Investigation of ck/MyosinVIIA interactors using affinity tagged C‐terminal binding domains

Abstract

Actin‐based protrusions play a variety of roles in cells from motility via filopodia to increasing surface area in microvilli to providing the molecular basis of hearing in humans and other vertebrates with stereocilia. Loss of function mutations in the unconventional myosin motor protein Myosin VIIA result in shortened stereocilia which lack correct polarity of the protrusion. Theses mutations are seen in seventy‐five percent of patients afflicted with Usher Syndrome, the leading genetic cause of hereditary deaf‐blindness. Drosophila melanogaster contain a homologous protein to Myosin VIIA called crinkled (ck/MyoVIIA). Mutations in ck/MyoVIIA result in shortened, split, and malformed hairs and denticles, actin based protrusions similar to vertebrate microvilli and stereocilia. Our project aims to identify proteins that interact with ck/MyoVIIA to help determine the molecular mechanism of ck/MyoVIIA in actin‐based protrusion formation. In this study, we are investigating the binding of ck/MyoVIIA to Cad99C. Cad99C is the Drosophila homolog to Protocadherin 15, a protein necessary in the formation of stereocilia that is known to interact with MyoVIIA in humans. In addition, Cad99C has been shown to interact with ck/MyoVIIA in the formation of follicle epithelial cells in Drosophila oocytes. We want to investigate whether this interaction occurs in embryos due to the known effect of loss of ck/MyoVIIA on actin‐based denticle formation in embryos. To test this protein interaction we used a transgenic Drosophila expressing the C‐terminal tail binding domains (MyTH4, FERM, and SH3) of ck/MyoVIIA fused to an N‐terminal triple affinity tag (FLAG, His, STREPII tags). The triple tag ck‐tail and HA‐Cad99C were expressed using a Squash‐GAL4 driver and isolated using a Talon metal affinity pull‐down procedure for the HIS tag. Proteins were separated using SDS‐page gel electrophoresis and identified using anti‐FLAG antibody for the ck‐tail and anti‐HA antibody for Cad99C.Support or Funding InformationBeta Beta Beta Undergraduate Research Grant