The cystic kidney disease, infantile nephronophthisis (iNPHP) results in end-stage renal failure within the first several years of life. iNPHP is caused by mutations in the ankyrin repeat-containing proteins inversin (INVS) and ANKS6, and the NIMA (never-in-mitosis gene A)-related kinase, NEK8. These proteins appear to form a complex that localizes near the base of primary cilia. Little is known about the function of the iNPHP proteins or the mechanisms through which mutations in these proteins result in the fibrosis and cyst formation. In this project, I aim to understand the function of the iNPHP proteins by studying their homologs in C. elegans. MLT-4 (inversin), MLT-2 (ANKS6), and NEKL-2 (NEK8) are expressed in the hypodermal tissue. They localize to the junctions between the hypodermis and the seam cells. Both the hypodermis and seam cells produce and secrete collagen proteins that comprise the C. elegans cuticle, or apical extracellular matrix (ECM). Although the complete loss of function of MLT-4, MLT-2, or NEKL-2 is lethal due to a molting defect, our lab has discovered that gain-of-function mutations in these proteins result in what appears to be the aberrant separation of the apical ECM from the hypodermis. This phenotype is reversed by human disease alleles, suggesting that our gain-of-function mutants represent a hyperactive form of the iNPHP complex. Additionally, I have found that the gain-of-function mutations result in a disorganization of collagen fibrils in the apical ECM. Using these gain-of-function mutations, I will assess the role of iNPHP proteins in directing the organization of the cuticle collagen proteins using several live imaging techniques (Aim 1). I will also interrogate the role of the iNPHP complex in regulating the cytoskeleton and junctional complexes that anchor the apical ECM to the hypodermal tissue (Aim 2). I will also begin to probe how the complex is activated using optogenetic activation techniques (Aim 3). Understanding NEKL-2, MLT-2, and MLT-4 in C. elegans could guide our understanding of how the vertebrate inversin compartment is activated and its conserved role in regulating the cytoskeleton and ECM.