FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF MYOSIN VI ISOFORMS

Myosin VI is unique among the many members of the myosin superfamily. The peculiarities of this motor protein reside on its ability to travel along actin microfilaments towards their pointed- end, as well as its capacity to act either as anchor or as processive motor. As a consequence, myosin VI has been implicated in clathrin-mediated endocytosis, vesicle trafficking, autophagy, cell migration and tumorigenesis. The current understanding of the myosin VI protein does not explain how it carries out these diverse processes, as functional mechanistic studies are lacking. Alternative splicing in the tail region generates myosin VI molecules with different features (myosin VIshort and myosin VIlong) but little is known about the impact of the variable region on physiological and pathological functions of myosin VI. In this study, we have analysed the myosin VI isoforms from a molecular and a functional perspective. Using quantitative mass spectrometry approach and modern NMR we identified and structurally characterized clathrin light chain as novel and isoform-specific interactor. Within the novel clathrin-binding domain that is unique to myosin VIlong, an isoform-specific regulatory helix, named \u3b12-linker, defines a specific myosin VIlong conformation. Its presence or absence determines the target selectivity of myosin VIshort and myosin VIlong isoforms, acting like a molecular switch that regulates their functional involvement in migratory or endocytic pathways, respectively. The adaptor-binding RRL motif is embedded in the clathrin-binding domain and is masked in the structural configuration adopted by the myosin VIlong. Consequently, the previously identified RRL interactors show selective binding to myosin VIshort. Thus, we can provide for the first time a mechanistic explanation of why the various isoforms show different localization and function (i.e. myosin VIlong selectively involved in clathrin-mediated endocytosis). We also found that alternative myosin VI splicing is aberrantly regulated in ovarian cancers, where exon skipping dictates myosin VIshort-only expression. Importantly, cancer cell lines selectively expressing the myosin VIshort isoform exhibit severe migration defects when myosin VI is knocked down