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Myosin learns to walk

Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA

View larger version (62K): [in a new window]   Fig. 1. (A) An actin filament moves over a surface coated with 3.6 myosin V molecules/µm2 in 2 mM ATP. The pointed (green dots) and barbed (yellow dots) ends of the filament are marked when the ends are in the image plane. Panels 1-10 (left to right) show the time course of a filament before (panel 1) and after (panels 2-9) it has bound the surface. The apparent point of surface contact (crosshair) appears pronounced in the average fluorescent intensity throughout the time course of movement (panel 10). Panels 11-20 show the time course of a shorter filament moving over 5.4 molecules/µm2. This filament encounters a second contact point before releasing its first. Nodal swiveling behavior reminiscent of seminal observations with kinesin (Howard et al., 1989; Hunt and Howard, 1993) provided the first hint of myosin V processivity (Wang et al., 2000; Mehta et al., 1999a). Bar, 5 µm. (B) The rate at which actin filaments land and move, as a function of myosin V surface density. A filament was considered ‘landed’ if it moved >0.5 µm and for >2 seconds. (C) The fraction of filaments that moved more than their length (as in panels a11-a20) before dissociating, as a function of surface density. The fit reflects the single molecule model prediction of P(n>1|n>0), where P(N) represents the density-dependent probability that N molecules populate an arbitrary area. Figure reproduced, with permission, from Mehta et al., 1999a (http.//www.nature.com).

View larger version (35K): [in a new window]   Fig. 2. (A) Illustration of force-clamp records. An optically trapped bead, decorated with myosin V molecules, is moved into close proximity with a surface-mounted actin filament. The bead is subjected to a force-clamp, in which a feedback circuit maintains a constant trap-bead separation and hence constant system tension (left). In some cases, the bead proceeded to step along the actin filament in 36-nm increments (right) as the trap followed. (B) The fraction of beads observed to step as shown in A, as a function of surface density. The solid line reflects a fit of a single molecule model; the dotted line reflects a model posing two molecules as a minimal agent of such stepping behavior. Reduced 2 values for the one- (0.04) and two-molecule (0.98) models rendered relative confidence in the former 25 times that in the latter. Figure reproduced, with permission, from Rief et al., 2000.

View larger version (39K): [in a new window]   Fig. 3. Single myosin V molecules, imaged by total internal reflection microscopy, move along isolated actin filaments. Myosin V molecules are shown in green; actin is shown in red. Fluorophores appear to accumulate at the barbed filament end. This suggests that, when a single moving myosin V encounters a problem ahead (an absent or occupied binding site), the trailing head remains anchored upon the filament while the leading head remains unbound. Leading head binding may thus induce trailing head dissociation, which is similar to proposed mechanisms for kinesin (Hancock and Howard, 1999). However, uninteresting phenomena, for instance motors moving off the actin filament end and then adhering nonspecifically to the surface substrate, could also underlie observed accumulation. Figure reproduced, with permission, from Sakamoto et al., 2000.

View larger version (18K): [in a new window]   Fig. 4. Load-dependent dwell times separating adjacent step transitions and preceding forward-directed steps. Open boxes represent 1 µM ATP, and closed circles 2 mM ATP. The line traversing the closed circles reflects a fit of Eqn 3 to the data. Figure reproduced, with permission, from Nature (Mehta et al., 1999a).

View larger version (85K): [in a new window]   Fig. 5. Electron micrographs of single myosin V molecules attached to single actin filaments. The shown molecules have two motor domains spaced by 13 subunits, which is the most common spacing observed. Several molecules appear to mimic a snowboarder’s riding stance, in which their leading neck arcs forward, as if under tension to move forward. Figure reproduced, with permission, from Nature (Walker et al., 2000).

View larger version (13K): [in a new window]   Fig. 6. Molecular model proposed by Rief et al. (Rief et al., 2000). Grey motor domains correspond to weak (low affinity) binding to actin, and black motor domains correspond to strong (high affinity) binding to actin.

View larger version (24K): [in a new window]   Fig. 7. Limping behavior, observed extremely rarely, under saturating ATP. Every second step occurs at 12 s-1, intervening steps require much longer periods. The existence of such data suggests that every second step occurs from a similar biochemical state, which is consistent with models predicting that the two heads alternate roles with each unitary advance. The step distance in this data, taken with the dual-bead trapping geometry, is probably compromised by elastic linkages in the system.