Coordination of opposite-polarity microtubule motors

Abstract

Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.

Figure 1.

Models for how opposite-polarity motors on single cargoes might interact. (A) In the tug of war model, opposite-polarity motors are active simultaneously. Net motion results when one set of motors successfully competes against the opposing motors. (B) In the motor coordination model, competition is avoided because when plus-end motors are active, minus-end motors are turned off and vice versa. For clarity, only the cargo and the motors are depicted; hypothetical molecules that allow the motors to assemble into complexes and that mediate interactions between motors are not shown.

Figure 2.

Droplet stalling forces for minus-end travel. The panels show the percentage of droplets stalled in different genetic backgrounds as a function of force applied by optical tweezers. (A) Wild- type versus Dhc64C ^6–10/+ and Dhc64C ^8–1/+; (B) Wild-type versus Gl¹/+. To avoid bias, force measurements were performed in a blind fashion, with the genotype of the embryo being measured unknown to the person performing the force measurement. Each data point is derived from measurements on six to seven embryos in phase II, with ∼30 minus-moving droplets tested per embryo. See Materials and methods for a discussion how applied force and stalling force are related.

Figure 3.

Droplet stalling forces for plus-end travel. As for Fig. 2, the panels show the percentage of droplets stalled as a function of applied force. (A) Wild-type versus Dhc64C ^6–10/Dhc64C^8–1; (B) wild-type versus Dhc64C ^6–10/+ and Dhc64C ^8–1/+; (C) wild-type versus Gl¹/+. Forces were determined as for Fig. 2, in the same embryos.

Figure 4.

Mean travel speed as a function of run distance, for minus- (A) and plus-end (B) motion. The average speed of short (30–100 nm) or long (500–1000 nm) runs is shown, for droplets moving in wild-type, Dhc64C ^6–10/Dhc64C^8–1, Dhc64C ^8–1/+, Dhc64C ^6–10/+, and Gl ¹/+ genetic backgrounds. Data are from six to seven phase II embryos per genotype, with 120 to 170 runs per embryo analyzed. The error bars are the standard error of the average.

Figure 5.

Distribution of lengths of droplet travel in the plus-end direction (35-nm bins). The location of individual droplets as a function of time was determined with nanometer-level resolution using centroid analysis, and periods of uninterrupted motion (runs) were automatically detected with a custom program (Gross et al., 2000). Histograms shown are of plus-end run distances in (A) wild-type and (B) Dhc64C ^6–10/Dhc64C^8–1 embryos. The general shape of the histograms was the same, well fit by the sum of two decaying exponentials (solid line; see Table I for values for these fits). For comparison, Panel A shows data previously published (Gross et al., 2000). Histograms are based on ∼950 total runs per genotype, from six to seven phase II embryos.

Figure 6.

Distribution of lengths of droplet travel in the Gl¹ background (35-nm bins), for (A) minus-end and (B) plus-end travel. Histograms and fits were done as for Fig. 5. The general shape of the histograms was again well fit by the sum of two decaying exponentials (solid lines, see Table I for values). The histograms are based on droplets tracked in six phase II embryos.