An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting.

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a mediator of molting. Loss of adm-2, including mutations that disrupt the metalloprotease domain, led to the strong suppression of molting defects in partial loss-of-function nekl mutants. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of apical epidermal LRP-1, ADM-2 overexpression caused a reduction in LRP-1 levels. Consistent with this, several mammalian ADAMs, including the meltrin ADAM12, have been shown to regulate mammalian LRP1 via proteolysis. In contrast to mammalian homologs, however, the regulation of LRP-1 by ADM-2 does not appear to involve the metalloprotease function of ADM-2, nor is proteolytic processing of LRP-1 strongly affected in adm-2 mutants. Our findings suggest a noncanonical role for an ADAM family member in the regulation of a lipoprotein-like receptor and lead us to propose that endocytic trafficking may be important for both the internalization of factors that promote molting as well as the removal of proteins that can inhibit the process.

Effectors of anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. At present, the factors that contribute to aECM function are mostly unknown, including the aECM components themselves, their posttranslational regulators, and the pathways required for their secretion. Here we showed that two proteins previously linked to aECM function, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function in a complex. Proteomics experiments also suggested potential roles for SYM-3/FAM102A and SYM-4/WDR44 family proteins in intracellular trafficking. Nonetheless, we found no evidence to support a critical function for SYM-3 or SYM-4 in the apical deposition of two aECM components, NOAH-1 and FBN-1. Moreover, loss of a key splicing regulator of fbn-1, MEC-8/RBPMS2, had surprisingly little effect on the abundance or deposition of FBN-1. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. We also characterized morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a similar phenotype to mec-8; sym double mutants. Collectively, these findings add to our knowledge of factors controlling embryonic morphogenesis.

Pathways that affect anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. We showed that two conserved proteins linked to this process, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function together in a complex. Proteomics data also suggested potential roles for FAM102A and WDR44 family proteins in intracellular trafficking, consistent with their localization patterns. Nonetheless, we found no evidence to support a clear function for SYM-3 or SYM-4 in the apical deposition of two aECM components, FBN-1 and NOAH. Surprisingly, loss of MEC-8/RBPMS2, a conserved splicing factor and regulator of fbn-1 , had little effect on the abundance or deposition of FBN-1 to the aECM. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. Lastly, we examined morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a related embryonic phenotype to mec-8; sym double mutants. Collectively, our findings add to our knowledge of pathways controlling embryonic morphogenesis. SUMMARY STATEMENT: We identify new proteins in apical ECM biology in C. elegans and provide evidence that SYM-3/FAM102A and SYM-4/WDR44 function together in trafficking but do not regulate apical ECM protein deposition.