12 research outputs found
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Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.
cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli
Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.
The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi
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Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses
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
Recommended from our members
Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses.
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
The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition
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
Recommended from our members
Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.
cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli
\u3ci\u3eC. elegans\u3c/i\u3e avoids toxin-producing \u3ci\u3eStreptomyces\u3c/i\u3e using a seven transmembrane domain chemosensory receptor
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
CLR-1/RPTP is required during late embryogenesis and the first larval stage.
<p>(A) <i>clr-1/RPTP(e1745)</i> animals shifted from the permissive temperature (16°C) to the restrictive temperature (20°C) or vice versa after hatching display defective behavior (n≥40). <i>clr-1/RPTP(e1745)</i> animals at 16°C during the 3-fold embryo and larval stage 1 (L1) respond normally to SDS. Animals at 20°C during these stages have defective responses (n>65). NS, not significant, ***<i>P</i><0.001, t-test, comparison to wild-type. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. Error bars are SEM. (B) Schematics and micrographs of wild-type and <i>clr-1/RPTP</i> animals kept at 20°C or 16°C during the 3-fold embryo and L1 stages. (C) Quantification of reduced NLG-1 GRASP fluorescence intensity in <i>clr-1/RPTP</i> animals kept at 20°C during these stages, in comparison with <i>clr-1/RPTP</i> animals kept at 16°C during these stages. ***<i>P</i><0.001, U-test, comparison to wild-type if directly over bar, or as indicated. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>.</p
CLR-1/RPTP acts in postsynaptic neurons, and is localized to the synaptic region.
<p>(A) Schematics and micrographs of normal PHB-AVA NLG-1 GRASP fluorescence in wild-type and <i>clr-1/RPTP(e1745)</i> mutant animals expressing a transgene that drives expression of the <i>clr-1</i> cDNA in AVA neurons (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTP</i>), and reduced PHB-AVA NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutant animals expressing either a construct that drives expression of the <i>clr-1/RPTP</i> cDNA in PHB neurons (<sub><i>p</i></sub><i>PHB</i>::<i>clr-1/RPTP</i>), a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted in AVA neurons (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTPΔxcd)</i>, or a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with a mutation that inactivates the phosphatase domain (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTPpd</i>). (B) Quantification of NLG-1 GRASP fluorescence. Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs restores NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully restore NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>100). Two or more lines were examined with each transgene, and combined in the graph above. Values for each individual transgenic line are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s006" target="_blank">S2 Table</a>. NS, not significant, ***<i>P</i><0.001, *<i>P</i><0.05, U-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (C) Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs, rescues the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully rescue the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n≥60). NS, not significant, ***<i>P</i><0.001, t-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. Error bars are SEM. (D) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>YFP</i> in AVA (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTP</i>::<i>YFP</i>). (E) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>mCherry</i> in AVA (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTP</i>::<i>mCherry</i>) and PHB-AVA NLG-1 GRASP, and overlay in a <i>clr-1/RPTP</i> mutant animal. (D-E), CLR-1 localization is brightest in the preanal ganglion (yellow box), and the majority of animals show localization in the anterior half of this region, where PHB-AVA synapses usually form (green fluorescence).</p
CLR-1/RPTP is sufficient to drive increased synaptogenesis.
<p>(A) Representative schematics and micrographs of NLG-1 GRASP fluorescence labeling PHB-AVA synapses in wild-type animals and animals overexpressing <i>clr-1/RPTP</i> in AVA neurons (<sub><i>p</i></sub><i>AVA</i>::<i>clr-1/RPTP OE)</i>. (B) Quantification of NLG-1 GRASP fluorescence. Overexpression of <i>clr-1/RPTP</i> in AVA neurons results in increased NLG-1 GRASP fluorescence (n>100). Two lines were examined with this transgene, and combined in the graph above. Values for each individual transgenic line are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s006" target="_blank">S2 Table</a>. ***<i>P</i><0.001, U-test, comparison to wild-type. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (C) Overexpression of <i>clr-1/RPTP</i> in AVAs results in a faster behavioral response (n = 80). *<i>P</i><0.05, t-test, comparison to wild-type. Error bars are SEM.</p