A nonprocessive class V myosin drives cargo processively when a kinesin- related protein is a passenger

Abstract

During secretory events, kinesin transports cargo along microtubules and then shifts control to myosin V for delivery on actin filaments to the cell membrane [1]. When kinesin and myosin V are present on the same cargo, kinesin interacts electrostatically with actin to enhance myosin V-based transport in vitro [2]. The relevance of this observation within the cell was questioned. In budding yeast, overexpression of a kinesin-family protein (Smy1p) suppressed a transport defect in a strain with a mutant class V myosin (Myo2p) [3]. We postulate that this is a cellular manifestation of the in vitro observation. We demonstrate that Smy1p binds electrostatically to actin bundles. Although a single Myo2p cannot transport cargo along actin bundles, addition of Smy1p causes the complex to undergo long-range, continuous movement. We propose that the kinesin-family protein acts as a tether that prevents cargo dissociation from actin, allowing the myosin to take many steps before dissociating. We demonstrate that both the tether and the motor reside on moving secretory vesicles in yeast cells, a necessary feature for this mechanism to apply in vivo. The presence of both kinesin and myosin on the same cargo may be a general mechanism to enhance cellular transport in yeast and higher organisms.

Figure 1. Smy1p binds to and diffuses on both microtubules and actin bundles

(A) The interaction between Smy1p-coated streptavidin Qdots and rhodamine-labeled microtubules was observed by TIRF microscopy. (B) Kymograph (position versus time) of a Smy1p-coated Qdot diffusing on a microtubule. (C) The mean squared displacement increases linearly with time (r² = 0.999), indicating a diffusive process with a diffusion coefficient of 0.11 ± 0.07 µm²/sec. Error bars indicate S.E.M. (N = 22). (D) The experiment was repeated with rhodamine-labeled actin bundles. (E) Smy1p-coated Qdots (red) bind to actin bundles (green). (F) The number of Qdots bound per micron of actin bundle decreases as the ionic strength is increased, indicating that the interaction is electrostatic in nature. Error Bars represent S.E.M.

Figure 2. Actin filaments binding to a Smy1p-coated coverslip as a function of ionic strength

(A) Diagram of experimental setup. A Smy1p-coated coverslip was incubated with rhodamine-labeled actin filaments, then rinsed with and imaged in buffers with varying ionic strength. (B) Actin binding to a Smy1p-coated coverslip in 100–500 mM K-Acetate. Actin filaments do not bind to the coverslip when Smy1p is omitted (Control, imaged in 100 mM K-Acetate).

Figure 3. A single Myo2p transports a Qdot long distances in the presence of Smy1p

(A) Streptavidin Qdots were bound to multiple Smy1p molecules and at most one Myo2p motor. Their interaction with actin bundles was observed by TIRF microscopy. (B) TIRF microscopy image of one Qdot (red, identified by the yellow arrow) undergoing continuous and unidirectional movement along an actin bundle (green). The quantum dot moved 7.2 µm before dissociating at the end of the bundle. The remaining Qdots were either stationary or underwent short-range diffusion (<1 µm in length). The time between frames is ~0.67 seconds. (C) Histogram of all processive runs of at least 1 µm in length.

Figure 4. Smy1p is transported on secretory vesicles

(A) Smy1p-mCherry co-localizes with Sec4p-GFP. Both proteins localize to particles representing single or small clusters of secretory vesicles. (B) The first 5 panels show snapshots of a Smy1p-GFP particle (small bright spot, tracked by arrow) as it travels in the mother cell, through the mother-bud neck, and into the bud. The particle moved at 1.8 µm/s in the mother cell, but slowed down to 0.4 µm/s as it passed through the neck. The last panel (SD) is a standard deviation projection of the image sequence, highlighting areas of maximum variation in the intensity (see Supplemental Methods). This projection shows the trajectory followed by the particle. (C) A Sec4p-GFP particle (small bright spot, tracked by arrow) moves through the mother cell at 2.0 µm/s to the emerging bud (large bright spot). The last panel (Max) is a maximum projection of the image sequence, showing the trajectory followed by the particle.