In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport

Abstract

Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (∼plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (∼minus-end) stalls in vivo were 2-3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.

Fig. 1.

In vivo stall force measurements differ in the outward and inward directions. Stall force histograms of outward (n = 64) (A) and inward (n = 36) (B) stalls for A549 lipid droplets in vivo. Stall force histograms for Dictyostelium phagosomes (C and D), another in vivo system, are quite similar to A549 measurements and also reveal an asymmetry (outward n = 53, inward n = 38). The inward and outward distributions for both cell types are significantly different as determined by a Wilcoxon rank-sum test to the following P values: A549, P = 0.029; Dictyostelium, P = 0.0126. Both systems reveal outward stall forces that differ from the stall force of kinesin in vitro, which is the motor expected to drive outward motion. These histograms are an accumulation of stall measurements from multiple cells, and each stall force is measured by multiplying the stiffness (calibrated during acquisition independently for each organelle) by the distance moved during the stall. (E) Example stall traces for both directions for both cell types, with red boxes indicating the stalls (a quarter second pause before and after a movement >20 nm; Methods). The first two are outward stalls (2.8 and 5.2 pN) and the last two are inward stalls (1.7 pN for the A549 vesicle and 2.6-, 2.1-, and 2.0-pN stalls for the Dictyostelium trace). Each of these traces is the entirety of a typical 7-s data acquisition, which is judged for stall force and stall directionality after acquisition (Methods). In vivo traces were in two dimensions, but only the dimension along which the majority of motion occurred is displayed here.

Fig. 2.

In vivo stall force behavior can be replicated by motor-coated beads in vitro. (A) Stall force histogram of single molecules of mouse kinesin-1 in vitro, mean of 6.3 ± 1.1 pN (1 SD). This histogram shows that outward stall behavior cannot be explained by kinesin-1 alone. (B) The in vitro stall forces for purified Dictyostelium phagosomes with only DdUnc104, a kinesin-3, or beads coated with a kinesin purified from A549 cells also cannot explain the outward stall force observed in vivo (6.8 ± 1.2 pN mean for phagosomes, n = 37, and 6.8 ± 1.1 pN, n = 43, for A549 kinesin on beads). However, the stall force for plus-end directed beads coated with both kinesin-1 and dynein in vitro (n = 84) (C) replicates outward stall behavior in vivo (here compared with the outward A549 stall forces seen in Fig. 1A). Stall forces were only measured for beads that had stalls in both directions to ensure both motors were present. This data implies that an interaction between kinesin family motors and dynein through the cargo is likely the cause of the broad outward stall forces in vivo. In vitro single dynein stalls are also quite different from inward stall behavior (D), with a mean stall force of 1.1 ± 0.28 pN. (E) Coating a bead with many dyneins in vitro comes close to replicating in vivo inward behavior, leading to a larger stall force and spread in stall forces. (F) The stall force for minus-end directed in vitro stalls of beads coated with both kinesin-1 and dynein is similar to that for dynein alone, indicating that kinesin has minimal effect on minus-end directed motion.

Fig. 3.

Model of intracellular transport. (A) Single dynein version. This simplified single-dynein model shows dynein always attached. Whether or not kinesin is engaged with the microtubule determines the directionality of the cargo. (B) Multiple dynein version. A more complex model showing how the spread in plus-end directed stall forces could occur due to different numbers of dyneins being attached to the cargo during plus-end directed motion. The arrows on the cargos indicate direction of motion, and the plus and minus signs indicate the polarity of the microtubules (plus = kinesin family motor ∼ outward, dynein = minus ∼ inward). The forces indicate the likely stall force for various combinations of motors. The higher stall forces seen in Fig. 1 could be caused by multiple motors, misaligned microtubules, and other rare or chance events (cellular movement, microtubule movement, an unusually large number of motors on an organelle).