Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Significance How the mechanical properties of myosin motors relate to their functions in cells is poorly understood. Myosin VI (MYO6) is the only myosin that moves to the minus end of actin filaments, but the cellular requirement of this reverse movement is unknown. To investigate this question, we generated a mechanical mutant of MYO6, MYO6+, which moves to the plus end of actin filaments. This mutant causes clustering of signaling endosomes coupled to reorganization of cortical actin filaments into elongated filopodia. These two phenotypes depend on the multimerization of MYO6+ on the endosomal membrane induced by binding to lipids and adaptor proteins. Our results highlight the importance of endosomes for myosin-dependent regulation of cortical actin filaments in mammalian cells. Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end–directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end–directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.