How to navigate the myosin-V motor through the actin network

Abstract

1Myosin-V (MyoV) is a ubiquitous motor protein that transports an astonishingly diverse 2 set of cargos on the actin network in eukaryotes. Phosphorylation-dependent 3 processes often regulate MyoV-mediated cargo transport, molecular details of which 4 remain largely unknown. We previously showed that phosphorylation regulates MyoV's 5 switching from microtubules onto actin filaments, not its motor activity. Regulation of 6 switching at reconstituted microtubule-actin-crossings in fact sufficed to recapitulate 7 the MyoVa-driven redistribution of pigment-organelles in amphibian melanophores. 8 However, in those cells, MyoVa also encounter many actin-actin crossings. Here, we 9 show that isolated MyoVa motors switch with equal probabilities at reconstituted actin-10 actin-crossings. Under the control of its adaptor-protein melanophilin (Mlph), however, 11 the motor differentiates between the actin filaments at crossing points in a 12 phosphorylation-regulated manner. Whereas phosphorylation of Mlph forced about 13 ~2/3 of MyoVa to ignore the intersections, dephosphorylation completely reversed this 14 behavior and forced ~2/3 to switch. We show that the filament-binding domain (FBD) 15 of Mlph controls this switching behavior. This property evolved in amphibians, but not 16 in the early vertebrate zebrafish. By protein engineering, we demonstrate that changes 17 of a few residues are sufficient to impart actin-binding capability onto the zebrafish 18 Mlph. We thus unmask the molecular beginnings of dual filament binding in Mlph that 19 allow it to control the switching behavior of MyoVa at cytoskeletal crossings. We 20 therefore propose a direct link between intracellular phosphorylation activity and the 21 adaptor-protein, not to regulate MyoVa activity, but to navigate the motor through the 22 entire cytoskeletal maze for correct positioning of cargo. 23Significance statement 25 In virtually all eukaryotic cells, numerous myosin motors have to navigate through an 26 elaborate actin network for timely transport of intracellular cargo. Here, we unmask an 27 unintuitive regulation of the myosin-Va motor that is involved in pigment organelle 28 transport. We demonstrate that myosin-Va differentiates between the same actin 29 filaments and displays regulated switching at reconstituted actin-actin crossings, an 30 unexpected behavior that has been predicted from previous theoretical work. We trace 31 this regulation back to the adaptor protein of the myosin-Va motor and show that this 32 regulation was present in amphibian but had not evolved in the early vertebrate 33 zebrafish. Notably, we demonstrate that the evolution of actin-binding capability is 34 achieved by changing a few residues in the adaptor protein.