The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.

The C. elegans hermaphrodite vulva serves as a paradigm for understanding how signaling pathways control organ formation. Previous studies have shown that Wnt signaling plays important roles in vulval development. To understand the function and evolution of Wnt signaling in Caenorhabditis nematodes we focused on C. briggsae, a species that is substantially divergent from C. elegans in terms of the evolutionary time scale yet shares almost identical morphology. We isolated mutants in C. briggsae that display multiple pseudo-vulvae resulting from ectopic VPC induction. We cloned one of these loci and found that it encodes an Axin homolog, Cbr-PRY-1. Our genetic studies revealed that Cbr-pry-1 functions upstream of the canonical Wnt pathway components Cbr-bar-1 (beta-catenin) and Cbr-pop-1(tcf/lef) as well as the Hox target Cbr-lin-39 (Dfd/Scr). We further characterized the pry-1 vulval phenotype in C. briggsae and C. elegans using 8 cell fate markers, cell ablation, and genetic interaction approaches. Our results show that ectopically induced VPCs in pry-1 mutants adopt 2° fates independently of the gonad-derived inductive and LIN-12/Notch-mediated lateral signaling pathways. We also found that Cbr-pry-1 mutants frequently show a failure of P7.p induction. A similar, albeit low penetrant, defect is also observed in C. elegans pry-1 mutants. The genetic analysis of the P7.p induction defect revealed that it was caused by altered regulation of lin-12 and its transcriptional target lip-1 (MAP kinase phosphatase). Thus, our results provide evidence for LIN-12/Notch-dependent and independent roles of Wnt signaling in promoting 2 degrees VPC fates in both nematode species.

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

SignificanceBasement membrane is a particular kind of sheet-like extracellular matrix that separates tissue compartments. Invasion of cells through basement membrane barriers is a key aspect of many normal and pathological processes, including organ development and cancer cell metastasis. Invasive protrusions are rich in actin, a cytoskeletal biopolymer, the self-assembly of which can produce force and remodel extracellular matrix. However, in the invasive protrusion, actin has been attributed a function in scaffolding and trafficking, and its mechanical role has not been explored. Here we show that invading cells in C. elegans apply forces via actin assembly to break through basement membrane, and that force production is one of the key features of invasion.

The nematode Caenorhabditis briggsae is an excellent model organism for the comparative analysis of gene function and developmental mechanisms. To study the evolutionary conservation and divergence of genetic pathways mediating vulva formation, we screened for mutations in C. briggsae that cause the egg-laying defective (Egl) phenotype. Here, we report the characterization of 13 genes, including three that are orthologs of Caenorhabditis elegans unc-84 (SUN domain), lin-39 (Dfd/Scr-related homeobox), and lin-11 (LIM homeobox). Based on the morphology and cell fate changes, the mutants were placed into four different categories. Class 1 animals have normal-looking vulva and vulva-uterine connections, indicating defects in other components of the egg-laying system. Class 2 animals frequently lack some or all of the vulval precursor cells (VPCs) due to defects in the migration of P-cell nuclei into the ventral hypodermal region. Class 3 animals show inappropriate fusion of VPCs to the hypodermal syncytium, leading to a reduced number of vulval progeny. Finally, class 4 animals exhibit abnormal vulval invagination and morphology. Interestingly, we did not find mutations that affect VPC induction and fates. Our work is the first study involving the characterization of genes in C. briggsae vulva formation, and it offers a basis for future investigations of these genes in C. elegans.

Immune responses to pathogenic microbes include activation of resistance and tolerance mechanisms in the host both of which are energetically expensive. In this study, we show that C. elegans exposed to Gram positive bacteria Enterococcus faecalis and Staphylococcus aureus, rapidly utilizes lipid droplets, the major energy reserve in the nematode. Feeding on E. faecalis causes developmental arrest in C. elegans larvae and growth arrest in adults, pointing to starvation response. We find that nematode's early response to infection entails upregulation of 25 genes involved in lipid hydrolysis and downregulation of 13 lipid synthesis genes as early as 8 hours following exposure. We also show that lipid droplets play a protective role in C. elegans during infection. NHR-49, a PPARα ortholog, is required for E. faecalis induced betaoxidation of fatty acids and immune effector production. It regulates an immunometabolic axis required for survival of the nematode on E. faecalis. Our findings reveal a facet of nutritional immunity wherein lipid droplet homeostasis plays a central role in nematode microbe interactions.

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