Abstract
Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30–36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.
Highlights
Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells
Individual Myo5c molecules in two-dimer complexes move stochastically in 30–36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells
In the current study, we found that the duty ratio of Myo5c-heavy meromyosin (HMM) was increased to 40%
Summary
Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. For a single dimer of myosin 5 to be an effective cargo transporter, it is important that the reattachment rate of its leading head to actin (Actin 1 M-ADP-Pi R ActoM-ADP-Pi R AM-ADP) is much faster than the dissociwww.nature.com/scientificreports ation rate of the rear head In this manner, at least one of the two heads remains attached, allowing continuous movement of the motor molecule along the actin filament. Further study using dimeric myosin 5a heavy meromyosin (HMM) fragment showed biphasic kinetics of ADP release in the presence of actin when both heads were strained by simultaneous binding to an single actin filament[32] This demonstrates that strain resulting from the two heads of myosin 5a binding to actin increases the run length by reducing the rate of ADP dissociation from the leading head. We show that two molecules of Myo5c-HMM dimers linked with each other on a DNA scaffold can move processively along actin filaments
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