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Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1) is a rare, autosomal recessive disorder caused by mutations in the NFU1 gene. NFU1 is responsible for delivery of iron-sulfur clusters (ISCs) to recipient proteins which require these metallic cofactors for their function. Pathogenic variants of NFU1 lead to dysfunction of its target proteins within mitochondria. To date, 20 NFU1 variants have been reported and the unique contributions of each variant to MMDS1 pathogenesis is unknown. Given that over half of MMDS1 individuals are compound heterozygous for different NFU1 variants, it is valuable to investigate individual variants in an isogenic background. In order to understand the shared and unique phenotypes of NFU1 variants, we used CRISPR/Cas9 gene editing to recreate exact patient variants of NFU1 in the orthologous gene, nfu-1 (formerly lpd-8), in C. elegans. Five mutant C. elegans alleles focused on the presumptive iron-sulfur cluster interaction domain were generated and analyzed for mitochondrial phenotypes including respiratory dysfunction and oxidative stress. Phenotypes were variable between the mutant nfu-1 alleles and generally presented as an allelic series indicating that not all variants have lost complete function. Furthermore, reactive iron within mitochondria was evident in some, but not all, nfu-1 mutants indicating that iron dyshomeostasis may contribute to disease pathogenesis in some MMDS1 individuals. Author summary: Functional mitochondria are essential to life in eukaryotes, but they can be perterbured by inherent dysfunction of important proteins or stressors. Mitochondrial dysfunction is the root cause of dozens of diseases many of which involve complex phenotypes. One such disease is Multiple Mitochondrial Dysfunctions Syndrome 1, a pediatric-fatal disease that is poorly understood in part due to the lack of clarity about how mutations in the causative gene, NFU1, affect protein function and phenotype development and severity. Here we employ the power of CRISPR/Cas9 gene editing in the small nematode Caenorhabditis elegans to recreate five patient-specific mutations known to cause Multiple Mitochondrial Dysfunctions Syndrome 1. We are able to analyze each of these mutations individually, evaluate how mitochondrial dysfunction differs between them, and whether or not the phenotypes can be improved. We find that there are meaningful differences between each mutation which not only effects the types of stress that develop, but also the ability to rescue deleterious phenotypes. This work thus provides insight into disease pathogensis and establishes a foundation for potential future therapeutic intervention. [ABSTRACT FROM AUTHOR]

Examines metaphase to anaphase transition-defective mutants in the nematode Caenorhabditis elegans. Failure to form pronuclei; Persistence of phosphohistone H3 and MPM-2 antibody staining; Perturbation of spermatocyte meiosis; Gene emb-27's encoding of the C. elegans CDC16 ortholog.

Topoisomerase II alleviates DNA entanglements that are generated during mitotic DNA replication, transcription, and sister chromatid separation. In contrast to mitosis, meiosis has two rounds of chromosome segregation following one round of DNA replication. In meiosis II, sister chromatids segregate from each other, similar to mitosis. Meiosis I, on the other hand, segregates homologs, which requires pairing, synapsis, and recombination. The exact role that topoisomerase II plays during meiosis is unknown. In a screen reexamining Caenorhabditis elegans legacy mutants isolated 30 years ago, we identified a novel allele of the gene encoding topoisomerase II, top-2(it7). In this study, we demonstrate that top-2(it7) males produce dead embryos, even when fertilizing wild-type oocytes. Characterization of early embryonic events indicates that fertilization is successful and sperm components are transmitted to the embryo. However, sperm chromatin is not detected in these fertilized embryos. Examination of top-2(it7) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during anaphase I of meiosis, resulting in anucleate sperm. top-2(it7) chromosome-segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not dependent on SPO-11-induced double-strand DNA breaks. Finally, we show that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7) mutants. [ABSTRACT FROM AUTHOR]

In the nematode Caenorhabditis elegans, temperature-sensitive mutants of emb-1 arrest as one-cell embryos in metaphase of meiosis I in a manner that is indistinguishable from embryos that have been depleted of known subunits of the anaphase-promoting complex or cyclosome (APC/C). Here we show that the emb-1 phenotype is enhanced in double mutant combinations with known APC/C subunits and suppressed in double mutant combinations with known APC/C suppressors. In addition to its meiotic function, emb-1 is required for mitotic proliferation of the germline. These studies reveal that emb-1 encodes K10D2.4, a homolog of the small, recently discovered APC/C subunit, APC16. [ABSTRACT FROM AUTHOR]

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activatorfzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-l, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis. [ABSTRACT FROM AUTHOR]