The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition

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.

Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses

“…We established that like NLG-1 GRASP, NLG-1 CLASP labels connections between correct synaptic partners, and has a spatial pattern similar to that predicted by electron micrograph reconstruction (11,12). As a validation, NLG-1 CLASP labeling is disrupted by loss of the clr-1 gene, which is required for synaptic partner recognition (13).…”

“…To determine if these puncta were indeed synaptic, we introduced NLG-1 CLASP into clr-1/RPTP synaptogenesis mutants. In clr-1/RPTP mutants, PHB-AVA NLG-1 GRASP fluorescence intensity is dramatically reduced, and a PHB circuit-specific behavior is disrupted, indicating a reduction in synaptogenesis between the two neurons (13). We found that NLG-1 CLASP fluorescence intensity was also dramatically reduced in clr-1/RPTP mutants ( Fig.…”

“…5A). Using NLG-1 GRASP, we discovered that the recognition between two synaptic partners, the PHB sensory neurons and the AVA interneurons, is mediated the secreted ligand UNC-6/Netrin, its canonical receptor UNC-40/Deleted in Colorectal Cancer (20), and the receptor protein tyrosine phosphatase (RPTP) CLR-1 (13). NLG-1 GRASP has also been adapted to many other systems, indicating that this technology is transferable (21).…”

“…Intensity of PHB-AVA NLG-1 GRASP and PHB-AVA NLG-1 CLASP was measured as previously described (13,35). Briefly, the intensity at each pixel within each synaptic puncta was measured using NIH ImageJ.…”

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans

“…FlincG3 allows visualization of cGMP production in PHB chemosensory neurons in response to the repellent SDS. (A) Diagram of the PHB circuit; this figure is reproduced from Figure 1C in Varshney et al (2018) under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). The primary postsynaptic partners of PHB neurons are the AVA backward command interneurons and the PVC forward command interneurons (Hilliard et al 2002; Park et al 2011; Tran et al 2017).…”