Biology subjects

Clancy, J. C.

Publications and source records attributed to Clancy, J. C..

3 recordsLinked to original sources

bioRxiv2025

MLT-11 is necessary for C. elegans embryogenesis and conserved sequences play distinct roles in cuticle structure

Apical extracellular matrices (aECMs) are associated with many epithelia and many form a protective layer against biotic and abiotic threats in the environment. Despite their importance, we lack a deep understanding of their structure and dynamics in development and disease. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. Here, we show that the poorly characterized putative protease inhibitor gene, mlt-11, is directly regulated by the NHR-23 transcription factor. We identify key cis-regulatory elements required for robust mlt-11 expression. An internal MLT-11::mNeonGreen translational fusion transiently localized to the aECM in the cuticle and embryo. MLT-11::mNeonGreen was also detected in lining openings to the exterior (vulva, rectum, mouth). mlt-11 is necessary to pattern all layers of the adult cuticle, and reduction of MLT-11 levels disrupted the barrier function of the cuticle. Deletion of conserved Kunitz protease inhibitor domains or intervening sequences produced a range of defects including either left or right rollers, and small separations of the cuticle along the length of the animal (microblisters). MLT-11 is processed into at least two fragments and internal and C-terminal mNeonGreen knock-ins display distinct localization patterns. Predicted mlt-11 null mutations caused fully penetrant embryonic lethality and elongation defects. Together, this work suggests that MLT-11 localizes similarly to pre-cuticle components and conserved sequences play distinct roles in promoting proper assembly of the aECM.

developmental biology↗

bioRxiv2022

Experimental considerations for study of C. elegans lysosomal proteins

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic disease. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a C. elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation was critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1) we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.

molecular biology↗

bioRxiv2022

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.

developmental biology↗