The conserved, secreted protease inhibitor MLT-11 is necessary for C. elegans molting and embryogenesis

Apical extracellular matrices (aECMs) are associated with all epithelia and form a protective layer against biotic and abiotic threats in the environment. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. Several protease inhibitors are implicated in molting, but their functions remain poorly understood. Here we characterize mlt-11, an unusual protease inhibitor with 10 conserved Kunitz domains. MLT-11 oscillates and is localized in the cuticle and in lysosomes in larvae and in the embryonic sheath starting at the 3-fold embryo stage. mlt-11 (RNAi) produced a developmental delay, motility defects, failed apolysis, and a defective cuticle barrier. mlt-11 null and C-terminal Kunitz domain deletion mutants are embryonic lethal while N-terminal deletions cause a rolling phenotype indicative of cuticle structure abnormalities. mlt-11 activity is primarily necessary in seam and hypodermal cells and accordingly mlt-11 (RNAi) causes defects in localization of the collagens ROL-6 and BLI-1 over the cuticle. mlt-11 (RNAi) molting phenotypes can be suppressed by genetically inhibiting endocytosis. Our model is that MLT-11 is acting in the aECM to coordinate remodeling and timely ecdysis.