Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection

Abstract

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin-actin) or mixed polarity (α-actinin-actin) bundles. The stepping dynamics of individual Qdot-labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor.

Figure 1

Experimental and Motor Design. (A) Schematic of expressed myoVI, myoVa and myoVI chimera used in this paper. The myoVI motor domain is followed by a unique insert-2 (IN2), a conventional IQ motif that binds an exchangeable calmodulin (CaM), a tail domain, eGFP, a leucine zipper (ZIP) for dimerization, and finally a FLAG tag at the C terminus. The myoVa N-terminal, motor domain is followed by six IQ motifs (IQX6) with their bound CaM, a coiled coil tail domain, YFP, and finally a FLAG tag at the C terminus. The myoVI chimera was generated by replacing the myoVI single IQ by the myoVa 6IQ. (B) An image of a unipolar fascin-actin bundle. (C) Illustration of an in vitro cytoskeletal intersection model. Actin filament intersections were created on glass surface by first attaching Alexa 660 (green) actin followed by TRITC-actin filaments (blue) (24). Stepping dynamics were monitored by imaging the Qdot (red star) attached to each motor (see Material and Methods). (D) Illustration of unipolar fascin-actin bundle with motors and their associated target zones (semi-circular shaded areas). Polarity of actin designated by plus and minuses.

Figure 2

Velocity and run lengths of motors on single actin filaments and bundles. (A) Velocities of motors on different tracks are: MyoVI: 146±51 nm/s, N=118 (single actin filament), 136±50 nm/s N=95 (unipolar bundles), 85±43 nm/s N=65 (mixed polarity bundles); MyoVa: 407±98 nm/s, N=84 (single actin filament), 330±133 nm/s, N=82 (unipolar bundles), 275±130 nm/s, N=47 (mixed polarity bundles); Chimera: 143±52, N=72 nm/s, N=72 (single actin filament), 120±52 nm/s, N= 65 (unipolar bundles), 73±42 nm/s N=44 (mixed polarity bundles). (B) Run lengths of motors on different tracks are: MyoVI: 620±110 nm, N=118 (single actin filament), 550±100 N=95 (unipolar bundles), 450±90 (mixed polarity bundles); MyoVa: 1120±130 nm, N=84 (single actin filament), 2200±150 nm, N=82 (unipolar bundles) 2070±230 nm, N=47 (mixed polarity bundles); Chimera: 440±110 nm, N=72 (single actin filament), 500±90 nm, N=65 (unipolar bundles), 420±90 nm N=44 (mixed polarity bundles). Asterisk sign (*) indicates statistical significance (p<0.05) in comparison to the velocity and run length on single actin filament.

Figure 3

High spatial and temporal resolution imaging of myoVI, myoVa and myoVI chimera stepping while travelling on unipolar and mixed polarity bundles. All three motors move in stepwise manner on fascin (A) and α-actinin bundles (B) with filament switching indentified (blue asterisk). The average step position is denoted by red square. Head tapping events between two filaments identified by black double arrow. (C) Example of myoVa head tapping on unipolar bundle. Gray points represent the head position at each frame (16.7 ms) with the open circles the average head position for all frames at a given step or tap. Sequence of events from left to right: 1) step (first black open circle; lifetime=1139 ms), 2) head tapping (multiple taps with time sequence color coded from left to right open circles; average tap lifetime= 198 ms), 3) step (last black open circle; lifetime=1440 ms).

Figure 4

Turning angles between successive steps. (A) A schematic diagram of angular displacement measurement (θ) relative the x-axis while a motor switches tracks (dashed line) on actin bundles. (B) Turning angles of myoVI, myoVa and myoVI chimera on unipolar bundles are −1±22 ° (N=88), 9±40° (N=114), 7±30° (N=54) respectively. As a control, turning angles of myoVI on single actin filament were measured as 1±11° (N=69) (black dotted line). (C) Similarly, the turning angles of myoVI, myoVa and myoVI chimera on mixed polarity bundle are 9±29° (N=68), 6 ±48° (N=64), 5±38° (N=56), respectively.

Figure 5

Stepping dynamics of myoVI, myoVa and myoVI chimera at 50μM ATP on actin filaments and bundles. Displacement versus time traces of myoVI (A, B), myoVa (D, E) and the myoVI chimera (G, H) on single actin and unipolar bundle, respectively. All three motors take occasional back steps (arrows in B, E(inset), H). MyoVa and myoVI chimera head tap between two defined binding sites (E, H). Step size distributions of all three motors were fitted to single Gaussians with values, (C) myoVI: 63±21 nm, N=155 (single actin filament), 57±27 nm, N=111 (unipolar bundles), 60±25 nm, N=64 (mixed polarity bundles); (F) myoVa: 72±10 nm, N=142 (single actin filament), 63±26 nm, N=127 (unipolar bundles), 64±24 nm, N=81 (mixed polarity bundles); (I) MyoVI chimera: 70±28nm, N=67 (single actin filament), 65±24 nm, N=99 (unipolar bundles), 64±32 nm, N=63 (mixed polarity bundles).