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1.
Commun Biol ; 2: 344, 2019.
Article in English | MEDLINE | ID: mdl-31552297

ABSTRACT

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.


Subject(s)
Gene Expression , Genes, Reporter , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Synapses/metabolism , Amino Acid Sequence , Fluorescent Dyes , HEK293 Cells , Humans , Luminescent Proteins/chemistry , Microscopy, Fluorescence , Models, Molecular , Molecular Imaging , Structure-Activity Relationship , Red Fluorescent Protein
2.
PLoS Genet ; 14(5): e1007312, 2018 05.
Article in English | MEDLINE | ID: mdl-29742100

ABSTRACT

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.


Subject(s)
Mutation , Recognition, Psychology , Synapses/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Interneurons/metabolism , Larva/genetics , Larva/metabolism , Locomotion/genetics , Locomotion/physiology , Microscopy, Confocal , Receptor-Like Protein Tyrosine Phosphatases/genetics , Receptor-Like Protein Tyrosine Phosphatases/metabolism , Sensory Receptor Cells/metabolism , Synapses/genetics , Synaptic Transmission/genetics , Synaptic Transmission/physiology
3.
Elife ; 62017 09 05.
Article in English | MEDLINE | ID: mdl-28873053

ABSTRACT

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator-prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic acid, which is sensed by SRB-6. This behavioral adaptation represents an important strategy for the nematode, which utilizes specialized sensory organs and a chemoreceptor that is tuned to recognize the bacteria. These findings provide a window into the molecules and organs used in the coevolutionary arms race between predator and potential prey.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , Chemoreceptor Cells/physiology , Neurons/physiology , Streptomyces/pathogenicity , Adaptation, Physiological , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/microbiology , Chemotaxis , Neurons/cytology , Neurons/microbiology , Phylogeny , Signal Transduction
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