and Fig. 2 A and B)undergo and efficiently through the meiotic program, but the normal pro- simultaneous mitosis (Fig. 2B), with spindles positioned such that gram of recombination-mediated interactions between homolo- one daughter nucleus from each parent is present in the crook gous chromosomes (including synaptonemal complex formation portion of the cell. Septa form on each side of the crook, resulting and crossover interference) (20) now occurs regularly and effi- in a basal and a lateral cell flanking the binucleate upper ascus- ciently between sister chromatids. These and other observations mother cell (Fig. S4). When the ascus starts growing, the two imply that the two haploid twin nuclei sense their change in status, nuclei move from a side-by-side disposition into a vertical ar- either by detecting absence of karyogamy or by directly detecting rangement (Figs. 1 and 2 C and D). Accordingly, MT dynamics are absence of a homolog partner, and appropriately alter the program WT-like: (i) In croziers (Fig. 2 A and B) and, when nuclei alter of effects to rescue a minimal level of gamete formation. position into a tandem arrangement, nuclei are surrounded by short MTs arising from the periphery of each nucleus (Fig. 2 C Results and D)likeinWT(Fig.2E and F). (ii) In the developing ascus of The Two Sordaria SUN-Domain Proteins Belong to Two Subfamilies. meiotic prophase, MTs are organized as cortical arrays of longi- Sordaria genomic sequence (http://c4-1-8.serverhosting.rub.de/ tudinal MTs converging on an apical microtubule organizing public/) (21) contains only two SUN-domain encoding genes: center (MTOC) in both WT (Fig. 2G)andslp1Δ (Fig. 2 H and I). SMAC_01167 and SMAC_08081 (Fig. S1). SMAC_01167 corre- Second, nuclear congression is also WT-like. Once they are sponds to the canonical SUN1/SUN2/MPS3/SAD1 genes identified vertically arranged, slp1Δ nuclei move toward each other as in WT, so far in all eukaryotes (Fig. S2). It is an essential gene in Sordaria. excluding any problem in either movement or “recognition” (Fig. 2 Sordaria SUN1 encodes an ∼111-kDa predicted protein with the D and I). Also, in both mutant and WT, nuclei are first surrounded three structural features characteristic of this family: a trans- by arrays of MTs (Fig. 2 C and E), which redisperse to form the membrane, a coiled-coil, and a C-terminal SUN/Sad1_UNC do- longitudinal arrays seen during prophase (Fig. 2 G and H). main (PF07738; Fig. S2). Sun1 protein localizes to both the NE Therefore, this latter MT network, which is involved in main- and the SPB. taining the ascus shape (23), forms independently of the ploidy of In the second protein, encoded by SMAC_08081, the SUN do- the nucleus and also independently of the presence of one or two main is located in the central region of the protein (Fig. S3). This nuclei in the ascus. ∼117-kDa predicted protein exhibits four structural features: an Moreover, slp1Δ exhibits normal SPB shapes and localization N-terminal signal peptide, a SUN/Sad1_UNC (PF07738), a coiled- as seen by Sme4–GFP and Sun1–GFP, which specifically mark coil, and a transmembrane domain (Fig. S3). This gene, which we the SPB (24). SPBs lead the nuclear movement and are located call SLP1, is a member of a family of orthologs present in all in the fusion area (Fig. 2 J and K) like in WT (23). However, eukaryotic kingdoms with names differing with organisms (SLP1/ although events immediately preceding nuclear fusion are nor- OPT/SUCO/SUNB; Fig. S1). Plants, in fact, harbor three such genes: mal, nuclear fusion is completely blocked in slp1Δ:(i) The two SUN3/4/5 (22), whereas only one member of this subfamily is found haploid nuclei approach each other as closely as in WT (Figs. 1 in the other organisms analyzed so far (Fig. S3)(13–15, 17, 19).

Slp1 Is Required for Normal Vegetative Growth and, During the Sexual Cycle, Is Essential for Karyogamy. The slp1Δ null allele (SI Materials and Methods) shows defects in both vegetative growth and sporulation: (i) It grows more slowly than the wild-type (WT) strain: 0.86 ± 0.01 cm per 12 h compared with 1.19 ± 0.02 cm for WT; (ii) it exhibits a 24-h delay in progression through the sexual cycle; and (iii) the number of asci per fruiting body is also reduced: ∼20–30 instead of over 100 in WT (n = 50 fruiting bodies). The mutant is also dramatically defective in a central step of the fungal sexual cycle: karyogamy. In filamentous ascomycetes like Sordaria, an ordered series of differentiation steps leads to the formation of sexual organs, fertilization, formation of the fruiting bodies (perithecia), and compartmentalization of dikaryotic cells that then undergo karyogamy, leading to diploid cells/asci that immediately enter meiosis (Fig. S4). In slp1Δ homozygous crosses, the first four steps are normal compared with WT crosses made in parallel, with formation of perithecia indicating that fertilization had occurred. However, young slp1Δ perithecia contain only asci with two nuclei (n = 100 perithecia), a clear indication of a karyogamy defect (Fig. 1). Prekaryogamy binucleate asci are also seen Fig. 1. Progression from crozier to meiotic pachytene in WT (Upper)and in young WT perithecia, but in very small numbers (<5%, n = 100) slp1 (Lower). Chromosome axes are visualized by Spo76/Pds5–GFP.

E4016 | www.pnas.org/cgi/doi/10.1073/pnas.1415758111 Vasnier et al. Downloaded by guest on September 24, 2021 suggest that the mechanisms that partition phospholipids (a major PNAS PLUS component of the NE) and proteins between the peripheral ER and NE is not the major defect in the mutant. This hypothesis is further supported by the fact that nuclei increase in size throughout meiotic prophase in the mutant as in WT (Fig. 1 and below). Thus, the requirement for coordinated growth between outer and inner NE membranes to maintain normal nuclear shape is not defective in absence of Slp1.

In the Absence of Karyogamy, Meiosis Proceeds, with Synchronous Parallel Development of the Twin Haploid Nuclei. In WT meiosis, S phase occurs just before karyogamy and is accompanied by loading of both the cohesin-associated Spo76/Pds5 protein and the meiotic-specific cohesin Rec8 (25, 26). Meiotic S phase occurs normally in slp1Δ, as indicated by the fact that mutant chromosomes exhibit two chromatids (Fig. 4A). The mutant is also in- distinguishable from WT with respect to pre- and postkaryogamy axis development and organization: both nuclei develop Spo76/ Pds5–GFP or Rec8–GFP lines along all chromosomes. Moreover, this loading occurs in perfect synchrony in the two partner nuclei (Fig. 4 B–E). Also, as in WT (Fig. 4B), Spo76–GFP makes bright and smooth lines in the mutant (Fig. 4C) when Rec8–GFP makes fainter dotty lines (Fig. 4D) like in WT (Fig. 4E). Fig. 2. Pre- and postkaryogamy in slp1 compared with WT. (A–I) Anti- Interestingly, all examined events of meiosis from the earliest tubulin staining. (A and B) MTs in crozier before (A) and during mitosis (B). slp1Δ Spindles (B) are positioned to leave the two upper nuclei in the crook, which, stages onward occur in perfect synchrony in the unfused after septum formation will become the ascus (see Fig. S4, Upper). (C–F) partner nuclei. This finding presumably reflects the fact that the Prekaryogamy nuclei are surrounded by MTs in both slp1 (C) and WT (E); two nuclei still share a common cytoplasm. In support of this D and F, corresponding DAPI. (G–I) Cortical MTs of young asci in WT (G) and possibility, in WT diploid meiosis, the nuclei are separated after slp1 (H and I). Red arrows point to the WT diploid nucleus (G, DAPI in red) the first meiotic division and nevertheless both second division and the twin nuclei of slp1 (I). (J and K) slp1 nuclei have moved very close and postmeiotic divisions (plus ascospore formation) occur syn- – with their SPBs marked by Sun1 GFP (green arrows) facing each other; (K) chronously, suggesting that a common cytoplasm may be suffi- corresponding DAPI. (L and M) NEs of the two slp1 nuclei are flattened and cient for synchrony of these latter events. parallel to one another (red arrows) when observed by Mad2–GFP (L)or ER-Tracker (M). (Scale bars, 5 μm.) Other cellular aspects of the meiotic process also occur normally in the slp1Δ mutant: (i) Asci elongate from 20 to 150 μm and reach their full lengths by the end of the first meiotic division as in WT. and 2 J–M, n = 100). (ii) Moreover, they are aligned with the (ii) Sme4–GFP, which loads on WT SPBs only from metaphase I “fusion portions” of the NEs flattened and parallel to one another on through postmeiotic mitosis (24), is also seen specifically at as seen by Mad2–GFP, which stains the nuclear envelope (Fig. these stages in slp1Δ.(iii) In diploid WT nuclei, at the first meiotic 2L) and by ER-Tracker dyes, which stain similarly the ER and the division, homologous chromosomes segregate to opposite poles. NE in both the WT and the mutant (Fig. 2M,andseebelow). In the slp1Δ haploid nuclei, there is no precocious sister–centro- During meiosis, the two nuclei remain either close or separated mere separation and chromosomes also segregate to opposite – μ A B

over a distance of 2 4 m (Fig. 1 and Fig. S5 and ). These GENETICS findings show that Slp1 is not required for nuclear congression, SPB localization, and prefusion nuclear shape remodeling, but its absence dramatically affects the last steps of NE fusion per se.

Slp1 Exhibits ER/Perinuclear Localization Before and After Karyogamy, Consistent with the Null Mutant Defect. Localization of Slp1 through the life cycle of Sordaria was monitored by fluorescent protein fusion Slp1–GFP in both WT and in slp1Δ backgrounds (SI Materials and Methods). As Slp1–GFP shows a reticular pattern reminiscent of an ER staining, we compared its localization with the ER-Tracker Red (known to be a highly sensitive and a specific probe for the ER) both in WT and mutant. In WT, from the crozier stage to the end of meiosis, the Slp1– GFP signal comprises an irregular network spanning over the entire cytoplasm. During early meiotic prophase, Slp1–GFP also makes a faint ring around the nucleus (Fig. 3 A and B) whereas ER-Tracker Red concomitantly makes a smoother and brighter ring around the nucleus (Fig. 3C). The same bright perinuclear ring is also seen in slp1Δ stained by ER-Tracker Red (Fig. 3 D and E). In WT, at midlate prophase, the Slp1–GFP perinuclear ring is Fig. 3. Slp1–GFP localization in WT. (A–C) Colocalization of Slp1–GFP with replaced by a cluster of net-like structures around the NE (Fig. 3 F ER-Tracker at the NE (red arrows) during early prophase; (A) corresponding G DAPI. (D and E)AsinWT(C), ER-Tracker makes a clear ring around slp1 twin and ). Similar perinuclear changes are also visible by ER-Tracker – – H I nuclei (red arrows in D);(E) corresponding DAPI. (F I) Colocalization of Slp1 Red staining (Fig. 3 and ). GFP with ER-Tracker at late prophase. They show a similar cytoplasmic re- Because the NE is part of the ER, similar localization, forma- ticular pattern and net-like structures (arrow) around the NE (G–I); (F)cor- tion, and disappearance of the perinuclear ring in WT and slp1Δ responding DAPI. (Scale bars, 5 μm.)

Vasnier et al. PNAS | Published online September 10, 2014 | E4017 Downloaded by guest on September 24, 2021 (Fig. 4J). Although random segregation of chromosomes occurs at anaphase I, no such block occurs in the slp1Δ single mutant (Fig. 4 K and L). Thus, Spo76 is strictly required for sister- chromatid cohesion and meiotic progression in slp1Δ as in WT, suggesting that the same regulation or control pathways occur in both diploid and haploid meiosis.

In slp1Δ Where a Homologous Chromosome Is Absent, Synaptonemal Complex Central Components Load Between Sister Chromatids. In WT, homologous pairing, which culminates in presynaptic align- ment, is mediated by double-strand break (DSB)-initiated recombination events via recombination complexes that are associated with their underlying chromosome axes (27). Synapsis, mediated by the synaptonemal complex (SC), then occurs, juxtaposing the chromosome axes at a distance of 100 nm. Correspondingly, SC central- region components Sme4 and Zip4 localize between homologous axes, progressively elongating through zygotene to form bright lines along all homologs at pachytene and disappearing at the diffuse stage (20, 24). Just as in WT, the loading of both Sme4 and Zip4 is dependent upon DSB formation, with no loading in slp1Δ spo11Δ double mutant, just as in spo11Δ simple mutant. Also, as Slp1 is not required for robust DSB formation (see below), both Zip4 and Sme4 (Fig. 5 A–F) load progressively along chromosomes during zygotene and disappear at the diffuse stage in slp1Δ as in WT. Zip4 and Sme4 foci appear synchronously in each haploid nucleus (Fig. 5 A–F) with the correct timing as defined by ascus size. Moreover, progressive loading is also perfectly synchronous in the partner nuclei as seen for Zip4–GFP (Fig. 5 A–C) and Fig. 4. Axis formation and Spo76/Pds5 requirement. (A) slp1 nuclei exhibit Sme4–GFP (Fig. 5 D–F) with both types of foci elongating as in two chromatids (arrows), clear indication of correct premeiotic S phase. WT (Fig. 5 G–I). They form finally long lines along all chro- – – (B and C)Spo76 GFPstaininginWT(B)andslp1 twin nuclei (C). (D and E)Rec8 mosomes (Fig. 5 J–M). Most importantly, in all cases, Sme4 and GFP staining in slp1 (C)andWT(E). (F and G) Diffuse chromatin in spo76-1 Zip4 loading and thus SC formation is occurring between sister (F)andspo76-1 slp1 (G) prophase nuclei. (H and I) Premature loss of sister J K cohesion in spo76-1 slp1 (H), whereas centromeres remain attached in slp1 chromatids as seen by overlay with DAPI (Fig. 5 and , simple mutant (I). (J) Degenerated spo76-1 slp1 nuclei after their anaphase I arrows). Correspondingly, when SC is fully loaded, the distance arrest. (K and L) Anaphase I onset in slp1 twin nuclei. Spindles form normally between sister chromatids is increased (Fig. 5K, arrows and also (K) despite random segregation of chromosomes (L). (Scale bars, 1 μm.) evident in Fig. 4A), compared with zygotene stages (Fig. 5 B and E) and to WT where sister chromatids are tightly juxtaposed as single units when SC is loaded between homologs (Fig. 5H). poles; however, the seven chromosomes segregate randomly with Moreover, as expected from localization along separated axes to respect to one another. Correspondingly, this random segregation either side of the SC, cohesin Rec8, which is present along all leads to variable partitioning of the seven chromosomes, and thus chromosomes from leptotene on (Fig. 4D), forms two lines at to aneuploidy (see upper spindle in Fig. 4L). Consequently, the early pachytene (Fig. S5 A–F) and a wide fuzzy signal when SC is number of eight-spored asci (meiocytes) per fruiting body is dra- completed (Fig. S5 G–J). matically reduced to 0.5% in the mutant compared with 100% Chromosomes remain separated through midpachytene (by eight-spored asci seen in WT. ascus size) without synapsis between unrelated chromosomes (Fig. 5 N and O). However, by late pachytene, 20% of nuclei exhibit one Cohesin-Associated Spo76/Pds5 Protein Is Strictly Required for Sister or two regions of nonhomologous synapsis (Fig. 5P). These and slp1Δ Sordaria Cohesion and Anaphase I Progression in ,asinWT. other findings (next section) imply that the same program of recom- Spo76/Pds5 protein is involved in chromosome axis morpho- bination-mediated synapsis that normally occurs between ho- genesis, sister-chromatid cohesiveness, and chromatin condensa- mologous chromosomes is now, in slp1Δ, occurring between tion. In a strain expressing a truncated Spo76 protein (spo76-1; sister chromatids. SPO76 is an essential gene), sister-cohesion defects, which are observed during early meiotic prophase, lead to premature sister- DSB-Mediated Recombination Progresses Synchronously and Efficiently chromatid separation at diplotene and a cell division arrest at Between Sister Chromatids of the Twin Haploid Nuclei. Meiotic re- anaphase I (25). combination initiates by programmed DSBs (reviewed in ref. 28). The slp1Δ spo76-1 double mutant shows the combined defects In Sordaria, sites of DSBs are marked by foci of Rad51, the only of the two component mutations. It exhibits the same karyogamy RecA homolog in this organism. Analysis of Rad51 foci shows defect as the single slp1Δ mutant and the resulting partner that Slp1 is not required for robust DSB formation (Fig. 6 A and B). haploid nuclei display the characteristic phenotype of spo76-1 Interestingly, each haploid nucleus (Fig. 6B) shows 21 ± 6 foci diploid meiotic nuclei (Fig. 4 F–J): (i) Chromosomes are kinky (n = 70), thus approximately half the number observed in the and chromatin remains diffuse through prophase (Fig. 4 F and G diploid WT nuclei (52 ± 8; n = 100; Fig. 6 C and D). Rad51 foci compare with A). (ii) At diplotene, a premature loss of sister- also show the same temporal and spatial dynamics pattern as in chromatid cohesion throughout arms and centromeres leads to WT: synchronous loading begins at early leptotene and foci are no the presence of 14 chromatids in each twin haploid nucleus (Fig. longer visible at early pachytene (by the ascus size). Moreover, the 4H) when seven chromosomes are seen in slp1Δ simple mutant foci are WT-like in size and do not form aberrant elongated haploid nuclei (Fig. 4I). (iii) Finally, slp1Δ spo76-1 double mu- structures like those seen in mer3Δ or msh4Δ recombination tant, like spo76-1 single mutant, arrests meiosis after metaphase I mutants (27).

E4018 | www.pnas.org/cgi/doi/10.1073/pnas.1415758111 Vasnier et al. Downloaded by guest on September 24, 2021 move to a between-axis position during synapsis (24, 27). In PNAS PLUS slp1Δ, Mer3 foci form efficiently, with perfect synchrony in the partner nuclei (Fig. 6 F and G) and like for Rad51 occur in a number corresponding to half the WT number: 38 ± 6 in each nucleus compared with 68 ± 10 pairs of foci in WT (n = 50 and 100 nuclei, respectively). Also, like in WT (Fig. 6E), Mer3 foci are located on axes and are evenly spaced (Fig. 6H) but they do not form initially in well-separated pairs as they do along coal- igned homologous axes in diploids (27). Msh4 foci (Fig. 6 I and J) also appear with regular timing, after Mer3 foci, localize to chromosomes, and occur in half the WT number (37 ± 6 compared with 81 ± 8; n = 50 and 100, respectively). These observations support two conclusions. First, the number of DSBs per pair of sisters is the same as in WT, with interactions between homologs not required for normal DSB induction. Second, the Mer3 and Msh4 steps of DSB-mediated recombination occur just as in diploid meiosis except that interactions occur between the two sister chromatids, via their individual axes, rather than between two homolog axes, ultimately leading to regular intersister SC formation (above). In WT, the E3-ligase Homo sapiens enhancer of invasion 10 (Hei10) protein localizes as foci along the SC central regions, in GENETICS

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 corre- Fig. 6. Synchronous loading of recombination proteins in slp1. Numbers of sponding cartoon (M). (N and O) Spread nuclei of slp1 showing clear in- foci are indicated on the twin slp1 nuclei in red and green and in red for WT. dividualization of the seven chromosomes (stained by Spo76–GFP) in both (A–D) Rad51 in the twin nuclei of slp1 (A and B)comparedwithWT(C and D). nuclei; (O) corresponding cartoon. (P) Late pachytene slp1 nucleus with (E–H) Mer3–GFP in WT (E) and slp1 (F–H). Arrows in H point to evenly spaced nonhomologous synapsis (arrows). (Scale bars, 2 μm.) 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 In WT meiosis, DSB formation is followed by loading of post- nonhomologous chromatids (arrow in K and L). Foci remain through the DSB helicase Mer3 during leptotene and of MutS homolog 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 Msh4 at late zygotene/early pachytene, both of which form foci chromosomes (P) is almost WT-like for the two longer chromosomes (Upper) on all DSB-initiated recombinational interactions. Mer3 (Fig. but much flatter for the smaller chromosomes (Lower). nu, nucleolus. (Scale 6E) and Msh4 foci initially occur along homolog axes and then bars, 5 μm.)

Vasnier et al. PNAS | Published online September 10, 2014 | E4019 Downloaded by guest on September 24, 2021 strict dependence on SC formation. Moreover, the number plus Discussion distribution of mid- and late pachytene Hei10 foci correspond to The presented results identify mid–SUN-domain protein Slp1 as both the sites of crossover (CO)-fated recombinational inter- an ER/NE-localized mediator of nuclear fusion for karyogamy actions and to the chiasma sites (20). Analyses of the formation during the S. macrospora sexual cycle, therefore providing pre- and distribution of the Hei10 foci along the chromosomes of the viously unidentified insights into that process in this large group slp1Δ haploid nuclei (Fig. 6 K–P) in comparison with WT (Fig. of Sordariales, which includes some notable taxa such as Neu- 6Q) provided several interesting findings: (i) As in WT, Hei10 rospora, Sordaria, and Podospora species. These findings expand loads first as small (T1) foci and as brighter (T2 and T3) foci also the known roles of the mid–SUN-domain proteins. The other during what should be pachytene by ascus size (Fig. 6 K and M, known members of this family show similar ER/NE localization compare with WT in Q) through the diffuse stage, where they and have been implicated in a diversity of processes, including remain attached to stretches of the SC as in WT (Fig. 6N, folding of integral membrane proteins in budding yeast (15, 16), arrows). As T2 foci were difficult to analyze in absence of an axis cell proliferation and differentiation in amoeba (14), regulation of marker and are not visible above the Spo76–GFP or Rec8–GFP bone formation in mouse (13, 17), and dehydration signaling in signals, we analyzed only the T3 CO-fated foci, which are visible chickpea (30). Sordaria represents thus the first instance, to our above the Spo76–GFP signal in both WT and mutant (Fig. 6 K–Q). knowledge, in which a member of this family is implicated in kar- (ii)Theslp1Δ T3 foci have the same diameter as the WT T3 yogamy. It would now be interesting to know whether this molecule foci at the same stage (303 ± 4 nm compared with 324 ± 3nmin might also play a role for karyogamy during fusion of sperm and WT) and, as in WT, are clearly visible in nuclei concomitantly egg nuclei before zygote formation in mammals and/or plants. expressing Hei10–GFP and Spo76–GFP (Fig. 5Q). (iii)Asforthe Analysis of meiosis in the absence of karyogamy further reveals CO precursors Mer3 and Msh4 (above), their number is half the that unfused nuclei proceed efficiently through meiosis, thus im- WT number in each nucleus: 11.4 ± 3 compared with 22 ± 3inWT plying absence of a “karyogamy checkpoint.” Moreover, the twin (n = 76 and 100, respectively; compare Fig. 6 M and Q). (iv)Re- haploid nuclei proceed through meiosis with perfect synchrony, markably, the number and distribution of Hei10 foci in slp1Δ presumably due to communication via their shared cytoplasm. Most pachytene nuclei (by ascus size) exhibit the two major CO hallmarks remarkably, those nuclei modify their interchromosomal inter- of diploid meioses: CO interference and the “obligatory CO.” actions such that the entire program of DSB-initiated recombination CO interference is reflected in a tendency for COs to be non- and recombination-mediated SC formation now occurs, efficiently randomly spaced along chromosomes. The presence of in- and in a timely manner, between sisterchromatidsratherthanbe- terference in slp1Δ is revealed by distribution of distances between tween homologous chromosomes. The nature of the signals for, adjacent Hei10 foci (as percentage of axis length or in micro- and mechanism of, this partner switch is an interesting question meters) along all chromosomes. The value of the gamma param- for future study. eter ν, which reflects the tendency for even spacing of COs along slp1Δ Possible Roles of Slp1 for Karyogamy. What could be the basis for chromosomes, is only slightly lower in compared with WT: slp1Δ 3.6 ± 0.4 for the long chromosomes and 3.9 ± 0.6 for the short the karyogamy defect? The mutant is not defective for cy- chromosomes versus 5.5 ± 0.3 and 4.9 ± 0.2 for the corresponding toskeleton, nuclear movement, and juxtaposition in preparation WT bivalents. The term “obligatory CO” refers to the fact that for karyogamy; it is only defective in the very last step of the the frequency of bivalents lacking even a single CO is very low. In process, nuclear fusion per se. The precise molecular basis for this defect remains unknown. Because Slp1 localizes into both ER slp1Δ haploid nuclei, each of the 309 chromosomes examined and NE, Slp1 could be required directly for disassembly of the NE exhibits at least one Hei10 focus (examples in Fig. 6 K–M). This or in protein targeting/import into the perinuclear space. Alter- finding is particularly notable because, compared with WT, the natively or in addition, it could be required indirectly to create or average number of COs per chromosome is reduced by 50%. transduce the signal that partner nuclei have been juxtaposed, Other features of CO distribution are also normal in slp1Δ. thus triggering onset of NE disassembly. Defects in ER forma- Like in WT, the number and distribution of Hei10 foci (Fig. 6O)is tion/function or protein targeting/import are also suggested to higher in the two long chromosomes than in the shorter chromo- explain the known SLP1/OPT/SUCO/SUN3/4/5/SUNB subfamily somes (average of 2.3 and 1.6 compared with 4.2 and 2.7 in WT). mutant phenotypes in other organisms (above). Also, like in WT, Hei10 foci show higher frequencies of terminal Sordaria P Upper Karyogamy may also involve Sun1, the second SUN- localization for chromosomes 1 and 2 (Fig. 6 , ) although domain protein. Proteins of the Sun1 family, in association with with less prominent peaks. In contrast, shorter chromosomes show P Lower KASH-domain proteins, comprise a physical connection between more internal localizations than in WT (Fig. 6 , ). This may the NE and the cytoplasmic cytoskeleton that facilitates processes reflect the fact that these latter chromosomes mostly experience like nuclear anchorage and migration (reviewed in refs. 12, 31). only a single CO. Sordaria Sun1 localizes to the NE as well as to the SPB before, Interestingly, whereas Hei10 foci are predominantly located during, and after karyogamy. Thus, Sun1 could potentially play K–M on individual chromosomes (Fig. 6 ), in 20% of nuclei that a role in karyogamy either alone, or in relationship with Slp1, for show synapsis between unrelated chromatids, one to two foci transducing signals from nucleus/cytoskeleton dynamics into the K also form in those regions, especially at late pachytene (Fig. 6 nuclear fusion process. Unfortunately this hypothesis could not L and , arrow). However, all Hei10 foci are still dependent on be tested because SUN1 is an essential gene in Sordaria.Wehave recombination, as are foci of Rad51, Mer3, and Msh4: no foci of established, however, that contrary to Saccharomyces cerevisiae spo11Δ slp1Δ any of these types are observed in a double mutant. Slp1/Mps3 (15), Sordaria Slp1 is not required for the localization In WT, DSBs primarily engage a homolog partner chromatid of Sun1 at the SPB (Fig. 2J). and undergo a detailed program of events that lead to crossover and noncrossover products (reviewed in ref. 29). In slp1Δ,no Recombination Between Sisters Instead of Homologs in Karyogamy- homolog is present. Nonetheless, chromosomes progress regu- Defective Meiotic Prophase. In WT meiosis, prophase interchro- larly to diplotene, with each nucleus exhibiting the normal hap- mosomal interactions occur preferentially between homologs. In loid number of seven condensed chromosomes (Fig. 4I), and with slp1Δ meiosis, this same program of interactions occurs between no evidence of chromosome fragmentation at anaphase I and II, sisters efficiently and in a timely manner. Absence of karyogamy in despite random segregation of chromosomes (Fig. 4L). Taken slp1Δ has no discernible effect on DSB formation, implying that together, these findings further imply that all DSBs are repaired presence of a homolog is not a crucial requirement for the initi- through interactions between sister chromatids. ation of meiotic recombination. This is not specific to Sordaria,

E4020 | www.pnas.org/cgi/doi/10.1073/pnas.1415758111 Vasnier et al. Downloaded by guest on September 24, 2021 because efficient DSB formation in haploid meiosis has also been One possibility is that SC itself tends to push sister axes apart PNAS PLUS reported in yeasts and in plants (32–37). while forming the appropriate structure. For example, in diploid The above data further suggest that the intersister interaction meiosis, particularly where normal interhomolog interactions are program seen in slp1Δ efficiently converts all DSBs into inter- defective, SC can form in the absence of nucleation at a specific sister recombination products by the normal program of events interaxis distance within self-assembling protein polycomplexes that includes CO designation and interference: (i) Following and also between nonhomologous chromosomes (reviewed in DSB formation, recombinational interactions all progress effi- refs. 47, 48). Nonhomologous SC formation also forms in hap- ciently through the steps of successive Mer3 and Msh4 loading. loid meioses where it can involve up to 75% of the chromosome Both types of foci appear with normal timing and in a number lengths, either as multiple associations or as foldbacks of single corresponding to total recombinational interactions and with chromosomes (34, 49–52 and references therein). even spacing as in the diploid case. (ii) Hei10 foci, which specifi- Another possibility is that absence of karyogamy alters the sister cally mark CO-fated recombination sites in diploid Sordaria mei- cohesion program, specifically loosening sister connections. Such osis (20), are not only present along all chromosomes in the loosening would only need to occur along the chromosome axes, haploid twin nuclei but do also show both interference and the rather than globally within the chromatin loops. Indeed, there are obligatory CO. We infer that CO designation has occurred nor- known situations in wild-type meiosis where, within the SC, sister mally at leptotene/zygotene as in diploid meiosis. This finding axes tend to separate without obvious loss of cohesin in chromatin reveals that CO interference can occur along a single pair of sisters loops (reviewed in ref. 48). In accord with such possibilities, SC as well as along a pair of homologs, each comprising a pair of formation between sisters has been observed by EM in diploid and sisters. Flexibility of the interference process with respect to sub- haploid budding yeasts defective for Pds5 with the distance be- strate is also shown by the fact that in budding yeast, interference tween sisters approximately the same as between homologs (53) occurs along pairs of homologous unreplicated chromatids (38). and, segmentally in mouse lacking cohesin Rec8 (54). Alterna- (iii) Furthermore, no broken chromosomes are observed at either tively, or in addition, SC installation may be facilitated by local diplotene or onset of anaphase I, implying efficient DSB repair. separation of sisters at recombination sites as observed in Sordaria There is also no evidence that DSBs frequently interact with in absence of Rec8 (26). Also, presence of a particular set of re- a nonhomologous chromosome. A few Hei10 T3-like foci can be combination intermediates like Mer3 and Msh4 might be sufficient observed in regions of nonhomologous synapsis, perhaps repre- to adjust engaged axes to the correct SC distance. senting recombination between ectopic homologies. However, these interactions do not yield CO products: we do not detect Does Karyogamy Direct the Meiotic Interchromosomal Interaction chiasmata between nonhomologous chromosomes at diplotene. Program to Occur Between Homologs Rather than Between Sisters? There is precedent for occurrence of CO recombination be- The current study to our knowledge represents the first case in tween sisters from genetic and cytological studies in Drosophila which meiotic events have been examined in a karyogamy- and budding yeast (39, 32, 40; reviewed in ref. 41) and from defective situation. This is also to our knowledge the first instance physical studies that identify intersister double Holiday junctions in which, in haploid meiosis, the entire meiotic prophase program and thus presumptively intersister COs in budding yeast (42–46 of interchromosomal events, including both recombination and and references therein). Also, in Arabidopsis, Mlh1 foci, which synapsis, is clearly switched from interhomolog to intersister mark the sites of interfering COs (analogously to Sordaria Hei10) mode. What could be the underlying basis for, and meaning of, and colocalize with Hei10, are observed on univalents of haploid this switch? meiosis and in a dmc1 mutant, which repairs DBSs almost exclu- One possibility is that the sexual cycle specifically detects the sively on sister chromatids (37). Finally, in budding yeast, DSBs presence or absence of karyogamy per se and adjusts the ensuing formed at hemizygous loci are repaired with the same efficiency program of meiotic recombination-related events appropriately. and timing as DSBs formed at homozygous loci (43), suggesting In accord with this possibility, a recent study of Candida lusitania that there is no intrinsic barrier to sister chromatid recombination. (55) reveals a fusion between the processes that regulate mating and meiosis. This might be expected in organisms like Sordariales GENETICS The Entire SC Formation Program Occurs Between Sister Chromatids with no stable diploid phase. Moreover, in accord with direct Instead of Between Homologs. In Sordaria diploid meiosis, SC communication between karyogamy and the meiotic program, in formation requires specific nucleation at sites of interhomolog Sordaria, both meiotic DNA replication and initiation of axial recombinational interactions (24, 27). Moreover, not only is SC chromosome structure occur in haploid nuclei before karyogamy formation nucleated at such sites but also Mer3/Msh4 foci shift (above). Two possibilities can be considered: (i) Intersister in- to an in-between-axes localization at exactly the time when SC is teraction could be the default option, with occurrence of kary- initiated (27). We show above that in haploid meiosis, SC for- ogamy sending positive signals directing a change to the mation is again dependent on DSB formation, implying that interhomolog mode; or (ii) interhomolog interaction could be recombinational interactions are again nucleating synapsis. Also, the default option, with absence of karyogamy sending signals Mer3 and Msh4 are present and thus likely play similar roles for that alter the program to intersister mode as a backup option. SC initiation in haploid meiosis as in the diploid case. The alternative possibility would be that, in absence of kary- Interestingly, in diploid meiosis, loading of SC central com- ogamy, the meiotic program initiates in the normal interhomolog ponents occurs only when homolog axes have approached one mode but then senses the absence of a homologous chromosome, another at a particular distance (200 nm), after which the SC irrespective of the karyogamy defect per se. Such sensing could itself links homolog axes at the evolutionarily conserved distance involve a specific regulatory feedback process or could simply be of 100 nm (24, 27). In haploid meiosis, both the homolog and thus a built-in time-dependent default mode (e.g., ref. 43). If the only the specific interhomolog distance are absent. Nonetheless, SC relevant feature is absence of a homolog partner, the same effi- appears to form regularly between sister-chromatid axes, to the cient progression of events between sisters seen in slp1Δ might be level of resolution of these studies. How sister separation arises, expected to occur when haploid meiosis arises in other ways. with concomitant positioning of sister axes at the appropriate However, this is not the case. In Arabidopsis and budding yeast, distance, is unclear. Interestingly, in slp1Δ, there is no dramatic both DSBs and intersister recombination occur efficiently, as separation of sister chromatids before SC installation, whereas, shown genetically in yeast and, in Arabidopsis, by Rad51 de- when SC has formed, sister chromatids are now separated, rather pendence of recombination and absence of either broken chro- than tightly conjoined as in diploid meiosis (above). Thus, sister mosomes or chiasmata between unrelated chromosomes (32, 34, separation arises concomitant with SC formation. 35, 37). Chiasmata are similarly absent in haploid meiosis of other

Vasnier et al. PNAS | Published online September 10, 2014 | E4021 Downloaded by guest on September 24, 2021 plants, except in rice where 0.25 chiasmata/cell are regularly allows all events to occur efficiently and to completion between formed, but in the majority, between two chromosomes with gene sisters rather than between homologs. Regardless of its basis, the duplications (50 and references therein). However, in contrast to mechanism for the observed partner choice change remains to be slp1Δ, none of the studied haploid meioses shows both regular elucidated, with alterations in sister cohesion and/or recombination SC formation and recombination (including CO interference) as components as the most obvious possibilities. observed in the present study. In haploid budding yeast, DSB turnover is delayed and exit from prophase is blocked, implying Materials and Methods activation of the “recombination/synapsis checkpoint” (32). In Strains, Cloning, and Transformation. S. macrospora WT is St-Ismier Fungal Arabidopsis, CO-correlated Mlh1 foci are highly reduced in num- Genetics Stock Center (FGSC) 4818. Sordaria SLP1 (SMAC_08081) gene was bers (1.6 vs. 9–10 expected; ref. 37). Furthermore, SC formation identified from the Sordaria genomic DNA sequence (21). slp1Δ null mutant occurs between nonhomologous chromosomes rather than be- was obtained by single step gene replacement: a hygromycin resistance tween sisters, extensively in budding yeast (34, 49) and in all cassette replaces the entire ORF. Additional information on plasmid con- studied haploid plants (50–52 and references therein), except in struction, mutant, and GFP tag is in SI Materials and Methods. Arabidopsis where no SCs are formed (37). Moreover, in these cases, as in cases where a homolog partner is absent in diploid Cytology. For cytological analysis, GFP and DAPI (0.5 mg/mL) signals were ob- meiosis, nonhomologous SC occurs late in pachytene, after the served, either on living material or after fixation in 4% (wt/vol) parafor- requirement for specific nucleation is relaxed (reviewed in ref. 48). maldehyde, with a Zeiss Axioplan microscope with a CCD Princeton camera as Indeed, in Sordaria slp1Δ, SC is first regularly polymerized only described (27). Staining was as follows: cells were incubated with 100 nM ER- between sisters and only later is it also seen between a few stretches Tracker Red (Molecular Probes) for 15 min at room temperature, and then of nonhomologous chromatids. washed with PBS two times for 15 min. Monoclonal anti-mouse tubulin DM1A Thus, regular, complete, timely, and efficient conversion of the (Abcam) at 1:1,200. Secondary antibody was FITC-conjugated polyvalent anti- entire recombination/synapsis program from an interhomolog mouse (Jackson) at 1:100. For both gamma analyses of interference, interfocus process to an intersister process is observed uniquely in the Sordaria distances were measured from the center of the first focus to the center of the karyogamy-defective case and not in haploid meiosis of budding next focus using public domain software ImageJ (SI Materials and Methods). yeast or plants. Organism-specific differences cannot be excluded ACKNOWLEDGMENTS. This work and C.V., E.E., A.d.M., S.T., and D.Z. were as the basis for these contrasting situations. Nonetheless, the cur- supported by grants from the Centre National de la Recherche Scientifique rent observations raise the possibility that absence of karyogamy (Unité Mixte de Recherche 8621) and by the National Institutes of Health per se triggers a basic change in the chromosomal program, which Grant GM044794 (to N.E.K.), which also supported N.E.K. and L.Z.

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