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

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

\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 acts in the UNC-6/Netrin and UNC-40/DCC pathway to direct SPR.

<p>In this model, limiting amounts of UNC-6/Netrin secreted from AVA interneurons bind UNC-40/DCC expressed in PHB neurons. CLR-1/RPTP expressed in AVA neurons acts genetically downstream of UNC-6/Netrin in the SPR pathway, and its extracellular domain is required for full SPR function, suggesting that it may interact with UNC-6/Netrin, UNC-40/DCC, or another unidentified ligand or receptor. The requirement of CLR-1/RPTP’s phosphatase domain for rescue indicates that phosphatase activity is also required for its function in SPR.</p

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

<i>clr-1/RPTP</i> mutants display defective synaptic partner recognition.

<p>(A) Schematic diagrams of PHB and AVA neurons. (B) Schematic diagrams of the trans-synaptic marker NLG-1 GRASP in pre- and postsynaptic neurites (red circles represent cross-sections of neurites in a region with <i>en passant</i> synapses). Split GFPs are linked to the synaptically localized protein NLG-1, so that specific synapses are labeled with green fluorescence in wild-type animals. If a neurite fails to form synapses with the correct partner, NLG-1 GRASP will not reconstitute. (C) Diagram of the PHB and ASH chemosensory circuits including synapses (triangles) connecting sensory neurons (ovals) and interneurons (rectangles) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref004" target="_blank">4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref010" target="_blank">10</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref052" target="_blank">52</a>]. (D) Outline of a behavioral assay that tests PHB circuit function. Backward movement is induced with a nose touch. Function of PHB-AVA synapses halts backward movement in response to 0.1% SDS. (E and F) Schematics and micrographs of mCherry-labeled PHB neurons and AVA neurites in wild-type (E) and <i>clr-1/RPTP(e1745)</i> mutant animals (F), displaying normal morphology and axon guidance to the ventral nerve cord, followed by anterior projection. (G) Schematics and micrographs of PHB-AVA NLG-1 GRASP in wild-type and <i>clr-1/RPTP(e1745)</i> mutant animals, showing reduced synapses in <i>clr-1/RPTP(e1745)</i> mutant animals. (H) Quantification of NLG-1 GRASP fluorescence demonstrates a reduction in <i>clr-1/RPTP</i> mutant animals including <i>clr-1/RPTP(e1745)</i>, <i>clr-1/RPTP(e2530)</i>, and <i>clr-1/RPTP(n1992)</i> as compared with wild type (n>80 except for the low-brood size allele <i>e2530</i>, (n = 38)). ***<i>P</i><0.001, **<i>P</i><0.01, Mann-Whitney U-test, comparison to wild-type. <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>. (I) <i>clr-1/RPTP(e1745)</i> mutants display a defect in the behavioral response to SDS (n>75). ***<i>P</i><0.001, t-test, comparison to wild-type. Error bars are standard error of the mean (SEM).</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 (_<i>p</i><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 (_<i>p</i><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 (_<i>p</i><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 (_<i>p</i><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 (_<i>p</i><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 (_<i>p</i><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 (_<i>p</i><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