Bernazzani, Sarina et al. (2017) International Worm Meeting "Functions, regulation, and composition of the epidermal apical extracellular matrix."

Fay, David, Bernazzani, Sarina

[International Worm Meeting, 2017]

During development, as well as later in life, the extracellular matrix (ECM) performs essential structural, protective, and physiological functions. We have shown that a fibrillin-like protein, FBN-1, is expressed in epidermal cells and is an important component of the apical extracellular matrix (aECM), termed the sheath in embryos and the cuticle in larvae and adults. FBN-1 acts in the embryonic sheath to prevent epidermal cell deformation caused by several distinct biomechanical forces, and is required in the cuticle for normal molting. Our studies previously demonstrated that FBN-1 functions within a network that includes the highly conserved proteins SYM-3/FAM102B, a predicted membrane-tethered protein, SYM-4/WDR44, a WD40 repeat protein, and MEC-8/RBPMS, a regulator of alternative splicing that is required for normal fbn-1 mRNA processing. Although viable as single mutants, animals that are doubly mutant for mec-8; sym-3, mec-8; sym-4, fbn-1; sym-3, or fbn-1; sym-4, display an anterior morphological defect termed the Pharynx Ingressed, or Pin, phenotype. Our recent studies indicate that FBN-1 may serve as a link between epidermal cells and the aECM via attachment to integrins. Notably, integrins have not previously been reported to act at the apical surface of epidermal cells in C. elegans. Consistent with our model, mutations of the two RGD (integrin-binding) sites in FBN-1 lead to molting defects. In addition, fbn-1; sym-3, or fbn-1; sym-4, mutants exhibit a "notched-head" phenotype, which is also observed in integrin mutants. Furthermore, sym-3 and sym-4 mutants are hypersensitive to partial inhibition of pat-3, the sole C. elegans b-integrin subunit. Our work previously indicated that SYM-3 and SYM-4 act in a parallel pathway to MEC-8 and may promote the trafficking of structural proteins to the apical surface of epidermal cells. Functional full-length SYM-3 and SYM-4 fluorescent reporters localize to vesicle-like structures at or near the plasma membrane in epidermal cells. Furthermore, SYM-3 and SYM-4 act in a common genetic pathway with the RAB-11 GTPase, a known regulator of endocytic recycling and exocytosis. Interestingly, mammalian WD44 physically interacts with Rab11, although no functional link between these proteins has been demonstrated. In other studies, we have sought to identify novel components of the aECM that may function with FBN-1. Partial inhibition of these candidate aECM components led to enhancement of the Pin phenotype in sym-4 mutants. Identified aECM components include, NID-1, an RGD-containing protein and, mlt-7, which encodes a collagen-linking enzyme that we have shown localizes to the aECM. Taken together, we have identified a network of genes that provide insight into the composition, regulation, and functions of the aECM during development.

Joseph, Braveen et al. (2021) International Worm Meeting "Loss of a conserved protease can suppress molting defects"

Joseph, Braveen, Fay, David, Edeen, Phil, Bernazzani, Sarina

[International Worm Meeting, 2021]

Molting is essential for the growth and life cycle of ecdysozoa, a large group of animals that includes arthropods and nematodes. During C. elegans molting, a new cuticle is synthesized underneath the old cuticle, which is eventually perforated and discarded. Protease activity is thought to be required for the partial degradation and release of the old cuticle, as loss of specific proteases can lead to molting defects. Interestingly, peptide protease inhibitors are also necessary for nematode molting. This has led to the model that the precisely timed activation and repression of proteases is essential for normal molting. In this study we show that clathrin-mediated endocytosis (CME) promotes downregulation of the matrix metalloprotease ADM-2/ADAM9/12/19/33, a conserved member of the ADAM (a disintegrin and metalloprotease) family of transmembrane metallopeptidases. In previous work we have shown that NEKL-2/NEK8/9 and NEKL-3/NEK6/7, members of the highly conserved family of NIMA-related kinases, are required for normal molting in C. elegans. Moreover, defective molting in nekl mutants is due to defects in CME, and partial restoration of CME by mutations in the AP2 clathrin-adapter complex, also restores normal molting. Through a non-biased genetic screen, we found that loss of ADM-2 function can suppress molting defects in nekl mutants. Unlike mutations in genes associated with AP2, however, loss of ADM-2 does not correct nekl trafficking defects, indicating that suppression by adm-2 occurs through a novel mechanism. Endogenously tagged ADM-2 is expressed in the epidermis and colocalization studies indicate that ADM-2 is trafficked via CME to the lysosome. Furthermore, loss of NEKL function leads to increased expression of ADM-2 at the epidermal plasma membrane. Importantly, CRISPR-derived mutations targeting the extracellular protease and disintegrin domains of ADM-2 strongly suppress molting defects in nekl mutants. Unlike AP2 mutations, however, loss of ADM-2 does not suppress null or strong loss-of-function nekl mutants, indicating that a failure to endocytose additional cargo, such as steroid hormone precursors required for molting, also contributes to nekl molting defects. Our findings lead to the model that excess ADM-2 activity at the plasma membrane impedes normal molting and that NEKL-mediated CME acts to restrict the activity of proteases during the molting process.