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, how- ever, 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: [email protected], 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.