Myosin Va maneuvers through actin intersections and diffuses along microtubules

Abstract

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.

Fig. 1.

Illustration of in vitro cytoskeletal intersection model and experimental design. (A) To model myoVa's ability to maneuver through the actin cytoskeleton, actin intersections were created on a coverslip by first attaching Alexa Fluor 660 (red) actin followed by TRITC-actin filaments (green). In addition, branched filaments were created by using Arp2/3 (see Materials and Methods). (B) Microtubular-actin intersections were created on a coverslip by first attaching Alexa Fluor 660 actin followed by rhodamine-labeled microtubules (see Materials and Methods).

Fig. 2.

Sequential images of Qdot-labeled myoVa encountering various forms of actin filament intersections. A single myoVa motor (red dot) travels along an Alexa Fluor 660 phalloidin-labeled actin filament (not visible due to rapid photobleaching) in the sequences labeled “crossover,” “right turn,” and “terminate” before encountering a TRITC phalloidin-labeled actin filament (green). The TRITC actin filaments are draped over the Alexa Fluor 660 actin filaments based on the order of actin filament addition to the flow cell. The time sequence is shown in the lower right corner. In the sequence labeled “Arp2/3,” a myoVa motor travels along a TRITC actin filament (green) before encountering an Arp2/3 branch, which it takes up to the top of the image.

Fig. 3.

High spatial resolution analysis of myoVa encountering actin filament intersections. (A) MyoVa X, Y-positional information are shown as it travels in 500 nM ATP along an Alexa Fluor 660 actin filament (red line) and either crosses over or turns onto a TRITC actin filament (green line). The myoVa position (black circles) was determined by using FIONA every 0.33 s and, when paused at each step, multiple data points were averaged to give a final position (red circles). The distance between these final positions are provided. For the crossover example, the myoVa takes a 99-nm step to make it over the TRITC actin filament while taking 83 ± 19-nm steps during its run. For the turning example, the myoVa motor took a 51-nm step to switch to the intersecting filament while taking 80 ± 22-nm steps over its entire run. (B) Simple model explaining turning versus crossover frequency. MyoVa (light purple) at final position on Alexa Fluor 660 actin (red) before encountering a TRITC actin intersection (green). The Qdot-labeled trailing head (red star), when undergoing its step to its new position as the leading head (dark purple), undergoes a diffusional search that samples the available actin monomers (30 yellow and 7 blue monomers) within a 50- to 95-nm target zone (gray arc), determined by the range of distances normally observed between heads while paused on actin. The ratio of yellow or blue monomers to the 37 total monomers within the target zone gives an estimate of the probability for turning versus crossing over. (C) Histogram of the final myoVa distance to an actin intersection that resulted in run termination. Distances >100 nm were considered spontaneous terminations. Distances <100 nm were well fit by a Gaussian (51 ± 3 nm), which most likely represents the myosin's center of mass (see text). With only one head Qdot labeled and with a probability of labeling being equal for the two heads, it may be possible with sufficient data to resolve two Guassians (hypothetical blue and red dotted lines) that are 36 nm apart representing each of the heads. (Inset) A histogram of the lifetime of the last step before termination, resulting in an effective termination rate of 0.6 s⁻¹.

Fig. 4.

One dimensional diffusive search of myoVa on microtubules. (A) Images of Qdot-labeled myoVa (red) diffusing on TRITC-labeled microtubule. The sequence of images was obtained at the following times in seconds starting from the left: 0, 6, 9.6, 14.6, 22.6, 27.9, 37.2, 40.2, 43.5, 57.5, 69.5, 82.2, 86.5. (B) Images as in A were analyzed to determine the displacement between successive image frames (three frames per s) and then plotted as a displacement histogram. A single Gaussian was fit to the data using the equation: y = a exp[−0.5((x − x_o)/b)²], with the following parameters: a = 37.39, b = 431.53 nm, and x_o = −34.7 nm. From these data, the variance = 2Dt, where D is the diffusion coefficient and t is the time interval between images, resulting in D = 0.28 μm²/s. (C) The mean square displacement (MSD) is plotted versus time, with the slope providing an estimate of D, as follows: D = 〈Δx²〉/2t, where Δx is the change in distance measured from the starting point of the record during time interval t. For this example, D = 0.33 μm²/s. (D) The number of myoVa per μm of microtubule length that were associated and diffusing on the microtubule was determined as a function of ionic strength by varying [KCl]. The [myoVa] in solution was 1.25 nM. Upon subtilisin treatment of tubulin to remove the charged E-hook, the number of associated myoVa/μm microtubule (red filled circle) decreased significantly at 25 mM KCl (57 mM ionic strength equivalent). (E) Cleavage of the C-terminal E-hook from microtubules. (Left) SDS/8% PAGE gel of undigested microtubules (lane 1), and after digestion with increasing concentration of subtilisin (lanes 2,3; see Methods). (Right) Immunoblots of these three lanes with anti-α or anti-β tubulin antibodies. Cleavage of the E-hook of β-tubulin results in loss of epitope reactivity.

Fig. 5.

Illustration of cargo transport model. Organelle transport from the cell center near the nucleus and microtubular organizing center (MTOC) to the cell periphery involves kinesin movement on microtubules with handoff to myoVa for delivery on actin filaments. (A) Kinesin, carrying cargo, moves toward the microtubule plus-end (+) and encounters a diffusing myoVa that either landed on or stepped onto the microtubule at a microtubule-actin intersection. The partially bent tail for the diffusing myoVa represents the possibility that myoVa can diffuse in either the extended conformation (see text) or in the folded, inhibited state in the absence of cargo. The processively moving myoVa on actin could be in the extended conformation due to changes in calcium concentration or to cargo-binding. (B) Upon encountering myoVa, kinesin and myoVa associate directly through tail–tail interactions or by myoVa binding to the cargo. Kinesin would then continue on toward the periphery. (C) Near the periphery, myoVa would then take over cargo delivery on actin tracks. MyoVa can then effectively maneuver through actin intersections and Arp2/3-branched filaments.