Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

Abstract

Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments.

Fig. 1.

Myosin V and VI motility on the actin cytoskeleton of extracted Drosophila S2 cells. (A) Preserved actin cytoskeleton in an extracted S2 cell visualized via rhodamine-phalloidin. The center point of the cell is indicated (+, red). The angle is defined as the orientation angle and measured from the centroid of the cell to the beginning of a motor run (arrow). (Scale bar, 10 μm.) (B) Trajectory map (white arrows) of all myosin V motor (green) runs on the preserved actin cytoskeleton (red). Each arrow represents a single moving motor run. A processive run is defined as a linear movement lasting at least 3 consecutive frames (≥ 1.5 s). Each frame is acquired with a 0.5-s exposure for 200 frames. The majority of motors (76%) move toward the cell periphery. Very few MV motors (n = 7/138) actually switch actin tracks throughout this analysis (cyan). (C) Orientation angle measurements for processive runs. The majority of MV motors have a trajectory angle of 100–180°, indicating that the motors move toward the cell periphery. (D) The zone of motility (yellow outline) is where most motors move toward the cell periphery with a similar velocity. This motility zone is directly adjacent to a central zone of slow motility. (E) Trajectory map (white arrows) of single myosin VI motors (green) moving on a preserved actin cytoskeleton (red). Each arrow represents a single motor run. The majority of motors (72%) move away from the cell periphery. (Scale bar, 10 μm.) (F) Orientation angles measured for all MVI motors making a processive run. Most motors have a trajectory angle of 0–80°, indicating that these motors move away from the cell periphery or toward the cell center. (G) MVI motors that switch actin tracks are depicted (white). There are more MVI motors that switch than MV motors (compare to panel B, cyan). (H) Tracking pattern of a MVI motor that switches actin tracks (boxed in G). The motor runs for 26 frames over a distance of 1.5 μm. Since this motor tracks with a sharp turn (>20°), it is characterized as a motor that switches actin tracks during motility.

Fig. 2.

Myosin X motors move on filopodia and around the lamella edge of permeabilized Drosophila S2 cells. (A) Multiple extracted S2 cells displaying filopodia, lamella edge (purple dotted line) and a cell edge (yellow dotted line). The actin cytoskeleton is visible with TMR-phalloidin. (Scale bar, 10 μm.) (B) Trajectory map (white arrows) of all moving MX motors throughout these cells. (C) MX motors that make runs on filopodia (red circles) and around the lamella edge (magenta) move at various velocities (Fig. S6) on this bundled actin. (D–F) Orientation angle measurements for all moving MX motors. Motors moving on filopodia (D) have a center angle of 100–180°, indicating that motors start at the base of filopodia and run out, toward the cell periphery. Center angles determined for motors moving on the lamella (E) indicated that these motors move circumferentially at an angle of 55–120°. All other motors moving throughout the cells revealed an uncorrelated center angle measurement (F), indicating that these motors move in all directions. (G) Some MX motors switch tracks (white) while making a run. These track switching events do not occur on the filopodia (red lines), the lamellar edge (purple dotted line), or at the cell edge (yellow dotted line).

Fig. 3.

Myosin V, VI, and X motility on extracted mammalian COS-7 and U2OS cells. Trajectory maps of moving myosin motors. MV runs in COS-7 (A) and U2OS (D) cells. MV motors in both cells move with the same velocity (Table 1 and Fig. S6), but considerably fewer runs are made in U2OS (D) compared with COS-7 (A) cells. MVI runs in COS-7 (B) and U2OS (E) cells. MVI motors move at a similar velocity and run length (Fig. S6 and Table 1) in both COS-7 and U2OS cells. Although, MVI runs track along the large stress fibers that run the length of U2OS cells (E). MX runs in COS-7 (C) and U2OS (F) cells. Few MX runs are detected in COS-7 cells, but even fewer are detected in U2OS cells. (Scale bars, 10 μm.)

Fig. 4.

Summary of unconventional myosin tracking in S2, COS-7, and U2OS cells. A cartoon of the spatial arrangement and properties of actin filaments or bundles in the 3 cell types examined here. The number of symbols displayed in each cell is directly proportional to the percentage of moving motors in that cell type (1 symbol = 1%). An arrow indicates a motor run. A lollipop arrow indicated a run in the opposite direction than expected. Chevrons indicate motors that switch tracks. MV motors in S2 cells show the highest percentage of moving motors compared to all cell types. These MV runs are primarily directed toward the cell periphery, but a percentage of motors do run toward the cell center (lollipop arrows). Runs that are made in the center of the cell move at a slower velocity (light blue arrows) than those made in the outer cortical region of the cell. There are also a good percentage of moving MV motors in COS-7 cells. These runs are not as directed as in S2 cells and are throughout the cell. In U2OS cells, few MV runs are seen. MVI motors in S2 cells primarily track toward the cell center, but a fraction move toward the cell periphery (lollipop arrows). A few runs are also found on a single filopodium (gray arrow). Isotropic MVI runs are seen in COS-7 cells, but fewer than MV. In U2OS cells, MVI is the primary moving motor. MVI makes significantly more runs than MV or MX on stress fiber bundles. MX takes the fewest runs in every cell type. In S2 cells, MX motors travel on filopodia and around the lamellar edge as expected, but overall MX makes significantly fewer runs than MV and MVI. Track-switching behavior was observed throughout this analysis, with the most track-switching behavior exhibited by MX in COS-7 cells.