Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We have examined the structure of actin-bound myosin V mutants in which the neck length was altered. These studies demonstrate that the neck of myosin V has evolved to allow it to take 36 nm strides along actin which matches the helical repeat. Atomic force microscopy was used to study the extensibility of the tails of myosin V from Drosophila and mouse. In both cases, the force-extension curve of these myosin tails differed dramatically from that of rabbit skeletal muscle myosin in that less force is needed to bring about extension. There were also differences in the behavior of the tail from the processive mouse myosin V and the nonprocessive Drosophila myosin V. Modeling studies have shown that an extensible myosin V tail would be very useful for processive movement in a viscous environment. We have mutated the switch-1 region of myosin V (S217A) and found that this mutant alters the duty ratio and effects processivity of the motor. Switch-2 mutants affect the speed of translocation and the ADP release rate. Optical trapping studies show that under certain levels of strain, the powerstroke of myosin V attached to actin can be reversed which may aid in maintaining myosin attachment during stall.
| Andrecka, Joanna; Ortega Arroyo, Jaime; Takagi, Yasuharu et al. (2015) Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy. Elife 4: |
| Ortega Arroyo, J; Andrecka, J; Spillane, K M et al. (2014) Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett 14:2065-70 |
| Hammer 3rd, John A; Sellers, James R (2012) Walking to work: roles for class V myosins as cargo transporters. Nat Rev Mol Cell Biol 13:13-26 |
| Oke, Olusola A; Burgess, Stan A; Forgacs, Eva et al. (2010) Influence of lever structure on myosin 5a walking. Proc Natl Acad Sci U S A 107:2509-14 |
| Sellers, James R; Veigel, Claudia (2010) Direct observation of the myosin-Va power stroke and its reversal. Nat Struct Mol Biol 17:590-5 |
| Nagy, Nikolett T; Sakamoto, Takeshi; Takacs, Balazs et al. (2010) Functional adaptation of the switch-2 nucleotide sensor enables rapid processive translocation by myosin-5. FASEB J 24:4480-90 |
| 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 |
| Forgacs, Eva; Sakamoto, Takeshi; Cartwright, Suzanne et al. (2009) Switch 1 mutation S217A converts myosin V into a low duty ratio motor. J Biol Chem 284:2138-49 |
| Sakamoto, Takeshi; Webb, Martin R; Forgacs, Eva et al. (2008) Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature 455:128-32 |
| Forgacs, Eva; Cartwright, Suzanne; Sakamoto, Takeshi et al. (2008) Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke. J Biol Chem 283:766-73 |
Showing the most recent 10 out of 12 publications
