• Actin;
  • CaM;
  • cell-free endosome fusion;
  • endosome recognition and fusion;
  • epithelial polarity;
  • myr4

In epithelial cells, endocytosed transferrin and its receptor, which cycle basolaterally, have been shown to transit through recycling endosomes which can also be accessed by markers internalized from the apical surface. In this work, we have used an in vitro assay to follow transfer of an endocytosed marker from apical or basolateral early endosomes to recycling endosomes labeled with transferrin. We show that calmodulin (CaM) function is necessary for transfer and identified myr4, a member of the unconventional myosin superfamily known to use CaM as a light chain, as a possible target protein for CaM. Since myr4 is believed to act as an actin-based mechanoenzyme, we tested the role of polymerized actin in the assay. Our data show that conditions which either prevent actin polymerization or induce the breakdown of existing filaments strongly inhibit interactions between recycling endosomes and either set of early endosomes. Altogether, our data indicate that trafficking at early steps of the endocytic pathway in Madin–Darby Canine Kidney cells depends on the actin-based mechanoenzyme myr4, its light chain CaM, and polymerized actin.