Woglar A et al. (2014) Chromosoma "Chromosome movement in meiosis I prophase of Caenorhabditis elegans."

Woglar A, Jantsch V

[Chromosoma, 2014]

Rapid chromosome movement during prophase of the first meiotic division has been observed in many organisms. It is generally concomitant with formation of the "meiotic chromosome bouquet," a special chromosome configuration in which one or both chromosome ends attach to the nuclear envelope and become concentrated within a limited area. The precise function of the chromosomal bouquet is still not fully understood. Chromosome mobility is implicated in homologous chromosome pairing, synaptonemal complex formation, recombination, and resolution of chromosome entanglements. The basic mechanistic module through which forces are exerted on chromosomes is widely conserved; however, phenotypic differences have been reported among various model organisms once movement is abrogated. Movements are transmitted to the chromosome ends by the nuclear membrane-bridging SUN/KASH complex and are dependent on cytoskeletal filaments and motor proteins located in the cytoplasm. Here we review the recent findings on chromosome mobility during meiosis in an animal model system: the Caenorhabditis elegans nematode.

Woglar A et al. (2022) PLoS Biol "Molecular architecture of the C. elegans centriole"

Jha K, Busso C, Pierron M, Schneider FZ, Woglar A, Gonczy P

[PLoS Biol, 2022]

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

Woglar A et al. (2018) Cell "Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination."

Villeneuve AM, Woglar A

[Cell, 2018]

Meiotic double-strand breaks (DSBs) are generated and repaired in a highly regulated manner to ensure formation of crossovers (COs) while also enabling efficient non-CO repair to restore genome integrity. We use structured-illumination microscopy to investigate the dynamic architecture of DSB repair complexes at meiotic recombination sites in relationship to the synaptonemal complex (SC). DSBs resected atboth ends are converted into inter-homolog repair intermediates harboring two populations of BLM helicase and RPA, flanking a single population of MutS. These intermediates accumulate until late pachytene, when repair proteins disappear from non-CO sites and CO-designated sites become enveloped by SC-central region proteins, acquire a second MutS population, and lose RPA. These and other data suggest that the SC may protect COintermediates from being dismantled inappropriately and promote CO maturation by generating a transient CO-specific repair compartment, thereby enabling differential timing and outcome of repair at CO and non-CO sites.

Woglar A et al. (2020) PLoS Biol "Quantitative cytogenetics reveals molecular stoichiometry and longitudinal organization of meiotic chromosome axes and loops."

Kohler S, Woglar A, Roelens B, Boettiger A, Villeneuve AM, Yamaya K

[PLoS Biol, 2020]

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence fluorescence in situ hybridization (FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

Woglar A et al. (2013) PLoS Genet "Matefin/SUN-1 phosphorylation is part of a surveillance mechanism to coordinate chromosome synapsis and recombination with meiotic progression and chromosome movement."

Habacher C, La Volpe A, Daryabeigi A, Jantsch V, Adamo A, Machacek T, Woglar A

[PLoS Genet, 2013]

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.

Yamaya, K. et al. (2019) International Worm Meeting "Investigating the Architecture of Meiotic Chromosome Pairing Centers."

Ramakrishnan, S., Woglar, A., Villeneuve, A., Yamaya, K.

[International Worm Meeting, 2019]

Pairing of homologous chromosomes is necessary for their faithful segregation at the meiosis I division. Failure to segregate chromosomes accurately during meiosis can result in embryonic lethality or severe developmental defects. In C. elegans, a region near the end of each chromosome known as the pairing center (PC) plays crucial roles in the pairing process, promoting local recognition and stabilization of pairing between homologs and licensing assembly of the synaptonemal complex. These functions require the activities of a family of zinc finger PC-binding proteins, which connect the chromosomes to the cytoskeletal motility apparatus through nuclear envelope-spanning protein complexes to mediate chromosome movements associated with the pairing process. However, it is not yet clear how homolog recognition is achieved and how the pairing centers facilitate this process. Understanding the physical organization of the pairing center could be important for shedding light on this question. To this end, we are using a combination of Hi-C sequencing-based methods and ORCA (Optical Reconstruction of Chromatin Architecture), a FISH-based imaging approach, to investigate how DNA is organized within the pairing centers and adjacent chromosome segments. Using a combination of these two strategies to analyze pairing center architecture, both during normal meiosis and in various meiotic mutants with pairing defects, we aim to gain a better understanding of how PCs may contribute to homolog recognition and pairing.

Pattabiraman D et al. (2017) PLoS Genet "Meiotic recombination modulates the structure and dynamics of the synaptonemal complex during C. elegans meiosis."

Woglar A, Pattabiraman D, Roelens B, Villeneuve AM

[PLoS Genet, 2017]

During meiotic prophase, a structure called the synaptonemal complex (SC) assembles at the interface between aligned pairs of homologous chromosomes, and crossover recombination events occur between their DNA molecules. Here we investigate the inter-relationships between these two hallmark features of the meiotic program in the nematode C. elegans, revealing dynamic properties of the SC that are modulated by recombination. We demonstrate that the SC incorporates new subunits and switches from a more highly dynamic/labile state to a more stable state as germ cells progress through the pachytene stage of meiotic prophase. We further show that the more dynamic state of the SC is prolonged in mutants where meiotic recombination is impaired. Moreover, in meiotic mutants where recombination intermediates are present in limiting numbers, SC central region subunits become preferentially stabilized on the subset of chromosome pairs that harbor a site where pro-crossover factors COSA-1 and MutS are concentrated. Polo-like kinase PLK-2 becomes preferentially localized to the SCs of chromosome pairs harboring recombination sites prior to the enrichment of SC central region proteins on such chromosomes, and PLK-2 is required for this enrichment to occur. Further, late pachytene nuclei in a plk-2 mutant exhibit the more highly dynamic SC state. Together our data demonstrate that crossover recombination events elicit chromosome-autonomous stabilizing effects on the SC and implicate PLK-2 in this process. We discuss how this recombination-triggered modulation of SC state might contribute to regulatory mechanisms that operate during meiosis to ensure the formation of crossovers while at the same time limiting their numbers.

Labella S et al. (2011) Dev Cell "Polo kinases establish links between meiotic chromosomes and cytoskeletal forces essential for homolog pairing."

Woglar A, Labella S, Zetka M, Jantsch V

[Dev Cell, 2011]

During meiosis, chromosomes must find and align with their homologous partners. SUN and KASH-domain protein pairs play a conserved role by establishing transient linkages between chromosome ends and cytoskeletal forces across the intact nuclear envelope (NE). In C.elegans, a pairing center (PC) on each chromosome mediates homolog pairing and linkage to the microtubule network. We report that the polo kinases PLK-1 and PLK-2 are targeted to the PC by ZIM/HIM-8-pairing proteins. Loss of plk-2 inhibits chromosome pairing and licenses synapsis between nonhomologous chromosomes, indicating that PLK-2 is required for PC-mediated interhomolog interactions. plk-2 is also required for meiosis-specific phosphorylation of SUN-1 and establishment of dynamic SUN/KASH (SUN-1/ZYG-12) modules that promote homolog pairing. Our results provide key insights into the regulation of homolog pairing and reveal that targeting of polo-like kinases to the NE by meiotic chromosomes establishes the conserved linkages to cytoskeletal forces needed for homology assessment.

Machovina, T. et al. (2013) International Worm Meeting "Evidence for a meiotic crossover surveillance system."

Jantsch, V., McGovern, O., Paouneskou, D., Machovina, T., Yanowitz, J., Woglar, A.

[International Worm Meeting, 2013]

During meiosis, each chromosome must receive a crossover (CO) to ensure its proper segregation into developing gametes or risk producing aneuploid progeny. Due to the critical importance of attaining this obligate crossover, it has long been speculated that a system exists to monitors that a CO has been made on each chromosome. Nevertheless, experimental support for such a system has been lacking, in part because most meiotic defects affect all chromosomes, and not one or two. Over the last several years, we have been studying the him-5 and xnd-1, genes that are required to ensure that a meiotic double strand break (DSB) is made on the X chromosome. Thus, in the mutants, CO frequency is dramatically reduced on the X. In both him-5 and xnd-1 mutants, the failure to receive the DSB on the X activates a delay in progression through early pachytene that alters SUN-1 phosphorylation and retains the chromosomes in a clustered morphology at the nuclear periphery. It also causes a premature dismantling of the synaptonemal complex (SC) on the X chromosome. Adding DSBs into the nucleus with gamma irradiation restores normal meiotic progression, SC morphology and crossover formation on the X, indicating that it is indeed the lack of a DSB which is responsible for the delay. In an effort to identify the signaling components responsible for this delay, we screened all the germline expressed kinases and phosphatases using RNAi knockdown. We show that components of the spindle assembly checkpoint have been co-opted for this meiotic checkpoint, and suppress the delay and SC phenotypes. We also have made double mutants with many of the known repair mutants and have shown that homolog engagement appears critical for checkpoint activation. Lastly, we show that the phenotypes we observe are not specific to defects in crossover on the X chromosome, but that defects in crossover formation on autosomes are all sufficient to activate this checkpoint. Thus, we believe that there exists a surveillance system that is monitoring crossover formation in a chromosome by chromosome specific manner.

Ramakrishnan, S. et al. (2017) International Worm Meeting "Analyzing DNA organization during meiotic chromosome pairing."

Woglar, A., Lasker, K., Villeneuve, A., Ramakrishnan, S.

[International Worm Meeting, 2017]

The ability of homologous chromosomes to locate, recognize and align with their correct pairing partners is a hallmark feature of meiosis, yet the nature of interactions between homologous DNA sequences is still poorly understood. In order to better understand the DNA organization within the chromosomal structures that we observe cytologically, we have developed and validated a robust experimental pipeline for Hi-C analysis of C. elegans meiotic prophase germ cell nuclei. Our overall goals are to understand: 1) how DNA is organized in the context of fully aligned homologs, and 2) how DNA is organized within chromosomes during the process of homolog pairing. Several features of C. elegans make it well-suited for using Hi-C technology to investigate chromosome organization during meiotic prophase, including a high abundance of germline nuclei in adult worms and robust tools for cytological validation of Hi-C data. To enrich for germ cell nuclei, we subject frozen worms to mild mechanical disruption followed by several filtering steps. Cytological analysis of the filtered nuclei shows that over 90% of the nuclei in our sample are germ cell nuclei; moreover, 80% of the nuclei are in meiotic prophase. Following crosslinking, nuclear lysis, restriction digestion, DNA ligation, junction capturing and high-throughput sequencing, computational analysis of the data yields Hi-C heatmaps of DNA interactions showing characteristic signatures of individual chromosomes. We were able to identify the chromosomal fusion points in worms carrying the two-chromosome fusion mnT12(1V;X), and we further observed that DNA interactions between the fused chromosomes spread far beyond the fusion points especially on the X-chromosomes. We also identified the translocation breakpoints in the reciprocal translocation nT1(IV;V), and our analysis further revealed additional cryptic chromosomal rearrangements in these balancer chromosomes. Moreover, we detected robust interactions between hetero-synapsed segments of chromosomes IV and V in worms heterozygous for nT1(IV;V), providing strong proof-of principle that our experimental pipeline is indeed capable of detecting chromosomal interactions that depend on meiotic synapsis. These experiments also provided evidence of synaptic adjustment, i.e. the ability to achieve apparent lengthwise synapsis between chromosomes with different lengths. We are currently comparing chromosomal organization between meiotic germ cells and tumorous mitotic germ cells. Finally, we are investigating interactions between homologous chromosomes using worms that are heterozygous for two distinct haplotypes.