[
International Worm Meeting,
2021]
Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. Dominant mutations in other channel components induce the necrosis of various neurons. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. We constructed the GCaMP5 Ca2+ reporter that is specifically expressed in touch neurons and and the MFG-E8::mCherry reporter for PS on cell surfaces. After monitoring the cytoplasmic Ca2+ signal simultaneously with the PS signal on the surfaces of touch neurons in real time during embryonic development, we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of two Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, our observations suggest that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F. Our work has revealed a novel Ca2+-triggered PS exposure mechanism active in necrotic neurons, a mechanism different from the caspase-activated PS exposure mechanism employed by apoptotic cells. We propose that this mechanism might be conserved in other organisms including mammals.
Chiao, Lucia, Wang, Ying, Gao, Lidan, Liu, Xianghua, He, Henry, Yao, Tianyou, Zhou, Zheng, Pena-Ramos, Omar
[
International Worm Meeting,
2021]
The autophagy pathway generates autophagosomes, double-membrane structures responsible for degrading protein aggregates, intracellular organelles, and other cellular components. This intracellular trafficking process is essential for the cell stress response, recycling the degraded material for use in different cellular processes. During C. elegans embryonic development, 113 somatic cells undergo apoptosis; these cells are then swiftly internalized by neighboring engulfing cells through phagocytosis, generating phagosomes. The newly formed phagosome then enters a maturation process during which acidic vesicles are fused to the phagosome promoting its degradation. Components of the autophagy pathway have previously been linked to phagosome maturation in mammalian cells in a process called LC3-associated phagocytosis (LAP). The autophagy machinery generates a single membrane-vesicle to conjugate LC3 directly onto the phagosome membrane promoting phagosome-lysosome fusion. Using live imaging on developing C. elegans embryos, we discovered that LC3-labeled puncta generated in engulfing cells are recruited to the surface of phagosomes and subsequently fuse to them, depositing their content into the phagosomal lumen. The observed fusion pattern suggests that the puncta are of a double-membrane nature, indicative of double-membranous autophagosomes and not single-membrane LAP vesicles. We also observed that these autophagosomes form two distinct populations, those that are labeled with LGG-1 and those marked with LGG-2, both homologs of yeast Atg8 and mammalian LC3. Lacking either group of autophagosomes significantly delay the degradation of apoptotic cells, indicating that both LGG-1 and LGG-2 function in the clearance of apoptotic cells. Finally, we identified that a signaling pathway with a previously known role in phagosome-lysosome fusion also facilitates the integration of autophagosomes to phagosomes. Our findings have collectively added autophagosomes to the list of intracellular organelles involved in phagosome maturation. Furthermore, we have demonstrated that in C. elegans, it is autophagosomes, not LAP vesicles, that facilitate phagosome maturation, and we have revealed a novel function of the autophagy pathway in the clearance of apoptotic cells.
Chitrakar, Rojin, Stevens, Lewis, Baugh, L. Ryan, Moya, Nicolas D., Walhout, Marian, Tanny, Robyn E., Dekker, Job, Na, Huimin, Andersen, Erik C.
[
International Worm Meeting,
2021]
Decades of research have led to the development of comprehensive genome resources that have been essential to study the Caenorhabditis elegans species. In parallel, the emergence of Caenorhabditis briggsae as a model system has been useful to make interspecies comparisons. Despite the importance of C. briggsae as a model, its genome resources have not been developed to the same extent as C. elegans. The current genome of C. briggsae reference strain AF16 contains thousands of unresolved gaps and numerous mis-assemblies. Because of these issues, C. briggsae gene models remain incomplete and have numerous structural errors in protein-coding genes. We sought to exploit the latest sequencing technologies and computational tools to provide the highest quality C. briggsae genome resources to date. First, we generated high-quality genome assemblies for two strains of C. briggsae: QX1410 (a "tropical" strain isolated in Saint Lucia that is closely related to AF16) and VX34 (a divergent strain isolated in China). These genome assemblies incorporate high coverage Oxford Nanopore PromethION long reads and chromosome conformation capture (Hi-C) data. Second, we genotyped 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34. Using these data, we produced a high-quality recombination map that validated the placement of scaffolds after genome assembly. Third, we sequenced the transcriptomes of each strain to high coverage using Pacific Biosciences SMRT and Illumina platforms. We developed a computational pipeline that leverages long and short RNA reads to generate a genome annotation for each strain. These new genome annotations have improved accuracy and completeness relative to the AF16 genome. Fourth, our research group currently maintains over 1,600 C. briggsae wild strains, comprising the largest collection worldwide. We sequenced the genomes of this entire collection to high coverage using the Illumina platform. We mapped the sequences of all wild strains to the QX1410 genome to call single nucleotide variants across the entire population. These high-quality genome resources will facilitate new avenues of research, including quantitative and population genetic studies of C. briggsae, and enable informative comparisons with C. elegans.