Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. We showed that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va and it is a non-processive, ensemble motor. We are also interested in what role myosin V plays in Drosophila. To do this, we have been localizing myosin V in various stages of development. Preliminary results suggest that myosin V in some cell types localizes to the nuclear envelope where it may interact with lamin B. We are in the process of raising new antibodies and are studying the localization of lamin B in myosin V deficient mutant Drosophila. The role of the rod fragment of myosin V molecules in the processive motion of these single molecule motors is largely unknown. We compared the mechanical stability of a processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. Rod fragments (coiled-coil regions) of mouse myosin Va and Drosophila myosin V were cloned into expression vector (pET-16b) and expressed in prokaryotic expression system (E. coli, BL21(DE3)pLysS). Recombinant proteins were purified using Ni-affinity column in denaturing conditions. The formation of proper coiled-coil structure during renaturation was monitored using circular dichroism (CD). Single molecule force spectroscopy measurements were performed using atomic force microscopy (AFM). In order to facilitate strong and specific binding, chemical handles were applied at the N- and C-terminal ends of the fragments. During the mechanical stretching, the extension of rod fragment occurs as two consecutive events: extension of flexible loops followed by extension and unfolding of coiled-coil motifs. We find that the processive and nonprocessive rod fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the AFM stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V. Additionally, we find that unfolding forces of the coiled-coil structures of mouse myosin Va is higher then those in Drosophila myosin V, suggesting that the former coiled-coil region is mechanically more stable. Heat-induced denaturation CD measurements show the same result. Our observations suggest that the tether between the cargo and motor in case of myosin V molecules consists of a mechanically strong coiled-coil region combined with elastic elements, and this connection might play an important role in sustaining processive motions of single molecule motors. We have also examined the structure of the myosin V tail region by atomic force microscopy and rotary shadowed electron microscopy.
| Nagy, Attila; Piszczek, Grzegorz; Sellers, James R (2009) Extensibility of the extended tail domain of processive and nonprocessive myosin V molecules. Biophys J 97:3123-31 |
