mec-15 encodes an F-box protein required for touch receptor neuron mechanosensation, synapse formation and development

Abstract

Selective protein degradation is a key regulator of neuronal development and synaptogenesis. Complexes that target proteins for degradation often contain F-box proteins. Here we characterize MEC-15, an F-box protein with WD repeats, which is required for the development and function of Caenorhabditis elegans touch receptor neurons (TRNs). Mutations in mec-15 produce defects in TRN touch sensitivity, chemical synapse formation, and cell-body morphology. All mec-15 mutant phenotypes are enhanced by mutations in a MAP kinase pathway composed of the MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAPK PMK-3. A mutation of the rpm-1 gene, which encodes an E3 ubiquitin ligase that negatively regulates this pathway to promote synaptogenesis, suppresses only the mec-15 cell-body defect. Thus, MEC-15 acts in parallel with RPM-1, implicating a second protein degradation pathway in TRN development. In addition, all mec-15 phenotypes can be dominantly suppressed by mutations in mec-7, which encodes a beta-tubulin, and dominantly enhanced by mutations in mec-12, which encodes an alpha-tubulin. Since mec-15 phenotypes depend on the relative levels of these tubulins, MEC-15 may target proteins whose function is affected by these levels.

Figure 1.—

mec-15 mutations produce temperature-sensitive defects in TRN mechanosensation, synapse formation, and cell-body morphology. (A) mec-15 mutants show a reduction in touch sensitivity that increases with temperature. Error brackets indicate SEM; n ≥ 15. (B) GFP∷RAB-3 expression in nerve ring, VNC, ALM, and PLM neurons in young adult wild type and mec-15(js414) animals grown at 20°. Bars, 20 μm.

Figure 2.—

mec-15 mutations and rescue. (A) Schematic of MEC-15 protein with notable domains and location of mutant lesions. tm2691 produces a deletion after the first 28 amino acids (black line), resulting in a frameshifted protein sequence that terminates shortly thereafter (red dashed line). (B) Touch sensitivity and (C) vulva Sam phenotype of mec-15(js414) animals are rescued with P_mec-18mec-15(+). Experiments were performed at 25°. Error brackets indicate SEM; n = 20.

Figure 3.—

MEC-15∷GFP is expressed in the TRNs in a mec-3-dependent and stage-specific manner. Immunostaining against GFP in (A) L2 larvae, (B) young adult, and (C) mec-3(e1338) L2 larva expressing uIs62(mec-15∷gfp). TRN expression (arrowheads) is absent in adults and mec-3 mutants (positions of TRN cell bodies are designated by brackets; “S” indicates expression in spermathecae). Bars, 20 μm.

Figure 4.—

Mutations in a p38 MAPK pathway enhance all mec-15 phenotypes. (A) Touch sensitivity experiments of animals grown at 20°. Error brackets indicate SEM; n = 40. Asterisks (*) indicate significant (P < 0.00001) decrease of touch sensitivity compared to mec-15(js414) animals. (B) Synapse formation and cell bodies visualized by GFP∷RAB-3. All animals for these experiments were grown at 20°. Bars, 20 μm.

Figure 5.—

mec-7 mutations dominantly suppress all mec-15 phenotypes. (A) Touch sensitivity experiments of animals grown at 20°. Error brackets indicate SEM; n = 40. (B) Synapse formation and cell bodies visualized by GFP∷RAB-3. The mec-15 allele in all experiments is js414, with the exception of mec-15(u75); mec-7 and mec-15(u75/js414); mec-7/+. All animals for these experiments were grown at 20°. Bars, 20 μm.

Figure 6.—

mec-12(e1607) dominantly enhances mec-15 touch insensitivity. The mean number of responses resulting from 10 touches to 20 animals grown at 20°. Error brackets indicate SEM. A single asterisk (*) indicates a significant decrease (P < 0.0001) of touch sensitivity compared to mec-15(u75) animals; double asterisks (**) indicate a significant decrease (P < 0.00001) of touch sensitivity compared to mec-15(u75)/+ and mec-12(e1607)/+ animals.