Biology subjects

Roelens, B.

Publications and source records attributed to Roelens, B..

6 recordsLinked to original sources

bioRxiv2026

PDZD-8 interacts with LGG-2/LC3 to promote autolysosome formation

LGG-2/LC3 and LGG-1/GABARAP are members of the Atg8 ubiquitin-like protein family, playing central roles in autophagy. Through a yeast two-hybrid screen, we identified the endoplasmic reticulum protein PDZD-8 as a novel interactor of the C. elegans autophagy protein LGG-2/LC3. We demonstrate that PDZD-8 binds specifically to LGG-2 and not LGG-1 via a canonical LC3-interacting region (LIR) and colocalizes with LGG-2-positive autophagosomes in vivo. Loss of PDZD-8 leads to a transient accumulation of enlarged LGG-2-positive autophagosomes that remain closely associated with lysosomes. Genetic disruption of core autophagy genes suppresses the pdzd-8 mutant phenotype, indicating that this phenotype is dependent on autophagy. Moreover, loss of rab-7, which blocks autophagosome-lysosome fusion, also suppresses the pdzd-8 phenotype, suggesting that the RAB-7-dependent fusion step is epistatic to PDZD-8 and that PDZD-8 acts downstream of autophagosome-lysosome maturation. Microscopy approaches (TEM, CLEM, and FIB-SEM) reveal an accumulation of fused autophagosome-lysosome structures, pointing to a transient block in autophagic flux in pdzd-8 mutants. Furthermore, PDZD-8 lacking either the SMP lipid-transfer domain or the LIR motif phenocopies the pdzd-8 null mutant, underscoring the importance of both domains. Our findings support a model in which PDZD-8, in conjunction with LGG-2/LC3, mediates ER-autophagosome/autolysosome contacts that are critical for subsequent autolysosome completion. Significance StatementAutophagy is a conserved cellular degradation pathway essential for maintaining homeostasis and responding to stress. Although the molecular machinery driving autophagosome formation and fusion with lysosomes is relatively well-characterized, the contribution of membrane contact sites to autophagic flux remains poorly understood. Here, we identify PDZD-8, an endoplasmic reticulum (ER) protein, as a novel interactor of the Caenorhabditis elegans LC3/Atg8 homolog LGG-2. We show that PDZD-8 regulates the completion of autolysosome maturation by bridging ER-autophagosome/autolysosome contacts. Loss of PDZD-8 results in the accumulation of enlarged autophagosomes adjacent to lysosomes and a transient block in autophagic flux, a phenotype that depends on the RAB-7-mediated tethering step. These findings reveal a previously unrecognized role for PDZD-8 in coordinating ER-autophagosome/autolysosome dynamics during autophagy.

cell biology↗

bioRxiv2021

C. elegans DSB-3 Reveals Conservation and Divergence among Protein Complexes Promoting Meiotic Double-Strand Breaks

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure for their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form meiotic crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2 and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have co-evolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery. Significance StatementFaithful inheritance of chromosomes during meiosis depends on the formation and repair of double-strand DNA breaks (DSBs), which are generated through the activity of a topoisomerase-like protein known as Spo11. Spo11 exhibits strong conservation throughout eukaryotes, presumably reflecting constraints imposed by its biochemical activity, but auxiliary proteins that collaborate with Spo11 to promote and regulate DSB formation are less well conserved. Here we investigate a cohort of proteins comprising a complex required for meiotic DSB formation in Caenorhabditis elegans, providing evidence for both conservation with and divergence from homologous complexes in other organisms. This work highlights the evolutionary malleability of protein complexes that serve essential, yet auxiliary, roles in fundamental biological processes that are central to reproduction.

genetics↗

bioRxiv2019

Quantitative Cytogenetics Reveals the Longitudinal Architecture of Meiotic Chromosome Axes

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 C. elegans. Analyses of WT chromosomes and de novo circular mini-chromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with two 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 three 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, immuno-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.

cell biology↗

bioRxiv2019

Spatial Regulation of Polo-like Kinase Activity during C. elegans Meiosis by the Nucleoplasmic HAL-2/HAL-3 Complex

Proper partitioning of homologous chromosomes during meiosis relies on the coordinated execution of multiple interconnected events: Homologs must locate, recognize and align with their correct pairing partners. Further, homolog pairing must be coupled to assembly of the synaptonemal complex (SC), a meiosis-specific tripartite structure that maintains stable associations between the axes of aligned homologs and regulates formation of crossovers between their DNA molecules to create linkages that enable their segregation. Here we identify HAL-3 (Homolog Alignment 3) as an important player in coordinating these key events during C. elegans meiosis. HAL-3 and the previously-identified HAL-2 are interacting and interdependent components of a protein complex that localizes to the nucleoplasm of germ cells. hal-3 (or hal-2) mutants exhibit multiple meiotic prophase defects including failure to establish homolog pairing, inappropriate loading of SC subunits onto unpaired chromosome axes, and premature loss of synapsis checkpoint protein PCH-2. Further, loss of hal function results in misregulation of the subcellular localization and activity of polo-like kinases (PLK-1 and PLK-2), which dynamically localize to different defined subnuclear sites during wild-type prophase progression to regulate distinct cellular events. Moreover, loss of PLK-2 activity partially restores tripartite SC structure in a hal mutant background, suggesting that the defect in pairwise SC assembly in hal mutants reflects inappropriate PLK activity. Together our data support a model in which the nucleoplasmic HAL-2/HAL-3 protein complex constrains both localization and activity of meiotic Polo-like kinases, thereby preventing premature interaction with stage-inappropriate targets.

genetics↗

bioRxiv2017

Interdependent and separable functions of C. elegans MRN-C complex members couple formation and repair of meiotic DSBs

Faithful inheritance of genetic information through sexual reproduction relies on the formation of crossovers between homologous chromosomes during meiosis, which in turn relies on the formation and repair of numerous double-strand DNA breaks (DSBs). As DSBs pose a potential threat to the genome, mechanisms that ensure timely and error-free DSB repair are crucial for successful meiosis. Here we identify NBS-1, the Caenorhabditis elegans ortholog of the NBS1 subunit of the conserved MRE11-RAD50-NBS1/Xrs2 (MRN) complex, as a key mediator of DSB repair via homologous recombination (HR) during meiosis. Loss of nbs-1 leads to: severely reduced loading of recombinase RAD-51, ssDNA binding protein RPA and pro-crossover factor COSA-1 during meiotic prophase progression; aggregated and fragmented chromosomes at the end of meiotic prophase; and 100% progeny lethality. These phenotypes reflect a role for NBS-1 in processing of meiotic DSBs for HR that is shared with its interacting partners MRE-11-RAD-50 and COM-1 (ortholog of Com1/Sae2/CtIP). Unexpectedly, in contrast to MRE-11 and RAD-50, NBS-1 is not required for meiotic DSB formation. Meiotic defects of the nbs-1 mutant are partially suppressed by abrogation of the non-homologous end-joining (NHEJ) pathway, indicating a role for NBS-1 in antagonizing NHEJ during meiosis. Our data further reveal that NBS-1 and COM-1 play distinct roles in promoting HR and antagonizing NHEJ. We propose a model in which different components of the MRN-C complex work together to couple meiotic DSB formation with efficient and timely engagement of HR, thereby ensuring crossover formation and restoration of genome integrity prior to the meiotic divisions.\n\nSignificance StatementDouble-strand breaks (DSBs) are deleterious DNA lesions, and impairment of the DSB repair machinery can lead to devastating diseases such as the Nijmegen Breakage Syndrome (NBS). During meiosis, DSBs represent a \"necessary evil\": they are required to promote formation of crossovers between homologous chromosomes. Crossovers in turn ensure correct chromosome inheritance during gamete formation, which is essential for viability and normal development of embryos. During meiosis, numerous DSBs are actively created, so meiotic cells must ensure that all breaks are properly repaired to ensure crossover formation and restore genomic integrity. Here we identify C. elegans NBS-1 as essential to properly process meiotic DSBs, both to promote crossover formation and antagonize an error-prone DSB repair pathway, thereby ensuring faithful chromosome inheritance.

genetics↗

bioRxiv2017

Meiotic recombination modulates the structure and dynamics of the synaptonemal complex during C. elegans meiosis

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{gamma} 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.

genetics↗