Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG.

The genetically encoded photosensitizer miniSOG (mini Singlet Oxygen Generator) can be used to kill cells in C. elegans. miniSOG generates the reactive oxygen species (ROS) singlet oxygen after illumination with blue light. Illumination of neurons expressing miniSOG targeted to the outer mitochondrial membrane (mito-miniSOG) causes neuronal death. To enhance miniSOG's efficiency as an ablation tool in multiple cell types we tested alternative targeting signals. We find that membrane targeted miniSOG allows highly efficient cell killing. When combined with a point mutation that increases miniSOG's ROS generation, membrane targeted miniSOG can ablate neurons in less than one tenth the time of mito-miniSOG. We extend the miniSOG ablation technique to non-neuronal tissues, revealing an essential role for the epidermis in locomotion. These improvements expand the utility and throughput of optogenetic cell ablation in C. elegans

Genetic Suppression of Basement Membrane Defects in Caenorhabditis elegans by Gain of Function in Extracellular Matrix and Cell-Matrix Attachment Genes.

Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulfilimine cross-links between type IV basement membrane collagens. Loss of function in the Caenorhabditis elegans peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we find that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of let-805, encoding the transmembrane protein myotactin, which suppress phenotypes of pxn-2 null mutants and of other basement membrane mutants such as F-spondin/spon-1 These let-805 suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (emb-9, let-2) and perlecan (unc-52), as well as a mutation affecting spectraplakin (vab-10), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of-function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix

Corrigendum: A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

[This corrects the article DOI: 10.3389/fncir.2018.00094.]

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond

Wounding triggers MIRO-1 dependent mitochondrial fragmentation that accelerates epidermal wound closure through oxidative signaling.

Organisms respond to tissue damage through the upregulation of protective responses which restore tissue structure and metabolic function. Mitochondria are key sources of intracellular oxidative metabolic signals that maintain cellular homeostasis. Here we report that tissue and cellular wounding triggers rapid and reversible mitochondrial fragmentation. Elevated mitochondrial fragmentation either in fzo-1 fusion-defective mutants or after acute drug treatment accelerates actin-based wound closure. Wounding triggered mitochondrial fragmentation is independent of the GTPase DRP-1 but acts via the mitochondrial Rho GTPase MIRO-1 and cytosolic Ca2+. The fragmented mitochondria and accelerated wound closure of fzo-1 mutants are dependent on MIRO-1 function. Genetic and transcriptomic analyzes show that enhanced mitochondrial fragmentation accelerates wound closure via the upregulation of mtROS and Cytochrome P450. Our results reveal how mitochondrial dynamics respond to cellular and tissue injury and promote tissue repair

C. elegans Kallmann syndrome protein KAL-1 interacts with syndecan and glypican to regulate neuronal cell migrations

AbstractThe anosmin-1 protein family regulates cell migration, axon guidance, and branching, by mechanisms that are not well understood. We show that the C. elegans anosmin-1 ortholog KAL-1 promotes migrations of ventral neuroblasts prior to epidermal enclosure. KAL-1 does not modulate FGF signaling in neuroblast migration and acts in parallel to other neuroblast migration pathways. Defects in heparan sulfate (HS) synthesis or in specific HS modifications disrupt neuroblast migrations and affect the KAL-1 pathway. KAL-1 binds the cell surface HS proteoglycans syndecan/SDN-1 and glypican/GPN-1. This interaction is mediated via HS side chains and requires specific HS modifications. SDN-1 and GPN-1 are expressed in ventral neuroblasts and have redundant roles in KAL-1-dependent neuroblast migrations. Our findings suggest that KAL-1 interacts with multiple HSPGs to promote cell migration

Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration