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Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens. Author summary: Exploring gene functions through chemical mutagenesis-driven genetic screens is pivotal, yet the cumbersome task of identifying causative mutations remains a bottleneck, limiting their high-throughput potential. In this investigation, we uncovered a non-uniform mutation pattern induced by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, highlighting the influence of proximate sequence context and chromatin status on mutation frequency. Leveraging these insights, we engineered a machine learning enhanced pipeline to construct a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are selectively enriched in genetic screens targeting specific phenotypes amid the backdrop of random mutations. Applying this mapping tool to Whole Genome Sequencing (WGS) data derived from genetic suppressors rescuing a C. elegans ciliary kinesin mutant, we achieved precise identification of causal mutations without resorting to the conventional generation of recombinant inbred lines. Our work not only advances understanding of mutation dynamics but also revitalizes the efficacy of forward genetic screens, contributing to the refinement of genetic exploration methodologies with implications for various organisms. [ABSTRACT FROM AUTHOR]

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies. GFP-tagged tubulins have been widely used to monitor and visualize tubulin isotypes, but this is known to interfere with their cellular functions. This study describes a functional labeling strategy, guided by AlphaFold2 structural predictions, to label endogenous tubulins across diverse species while preserving functionality. [ABSTRACT FROM AUTHOR]

Background Information: Microtubules serve as integral components in cellular operations such as cell division, intracellular trafficking, and cellular architecture. Composed of tubulin protein subunits, these hollow tubular structures have been increasingly elucidated through advanced cryo-electron microscopy (Cryo-EM), which has unveiled the presence of microtubule inner proteins (MIPs) within the microtubular lumen., Results: In the present investigation, we employ a synergistic approach incorporating high-pressure freezing, cryo-focused ion beam milling, and Cryo-electron tomography (Cryo-ET) to interrogate the in situ architecture of microtubules in Caenorhabditis elegans larvae. Our Cryo-ET assessments across neuronal cilia and diverse tissue types consistently demonstrate the formation of annular configurations within the microtubular lumen., Conclusions: In concert with recently characterized MIPs, our in situ observations within a living organism corroborate the hypothesis that intricate luminal assemblages exist within microtubule scaffolds. These findings necessitate further exploration into the molecular constituents and functional ramifications of these internal microtubular configurations in both cellular physiology and pathophysiology., (© 2024 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans . The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H + -adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates., Competing Interests: ZX, YC, ZZ, ZS, ZG, ZZ, LX, ZD, GO, WL No competing interests declared, (© 2023, Xie et al.)

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the Caenorhabditis elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms' movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND., Competing Interests: Competing interests statement:The authors declare no competing interest.