SC Pichler et al. (1999) International C. elegans Meeting "ooc-3, a novel gene required for rotation of the centrosome-nucleus complex in P1"

R Schnabel, H Schnabel, AA Hyman, SC Pichler, P Goenczy

[International C. elegans Meeting, 1999]

During the asymmetric cell division of the P 1 blastomere, proper positioning of the mitotic spindle along the antero-posterior axis relies on a 90 deg rotation of the centrosome-nucleus complex. This P 1 rotation depends on astral microtubules, actin and a site in the anterior cortex enriched in actin, dynactin and the actin-capping protein (CP). During rotation, astral microtubules emanating from one centrosome attach at the cortical site und subsequent microtubule shortening places this centrosome close to the cortical site leading to a 90 deg rotation of the centrosome-nucleus complex. To elucidate the mechanisms underlying such rotation we searched for mutants defective in P 1 rotation by screening an extensive collection of maternal-effect embryonic lethal mutations on chromosome II by time-lapse DIC videomicroscopy. We identified one mutant, t1308 in which rotation in P 1 is defective in 100% of the embryos and in P 0 in 50% of the embryos. In addition, this mutation causes non-penetrant meiotic and mitotic defects leading to an abnormal number of female pronuclei in P 1 and multiple nuclei in both AB and P 1 . By investigating the localisation of the polarity markers PAR-3 and the P-granules we found that the overall embryonic polarity is properly established in ooc-3 mutant embryos. This suggests that ooc-3 is a gene more specifically required for rotation, possibly for the organization of the cytoskeleton. Tubulin staining, however, showed that astral microtubules are unaffected in mutant embryos. We are currently investigating whether the anterior cortical site is properly assembled by performing actin-, CP- and dynactin stainings. Complementation analysis revealed that ooc-3 is allelic to the previously identified locus ooc-3 (mn241) . ooc-3 had been mapped by deficiency between unc-53 and age-1 . We have identified a candidate gene by rescuing the phenotype with entire cosmids and cosmid fragments. RNAi and sequencing of the two mutant alleles confirmed the candidate gene to be ooc-3 . Sequence analysis revealed that ooc-3 is a novel gene coding for a putative transmembrane protein. We are currently preparing antibodies to investigate the localisation of OOC-3.

Pichler SC et al. (1998) European Worm Meeting "ooc-3 is required for rotation of the centrosome-nucleus complex in P1"

Schnabel H, Pichler SC, Gonczy P, Schnabel R, Hyman T

[European Worm Meeting, 1998]

One of the mechanisms underlying the generation of cell diversity during the development of an organism are asymmetric cell divisions. Asymmetric cell divisions depend on two steps: first, polarization of fate determining factors, and second, positioning of the mitotic spindle along a previously established axis of polarity. Such a positioning of the mitotic spindle allows then the placement of the cleavage furrow perpendicular to the axis of polarity which is a prerequisite for a cell to divide into two daughter cells that differ in their fate. During the asymmetric divisions in the P-lineage of the C.elegans embryo, proper positioning of the mitotic spindle along the antero-posterior (A-P) axis relies on a 90o rotation of the centrosome-nucleus complex. In P1, the centrosome-nucleus complex is initially aligned transverse to the A-P axis, but then aligns on the A-P axis just prior to mitosis. During this rotation, astral microtubules emanating from both centrosomes are attached to a site in the anterior cortex which shows an enrichment in actin and the actin-capping protein (CP). To elucidate the mechanisms underlying rotation of the centrosome-nucleus complex, we searched for mutants defective in rotation in P1 by screening an extensive collection of maternal-effect embryonic lethal mutations on chromosome II by time-lapse DIC videomicroscopy. We identified one locus, t1308, in which the centrosome-nucleus complex fails to rotate in P1 (10/10 embryos) and in P0 (5/10 embryos). Besides these rotation defects, this mutant has additional non-penetrant phenotypes such as an abnormal number of pronuclei (6/9) and karyomeres in both the AB and the P1 daughter cells (2/9).To better understand the function of ooc-3 during rotation we are currently investigating the localisation of segregation markers such as the P-granules, and markers of the A-P polarity such as the PAR proteins. These experiments will reveal whether ooc-3 is implicated in overall embryonic polarity or whether it is a more specific component of the rotation machinery. Preliminary genetic interaction experiments of ooc-3 with par-3 suggest that ooc-3 is indeed a gene specifically required for the rotation event. If this were true, one hypothesis might be that ooc-3 is involved in the organisation of the cytoskeleton. To test this hypothesis, we are currently investigating the nature of astral microtubules and the anterior cortical site in mutant embryos by tubulin-, actin- and CP stainings. Complementation analysis showed that t1308 is allelic to a previously identified locus called ooc-3 but not to mel-19 which has been suggested to be allelic to ooc-3. ooc-3 has previously been mapped by deficiency between unc-53 and age-1. Using germline transformation we rescued the ooc-3 maternal-effect lethality with a single cosmid residing in this region. We are currently trying to narrow down the locus to a single gene using subcloning and RNAi techniques.

Gordon, S. et al. (2019) International Worm Meeting "Novel separation-of-function mutants of the synaptonemal complex specifically disrupt alignment of homologous chromosomes during meiotic prophase."

Rog, O., Shea, R., Kursel, L., Xu, K., Gordon, S.

[International Worm Meeting, 2019]

During meiosis, chromosomes pair and align with their homologous partner, and exchange genetic information in a non-conservative manner via crossovers. Both of these processes require a structurally conserved proteinaceous interface called the Synaptonemal Complex (SC) that assembles lengthwise between homologous chromosomes. Despite its ordered appearance in electron micrographs, the SC exhibits liquid-like characteristics that are conserved in yeast, flies and worms. This raised the possibility that liquid-liquid phase-separated SC compartments propagate signals that distribute crossovers and monitor their formation. How does the SC mediate crossover signaling and alignment of homologous chromosomes remains unknown partly because many SC mutants result in complete loss of the SC. We took an evolution-guided approach to subtly perturb specific SC functions. We identified a conserved five amino acid domain in SYP-1 (one of the four SC proteins in C. elegans) and used Deep Mutational Scanning to systematically mutagenize each amino acid in this domain. From this library of mutants, we identified multiple separation-of-function mutations that perturb at least one function of the SC. We will present our progress in characterizing two mutants that support some of the crossover-related functions of the SC but are unable to fully synapse homologous chromosomes. Unlike wildtype SC, which only associates with paired homologous chromosomes aligned along their lengths, these mutants display aberrant SC interactions with paired, unpaired and partially aligned chromosomes. Interestingly, these mutations differentially lessen the tendency of SC subunits to associate with one another. We propose a model that links the ability of SC subunits to self-associate with the SC's ability to enforce alignment of paired chromosomes and to propagate signals inside a liquid-liquid phase-separated compartment.

Pattabiraman, Divya et al. (2015) International Worm Meeting "Meiotic Progression and Recombination Modulate the Dynamic State of the Synaptonemal Complex."

Pattabiraman, Divya, Roelens, Baptiste, Presler, Marc, Villeneuve, Anne, Chen, Grace

[International Worm Meeting, 2015]

The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles at the interface between aligned homologous chromosome pairs during meiotic prophase. The SC is integral to most aspects of the meiotic program, with demonstrated roles in stabilization of homolog pairing, in promoting the formation of crossovers, in enabling meiotic progression and in crossover interference. Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent studies suggest that the SC may be much more dynamic than previously appreciated. We have investigated the dynamics of the SC structure by using FRAP (Fluorescence Recovery After Photobleaching) analysis to visualize the exchange/turnover of SC components within fully assembled SCs. This was done in pachytene nuclei of intact worms expressing a GFP-tagged version of SYP-3, a component of the SC central region.This approach has revealed a previously hidden dynamics of the SC, demonstrating that the SC is a malleable structure rather than a fixed scaffold. We observe significant recovery of GFP::SYP-3 by 10-15 minutes post-bleach, and recovery approaches a maximum by 1-1.5 h. Although this recovery time scale is slower than those observed for nucleoplasmic proteins (seconds) or spindle microtubules in the first mitotic spindle (a few minutes), it is short relative to the length of the pachytene stage when full-length SCs are present (18-24 h). Thus, the observed dynamics indicate that the SC has the potential to undergo substantial remodeling and reorganization in response to ongoing events of meiotic prophase. Based on our analyses, we can draw several conclusions: 1) Recovery occurs primarily by redistribution/exchange of existing SC subunits within the nucleus, and minimally by import of new SC subunits; 2) During wild-type meiosis, the extent of SC dynamics decreases during normal progression through the pachytene stage; and 3) In crossover defective mutants (spo-11, zhp-3 and cosa-1), the more dynamic state of the SC characteristic of early pachytene, is prolonged, suggesting a role for recombination in regulating SC dynamics. These conclusions have been corroborated by experiments conducted using two very different imaging systems and two different approaches for data analysis. Together our findings indicate that the dynamic state of the SC structure is an actively regulated feature of the meiotic program. Moreover, we propose that SC dynamics is likely modulated by the same network that regulates meiotic progression in response to monitoring the status of recombination intermediates.

Pattabiraman, Divya et al. (2013) International Worm Meeting "Visualizing dynamics of meiotic prophase chromosome structures."

Chen, Grace, Pattabiraman, Divya, Villeneuve, Anne, Presler, Marc, Roelens, Baptiste

[International Worm Meeting, 2013]

The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles at the interface between aligned homologous chromosome pairs during meiotic prophase. Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent studies suggest that the SC may be much more dynamic than previously appreciated. We are investigating the dynamics of the SC structure by using FRAP (Fluorescence Recovery After Photobleaching) to visualize the exchange of SC components within fully assembled SCs in pachytene nuclei of intact worms expressing a functional GFP-tagged version of SYP-3, a component of the SC central region. This approach has revealed a previously hidden dynamics of the SC, demonstrating that the SC is a malleable structure rather than a fixed scaffold. We observe half-maximal FRAP for GFP::SYP-3 by 10-15 minutes post-bleach, and recovery approaches a maximum by 1-1.5 h. Although this recovery time scale is slower than those observed for nucleoplasmic proteins (seconds) or microtubules in the first mitotic spindle (a few minutes), it is short relative to the length of the pachytene stage when full-length SCs are present (18-24 h). Thus, the observed dynamics indicate that the SC has the potential to undergo substantial remodeling and reorganization in response to different ongoing events of meiotic prophase. Based on our analyses, we can draw several conclusions: 1)Recovery occurs primarily by redistribution/exchange of SC subunits within the nucleus, and minimally by import of new SC subunits 2)The extent of SC dynamics decreases during pachytene progression and 3)SC dynamics characteristic of wild-type early pachytene were observed in mid and late pachytene nuclei in a zhp-3 mutant, wherein chromatin and nuclear envelope features that are normally limited to early pachytene are prolonged. Together our data suggest that the dynamic state of the SC structure is an actively regulated feature of the meiotic program.

Bilgir, Ceyda et al. (2009) International Worm Meeting "Regulation of the synptonemal complex assembly during meiosis in C. elegans ."

Bilgir, Ceyda, Dombecki, Carolyn, Nabeshima, Kentaro, Villeneuve, Anne, Skodack, Joshua

[International Worm Meeting, 2009]

During sexual reproduction, haploid gametes are generated by a specialized cell division program (meiosis) in which homologous chromosomes pair and their association is stabilized by a protein structure, the synaptonemal complex (SC). SC formation between homologous chromosomes is essential for reciprocal recombination that is required for faithful segregation of homologous chromosomes. In order to identify the genes responsible for proper homologous chromosome association during C. elegans meiosis, we have been conducting a genome-wide screen using: 1) a system for visualizing homologous pairing in live animals, and 2) a feeding RNAi library to knock-down gene function. This screen has allowed us to identify genes that were not previously known to function in pairing and/or synapsis. Here we report novel aspects of SC assembly discovered through the analyses of some of the newly identified genes. First, we found that pgl-1, a gene encoding a protein associated with P granules, was transiently required for the initiation of SC assembly after the shift to a high temperature. We found that the initiation of SC assembly was heat sensitive even in the wild type, which was further sensitized in pgl-1 mutant, suggesting that PGL-1 is regulating a factor(s) for the initiation of SC assembly. Second, we found that gld-2 and gld-3, components of a cytoplasmic poly(A) polymerase that is known to function in a mitosis-meiosis switch, were required for prompt SC assembly. In these mutants, SC formation was significantly delayed, and non-pairing center (PC) ends of chromosomes rarely showed homologous association. Interestingly, PCs showed stable homologous association during meiosis in these mutants, indicating SC was assembled at least at pairing centers. This observation suggests that initiation and progression of SC assembly are separable and gld-2 and gld-3 are involved in the latter process. Third, we found that mrg-1, a chromo-domain protein required for germ line development and X chromosome silencing in the germline, was required for proper synapsis establishment. The mrg-1 mutants showed partial synapsis and pairing defect at the non-PC ends (but not at the PC ends) of chromosomes. SC assembly initiated and progressed with the apparently normal timing and kinetics. This finding suggests that there is a mechanism to establish and/or maintain SC that is dedicated to non-PC region of chromosomes, possibly through chromatin modification. We are currently investigating how this mechanism is operating using cytological and genetic analyses.

Sarit Smolikov et al. (2007) International Worm Meeting "The uncoupling between chromosome synapsis and double-strand break formation is mediated by CRA-1 during meiosis in C. elegans."

Monica Colaiacovo, Sarit Smolikov

[International Worm Meeting, 2007]

Sexually reproducing organisms reduce their chromosome number by half and generate haploid gametes through the specialized cell division process known as meiosis. This involves segregation of homologous chromosomes away from each other at meiosis I and segregation of sister chromatids at meiosis II. At prophase of meiosis I, homologous chromosomes align and a protein complex known as the synaptonemal complex (SC) assembles between them stabilizing their interactions. The SC consists of a pair of lateral elements assembled along the axes of the homologs, interconnected by proteins forming the central region of this structure. The SC is required for crossover formation, leading to physical connections between the homologs (chiasmata), and therefore is crucial for faithful chromosomal inheritance. Although the SC is required for completion of recombination in yeast, flies, worms, plants and mammals, SC assembly is double-strand break (DSB)-independent in worms and flies. However, through the characterization of a novel meiotic protein, CRA-1 (central region assembly 1), we present evidence that this aspect of C. elegans meiosis has more in common with other organisms, than previously expected. cra-1 encodes for a protein which is highly conserved from worms to humans. A null allele of cra-1 results in chromosome segregation defects as evident by an increase in embryonic lethality. Early aspects of entrance into meiosis such as chromosomes acquiring a clustered organization, SC axis morphogenesis and initiation of chromosomal pairing are mostly intact. In contrast, later steps of meiotic prophase are largely affected in cra-1 mutants. Chromosomes persist in a clustered organization longer than in wild-type and central region components of the SC localize along both synapsed and unsynapsed chromosomes. As a result, homologous pairing interactions are not stabilized, DSB repair is severely delayed and achiasmate chromosomes are formed later in prophase. Interestingly, these defects are more commonly observed in autosomes, suggesting that the X chromosome is less dependent on CRA-1 function. Unexpectedly, in cra-1; spo-11 double mutants where the formation of programmed meiotic DSBs is abolished, SC formation is now abrogated. Extensive SC formation in this double mutant, however, can be rescued by inducing exogenous double-strand breaks via gamma-irradiation. This suggests that in the absence of CRA-1, SC assembly is DSB-dependent. This is the first time that a direct dependence on DSB formation for SC assembly is demonstrated for this organism, suggesting the presence of an evolutionarily conserved process which had previously gone unnoticed.

Monica P Colaiacovo et al. (2002) West Coast Worm Meeting "Relationship between the synaptonemal complex and progression of meiotic recombination in C elegans"

JoAnne Engebrecht, Adriana La Volpe, Cinzia Rinaldo, Amy J MacQueen, Anne M Villeneuve, Monica P Colaiacovo

[West Coast Worm Meeting, 2002]

Meiosis is the specialized cell division process by which diploid organisms generate haploid gametes. In preparation for the meiosis I division, homologous chromosomes recognize each other, pair, and undergo recombination. During this process a proteinaceous structure known as the synaptonemal complex (SC) forms at the interface between paired and aligned homologs. Crossing over is completed in the context of the SC, leading to the formation of chiasmata that connect the homologs and allow them to orient toward opposite poles of the meiosis I spindle. Despite the ubiquitous presence of the SC structure from yeast to mammals, its function(s) in meiosis are poorly understood and a matter of much debate. In order to investigate proposed roles for the SC during meiotic prophase and to understand how its components interact in a macromolecular assembly we set out to identify genes involved in synapsis.

Hurlock, M. E. et al. (2019) International Worm Meeting "Identification of novel synaptonemal complex components in C. elegans."

Nizami, Z., Kim, Y., Hurlock, M. E., Turniansky, R., Haversat, J. M., Gall, J. G.

[International Worm Meeting, 2019]

Proper chromosome segregation during meiosis requires that homologous chromosomes pair and undergo crossover recombination. In most eukaryotes, crossover formation depends on the assembly of the synaptonemal complex (SC), a zipper-like protein structure that forms between two paired homologs. The SC in C. elegans is known to be comprised of four coiled-coil domain proteins, SYP-1, SYP-2, SYP-3, and SYP-4. However, it is not clear whether the entire set of SYP proteins have been discovered or how SYPs interact with each other to assemble into higher order structure. Here we report the identification of two new SC components, SYP-5 and SYP-6. Using super-resolution microscopy, we demonstrate that SYP-5 and SYP-6 are localized along the SC between the two chromosome axes. SYP-5 and SYP-6 require the other SYP proteins for their localization and form polycomplexes in the absence of chromosome axes. Interestingly, single mutants of syp-5 or syp-6 do not show strong defects in meiosis. However, synapsis and crossover formation completely fail in mutants that lack both SYP-5 and SYP-6, indicating that these two paralogous proteins play redundant roles in SC assembly. We further find that truncating conserved low complexity regions from the C-termini of SYP-5 and SYP-6 leads to aberrant synapsis and attenuation of crossover interference. Taken together, our findings establish SYP-5 and SYP-6 as bona fide components of the SC and will help advance our understanding of SC structure and function.

Alleva, Benjamin et al. (2017) International Worm Meeting "The CUL-4 E3 Ligase complex plays two roles in meiotic prophase I."

Alleva, Benjamin, Clausen, Sean, Smolikove, Sarit

[International Worm Meeting, 2017]

Meiosis is the specialized cellular division that starts from a diploid meiocyte leading to the formation of 4 haploid gametes. The first meiotic division uses DNA damage repair to pair homologous chromosomes for proper segregation. Once paired, the Synaptonemal Complex (SC), a tripartite proteinaceous structure, assembles along homologous chromosomes to provide pairing stabilization. Stabilization allows for recombination to fix the DNA damage and create crossovers between homologs. The SC disassembles leaving the crossover holding together homologs up to their segregation. Previously, our lab has shown that the CSN/COP-9 complex plays a crucial role in SC assembly. CSN/COP-9 has deneddylation activity which is specific to Cullin E3 ligases. E3 ligases are activated by the addition of NED-8 and deactivated by the release of NED-8. Mutants of the complex, as well as NED-8 knockdown, lead to SC polycomplex formation. Due to uncompleted synapsis, DNA damage accumulates in these nuclei leading to a decrease in overall crossovers. We have previously shown that the CUL-4 E3 ligase is the likely target of CSN/COP-9, but further investigation of the CUL-4 complex was lacking. Here we show that the CUL-4 complex plays a role in both SC assembly and DNA damage repair. Mutants of the CUL-4 complex have SC polycomplex formation throughout pachytene. These mutants also have defects in DNA damage repair as RAD-51 immunofluorescent staining shows high levels within nuclei. We also show that not all canonical components of the CUL-4 complex are required for SC assembly. Through SYP-1 immunofluorescent staining, we found that DDB-1, a protein that connects CUL-4 to an adapter protein, and GAD-1, the adapter protein, are both involved in SC assembly. Interestingly, the RING proteins, RBX-1 and RBX-2, docking sites for E2 conjugating enzymes, are not required for SC assembly. During Cullin E3 ligase cycling, CAND-1 binding prevents reactivation until it's released which allows for reactivation. We found that loss of CAND-1 had no effect on SC assembly, suggesting that cycling but not sequestering of the inactive complex is necessary for SC assembly. The function of the Cullin E3 ligases are to ubiquitinate targeted proteins either for degradation or other functions such as signaling. We show that in csn-5 mutants, there is an increase in the localization of LYS-48 ubiquitination to nuclei, the residue associated with degradation. Whereas in cul-4 mutants, there is a lower nuclear signal of LYS-48 as compared to csn-5 mutants. This suggests that over-ubiquitination of target proteins or over-self-ubiquitination of CUL-4 is occurring in these mutants. Altogether, we suggest that the CUL-4 E3 ligase complex functions in both SC assembly and DNA damage repair through its ubiquitination activity.