Mutation in F-actin Polymerization Factor Suppresses Distal Arthrogryposis Type 5 (DA5) PIEZO2 Pathogenic Variant inCaenorhabditis elegans

Abstract

AbstractThe mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variantpezo-1[R2405P]that in humanPIEZO2causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate thatpezo-1(R2405P)is a gain-of-function allele. Using genomic mapping and whole genome sequencing approaches, we identified a candidate suppressor allele in theC. elegansgenegex-3.This gene is an ortholog of humanNCKAP1(NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion ofgex-3by RNAi, or with the suppressor allelegex-3(av259[L353F]), significantly restored the small brood size and low ovulation rate, as well as alleviated the crushed oocyte phenotype of thepezo-1(R2405P)mutant. Auxin-inducible degradation of GEX-3 revealed that only somatic-specific degradation of GEX-3 restored the reduced brood size in thepezo-1(R2405P)mutants. Additionally, actin organization and orientation were disrupted and distorted in thepezo-1mutants. Mutation ofgex-3(L353F)partially alleviated these defects. The identification ofgex-3as a suppressor of the pathogenic variantpezo-1(R2405P)suggests that the cytoskeleton plays an important role in regulating PIEZO channel activity and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.