ZP Domain Proteins and Epithelial Integrity

Boston Children'S Hospital, Boston MA

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Abstract

DESCRIPTION (provided by applicant): ZP domain proteins and epithelial integrity Significance: Epithelia are sheets of cells that create outer (apical) and inner (basal) compartments. ZP domain proteins are found in the apical extracellular matrix (ECM) of nearly all epithelia, and are present from jellyfish to humans, but no shared activity has been assigned to them. Many human ZP domain proteins are mutated in disease, including the nearly-ubiquitous hensin (cancer), inner ear tectorins (deafness), and renal uromodulin (nephropathy). Using the C. elegans amphid, a sensory epithelium composed of neurons and glia, we identified two ZP domain proteins, DYF-7 and FBN-1, required to prevent rupture of epithelial cell junctions. These results suggest a new role for ZP domain proteins, namely maintenance of epithelial integrity, and raise the possibility that ZP domain protein diseases might be treated as cell junction disorders. Innovativeness: In addition to our innovative hypothesis, the use of the C. elegans amphid as a model epithelium is technically innovative. It allows genetic manipulation of junction components with single-cell resolution; and, unlike other epithelia, rupture of sensory epithelia is not lethal, so null mutants are viable. Hypothesis: We hypothesize that DYF-7 prevents cell junctions from rupture by forming a filamentous meshwork attached to the apical surfaces of epithelia. Preliminary data: Loss of the ZP domain protein DYF-7 causes rupture of the amphid sensory epithelium. DYF-7 localizes with exquisite precision to extracellular caps adjacent to epithelial cell junctions. DYF-7 is required by all neurons that form junctions with glia, but not by others. Ectopic DYF-7 localizes adjacent to cell junctions in all epithelia. DYF-7 expressed in vitro spontaneously assembles bundled filaments, similar to filaments seen by electron microscopy (EM) at the surface of the embryonic amphid epithelium. Loss of the ZP domain protein FBN-1 or the putative ZP-domain-binding protein DEX-1 mimic loss of DYF-7. Aims: First, we will label, deplete, and overexpress epithelial components with single-cell resolution in a dyf-7 mutant to test the hypothesis that cell junctions rupture, and determine the mechanism. Second, we will test the hypothesis that DYF-7 assembles a filamentous meshwork attached to apical surfaces, using EM of DYF-7 filaments in vitro and in vivo, analysis of DYF-7 secretion and localization in an epithelial cell culture model, and a genetic screen for mutants that disrupt its localization in vivo. Third, we will elucidate the new epithelial integrity pathway we identified by screening for factors upstream or downstream of DYF-7 and by using genetics, biochemistry, and microscopy to assay interactions between DYF-7, FBN-1, and DEX-1.