Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to Actin

Abstract

Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of ϳ30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was ϳ60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position,whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin.The myosin superfamily is composed of 18 classes of molecular motor proteins, the vast majority of which traffic toward the barbed (ϩ) end of actin filaments (1). Class VI myosins were the first of the superfamily identified to traffic toward the pointed (Ϫ) end of the actin filament (2). They were first identified in Drosophila melanogaster (3) and are expressed from Caenorhabditis elegans to human (4 -6). In addition to its unusual directionality, myosin VI has a number of additional unusual features. Single molecules of two-headed myosin VI, like myosin V, are capable of taking multiple steps (processive movement) on an actin filament without detachment (7). However, while myosin V has been demonstrated to move along actin filaments in 36-nm steps using its long lever arm (containing six calmodulins (CaMs) 1 ) via a hand-over-hand mechanism (8 -12), myosin VI does not appear to use a simple lever arm mechanism for its motility (7, 13). Myosin VI has been shown recently to have only two CaMs bound to each head (14), which should result in a short effective lever arm and small step size. Surprisingly, myosin VI has a step size that is highly variable (7) but on average nearly as large as that of myosin V, which has a lever arm that is three times longer. The variability of the step size has led to the postulate that myosin VI contains a long elastic element that allows it to undergo biased diffusion on an actin filament. The elastic element could be attached to the short lever arm, and/or the lever arm itself could become loosely attached to the motor at some point in the motile ATPase cycle. It was initially postulated that the unique insert of 39 amino acids in myosin VI that precedes the IQ motif (CaM-binding site) might come apart to form this flexible linker during the motile cycle on actin (7). This would result in the CaM being significantly displaced from the motor domain.EM images of two-headed myosin V bound to actin s...

The structure of the myosin VI motor reveals the mechanism of directionality reversal

“…Consistent with this hypothesis is data that placed fluorescent probes either on the IQ-CaM or on the N-terminal subdomain of two-headed myosin VI 32 . Using FIONA (fluorescence imaging with one nanometer accuracy), large fluctuations in the position of the IQ-CaM, but not in the position of the motor domain, were observed during processive movement.…”

Myosin VI Lever Arm Rotation: Fixed or Variable?

“…This point is confirmed by ours and earlier data [7][8][9] . Thus, the geometrical relationship between the two lever arms should depend on the distance between the two heads after any given step, a point made previously by both groups 2,9 .…”

“…This point is confirmed by ours and earlier data [7][8][9] . Thus, the geometrical relationship between the two lever arms should depend on the distance between the two heads after any given step, a point made previously by both groups 2,9 . In Reifenberfer et al 1 , the variable step size is dismissed by postulating that each molecule walks straight, rarely taking sideways steps, and the overall distance distribution is broad due to contributions from different molecules.…”

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

“…Kinetic analysis again highlights the presence of 0 nm hidden steps, giving an additional support to hand-over-hand motion ( Figure 8B). 52 Unexpectedly, Cy3-calmodulin showed significant fluctuation in its ATP state while it is highly immobile in the rigor and ADP state. This implies that in some part of myosin VI's ATP state, the lever arm uncouples from the motor, which may potentially arise from the proposed elongation of the lever arm.…”

“…Labeled calmodulins exchange only with the second binding site on the lever arm. 52 The myosin VI FIGURE 6. The dwell time histograms for 74-0 nm steps (red crosshatched) displays an initial rise and then decays slowly.…”