612B Poster - 08. Patterning, morphogenesis and organogenesis
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Rachel Szymanski; Noah Gurley; Zuhayr Alam; Mark Peifer

Affiliation: University of North Carolina at Chapel Hill

Keywords:
r. cell-cell interactions; t. cell junctions and adhesion

The remarkable ability of cells to change shape and move without disrupting tissue integrity is a hallmark of embryonic development. To do so cells must link the contractile actomyosin cytoskeleton to cell-cell and cell-matrix junctions, and this linkage must be robust and dynamic. We have focused on the multidomain scaffolding protein Canoe, homolog of mammalian Afadin, which links the cadherin-catenin complex to actin. It includes two N-terminal RA domains, which bind the small GTPase Rap1, followed by FHA, Dilute and PDZ domains. These are separated from the C-terminal F-actin binding domain by a long intrinsically disordered region (IDR). IDRs are now recognized as important players in the multivalent interactions that assemble multiprotein complexes, including those that form phase-separated biomolecular condensates. One of our tasks is to define Canoe’s mechanistic role, by taking it apart as a machine. We began by exploring the roles of its PDZ and F-actin binding domains, thinking they provided the direct linkage between cadherin and actin. However, deleting each domain had only modest effects on protein function. We now have returned to this analysis, exploring a surprising result from earlier work—protein null alleles of canoe have a milder zygotic phenotype than the “canonical” canoe alleles, whose defects in dorsal closure gave the gene its name. To explore this, we have sequenced a series of 22 EMS-induced canoe alleles. Most result from premature stop codons, arrayed across the coding sequence. Intriguingly, the two strong “canonical” alleles carry stop codons early in the IDR. We suspect they encode truncated proteins that act in a dominant negative fashion, interfering with maternally-contributed Canoe protein. We are now exploring the function of this diverse set of Canoe proteins, examining both zygotic and maternal/zygotic mutants. We think this will help define the role of Canoe’s IDR in protein function.