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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]

Autophagy is a catabolic process through which damaged organelles and protein aggregates are delivered to lysosomes for degradation. Autophagy genes are reported to promote exposure of "eat me" signals on the surface of apoptotic cells, but whether they function in engulfing cells is not clear. Recently, we found that the autophagy mutants atg-18 and epg-5 are defective in removing apoptotic cells derived from the C. elegans Q neuroblast, a phenotype that can be fully rescued by expression of ATG-18 and EPG-5 in the engulfing cell. Loss of ATG-18 or EPG-5 does not affect cell corpse engulfment but causes defects in phagosomal recruitment of RAB-5 and RAB-7 and formation of phagolysosomes. EPG-5, ATG-18 and LGG-1 are sequentially recruited to phagosomes, suggesting that they function at different steps of phagosomal maturation. Our studies indicate that autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell.

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin alpha subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3-kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein-dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a 'canonical' IFT motor, whereas OSM-3 is an 'accessory' IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

Author(s): Guangshuo Ou [1]; Oliver E. Blacque [2]; Joshua J. Snow [1]; Michel R. Leroux [2]; Jonathan M. Scholey (corresponding author) [1] Cilia have diverse roles in motility and sensory [...]