Transcriptome dataset of Caenorhabditis elegans responses to varied microbial pathogens.

Transcriptome analysis through next-generation sequencing (NGS) is an invaluable tool for investigating changes in gene expression across diverse organisms. The nematode Caenorhabditis elegans (C. elegans) serves as an excellent model organism for dissecting host responses to bacterial infections. Here, our dataset obtained from bulk RNA-sequencing (RNA-seq) can be used to provide in-depth characterization of the mRNA transcriptome profiles of wild-type N2 animals and null mutants of the cytoskeletal regulatory gene unc-53/Nav2 following exposure to distinct bacterial environments: their natural laboratory food source, Escherichia coli OP50, the human and nematode pathogen Pseudomonas aeruginosa PA14, and the emerging pathogen Elizabethkingia anophelis Ag1. As proof of the dataset quality, downstream differential gene expression analysis reveals significant shifts in gene expression patterns within N2 and unc-53 mutants under varying bacterial conditions that will be useful for our companion studies investigating these pathways. These data provide an effective methodological framework for future investigators to investigate the interplay between cytoskeletal proteins and the innate immune response. The raw FASTQ files generated from our transcriptome experiment is deposited in the publicly available NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1010192, for further exploration and validation by the C. elegans research community.

Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C. elegans.

Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two-hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.

Live cell imaging of the cytoskeleton.

The cytoskeleton is the network of cytoplasmic protein filaments, composed of microtubules (MTs), actin filaments, and intermediate filaments, that provides an internal scaffold to give the cell shape. The organization of the cytoskeleton is not static but rather rearranges to enable a variety of fundamental cellular processes including chromosome segregation, cytokinesis, cell migration, cell polarity, cell adhesion, neuron outgrowth, chemotaxis, muscle contraction, cytoplasmic streaming, locomotion by flagella, subcellular organelle distribution, and intracellular trafficking. Given this multifunctional role, it is not surprising that cytoskeletal defects have been associated with a large variety of human diseases including neurodegenerative disorders, cancer, muscular dystrophies, and cardiac disorders. Therefore, understanding the molecular basis of cytoskeleton dynamics and its impact on cell biology is of vital importance. In this chapter, we provide an overview of some of the methods used to image cytoskeleton dynamics in live cells, placing an emphasis on recent advances in the visualization of the MT and the actin cytoskeleton in multicellular organisms.

Distinct cell guidance pathways controlled by the Rac and Rho GEF domains of UNC-73/TRIO in Caenorhabditis elegans.

The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1.

Navigating the cell: UNC-53 and the navigators, a family of cytoskeletal regulators with multiple roles in cell migration, outgrowth and trafficking.

Changes in cell shape are associated with a variety of processes including cell migration, axon outgrowth, cell division and vesicle trafficking. C. elegans UNC-53 and its vertebrate homologs, the Navigators, are required for the migration of cells and the outgrowth of neuronal processes. The identification of novel molecular interactions and live imaging studies have revealed that UNC-53/NAVs are signal transducers associated with actin filaments, microtubules and intermediate filaments. In addition to modulating cytoskeletal dynamics at the leading edge of migrating or outgrowing cells, both UNC-53 and the navigators are expressed in adult cells, conspicuously those with specialized roles in endocytosis or secretion. Collectively, these results suggest that UNC-53/NAVs may be a central regulator of cytoskeletal dynamics, responsible for integrating signaling cues to multiple components of the cytoskeleton to coordinate rearrangement during cell outgrowth or trafficking.

The cell migration molecule UNC-53/NAV2 is linked to the ARP2/3 complex by ABI-1.

The shape changes that are required to position a cell to migrate or grow out in a particular direction involve a coordinated reorganization of the actin cytoskeleton. Although it is known that the ARP2/3 complex nucleates actin filament assembly, exactly how the information from guidance cues is integrated to elicit ARP2/3-mediated remodeling during outgrowth remains vague. Previous studies have shown that C. elegans UNC-53 and its vertebrate homolog NAV (Neuronal Navigators) are required for the migration of cells and neuronal processes. We have identified ABI-1 as a novel molecular partner of UNC-53/NAV2 and have found that a restricted calponin homology (CH) domain of UNC-53 is sufficient to bind ABI-1. ABI-1 and UNC-53 have an overlapping expression pattern, and display similar cell migration phenotypes in the excretory cell, and in mechanosensory and motoneurons. Migration defects were also observed after RNAi of proteins known to function with abi-1 in actin dynamics, including nck-1, wve-1 and arx-2. We propose that UNC-53/NAV2, through its CH domain, acts as a scaffold that links ABI-1 to the ARP2/3 complex to regulate actin cytoskeleton remodeling.