Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VI

Abstract

Myosin VI (Myo6) is unique among myosins in that it moves toward the minus (pointed) end of the actin filament. Thus to exert tension on, or move cargo along an actin filament, Myo6 is working against potentially multiple plus (barbed)-end myosins. To test the effect of plus-end motors on Myo6, the gliding actin filament assay was used to assess the motility of single-headed Myo6 in the absence and presence of cardiac myosin II (Myo2) and myosin Va (Myo5a). Myo6 alone exhibited a filament gliding velocities of 60.34 ± 13.68 nm/s. Addition of either Myo2 or Myo5a, at densities below that required to promote plus-end movement resulted in an increase in Myo6 velocity (~100-150% increase). Movement in the presence of these plus-end myosins was minus-end directed as determined using polarity tagged filaments. High densities of Myo2 or Myo5a were required to convert to plus-end directed motility indicating that Myo6 is a potent inhibitor of Myo2 and Myo5a. Previous studies have shown that two-headed Myo6 slows and then stalls in an anchored state under load. Consistent with these studies, velocity of a two headed heavy mero myosin form of Myo6 was unaffected by Myo5a at low densities, and was inhibited at high Myo5a densities.

Fig. 1

EDC crosslinking of human Myo6 and Myo5a. Immunoblot analysis of 25 μg/ml Myo6 and Myo5a without (-) and with (+) EDC crosslinking. Note the three bands in the Myo6 lane after EDC treatment presumably representing Myo6 heavy chain with 0-2 crosslinked CaM light chains. In contrast EDC treatment of Myo5a results in complete loss of monomeric heavy chain and a very high molecular weight smear is present, presumably representing Myo5a heavy chain dimer and variable numbers of light chains.

Fig. 2

Gliding filament velocities of Myo2, Myo5a, Myo6, and Myo6 HMM as a function of motor density. Velocities of rhodamine-labeled actin filaments propelled by directly adsorbed Myo2 purified from mouse cardiac tissue (A), Myo5a purified from chick brain (B), baculo-virus expressed Myo6 (C) and Myo6 HMM (D) in the motility assay are shown. The concentrations of myosin used to coat the surfaces of the motility chambers are shown on the x-axes. Filament velocity averages were obtained from a minimum of 2 days of experiments. Values are represented as mean ± SD. The number of filaments for which velocities were measured ranged from 4-23 (A), 5-17 in (B) and 10-20 in (C) and 7-27 in (D).

Fig. 3

Single headed Myo6, but not Myo6 HMM velocity increases in the presence of plus-end directed motors. (A,B). Percentage change (relative to Myo6 alone) in gliding velocity of filaments by 0.014 μM Myo6 with the addition of Myo2 (A),Myo5a (B) is shown as a function of adsorption concentration of either Myo2 or Myo5a. (C). Motility of 0.2μM Myo6 HMM in the presence of increasing concentrations of Myo5a. Motility of either 0.014 μM Myo6 or 0.2 μM Myo6 HMM in the absence of plus-end motor on each day was considered baseline. Bars indicate average of percent-differences obtained from 2-3 days of experiments in (A and C) and 2-4 days of experiments in (B). Values are expressed as average ± SD. Number of filaments for which velocities were measured ranged from 10-66 in (A), 30 – 127 in (B) and 9-67 in (C).

Fig. 4

Myo6 motility is minus-end directed in the presence of Myo2 (A) and Myo5a (B). (A) Velocity distribution of two-color polarity tagged filaments by 0.014μM Myo6 in the absence (open bars) and presence of 0.5nM (hatched bars), 125 nM (gray bars) and 2.0 μM (black bars) Myo2 is shown. (B). Velocity distribution of polarity tagged filaments in the absence of Myo5a (open bars) and presence of 0.17 nM (hatched bars), 0.93 nM (grey bars) and 37 nM (black bars) Myo5a is shown. For (A) and (B) negative and positive numbers on the x-axis indicate minus-end and plus-end movement of filaments respectively. Only at high densities of Myo2 or Myo5a were plus-end directed movements observed.

Fig. 5

Distribution of velocities for Myo6 in the absence and presence of plus-end myosins at Myo2 (A) and Myo5a (B) concentrations that exhibit maximum increases in Myo6 motility. Gliding velocities as a function of number of filaments observed by 0.014 μM Myo6 in the absence (open bars) or presence (hatched bars) of either 0.5 nM Myo2 (A) or 0.9 nM Myo5a (B) are shown. Note that there is no overlap in velocities in the absence and presence of plus-end motor, indicating that the increased velocities is not an average of slower Myo6-minus end movement and faster plus-end motor mediated movement.

Fig. 6

NEM S1 does not increase Myo6 velocity. The histogram depicts velocity (open bars) and number (grey bars) of landing filaments moved by 0.014 μM Myo6 in the absence and presence of NEM S1. Values in the open bars are represented as mean ± SD. No increases in velocities are observed and at the higher NEM-S1 concentrations but movement is inhibited with respect to numbers of filaments moving (7.7 nM), and then completely blocked (19 nM).