Dynein-mediated cargo transport in vivo. A switch controls travel distance

Abstract

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

Figure 1

Physical association between cytoplasmic dynein and lipid droplets. Lipid droplets were diluted away from other organelles and unattached motors by squashing single embryos in buffer. Preparations were fixed and treated with the droplet-specific dye Nile red (red) and an antibody against the intermediate chain of dynein (Dic, green; Dillman and Pfister 1994). In many instances, lipid droplets had a punctate Dic signal associated with them, with little Dic staining in the general background. This association was ∼100-fold more frequent than expected by chance alone. Otherwise identically treated preparations for which incubation with the primary antibody was omitted showed no punctate staining on lipid droplets (not shown). Although our previous force measurements suggest that single droplets carry multiple dyneins in multimotor complexes (Welte et al. 1998), the apparent size of the Dic signal does not provide insight into the structure of these complexes because under the experimental conditions used even a single dynein molecule would be expected to yield a signal spread over a region ∼0.3 μm in diameter (see Materials and Methods). Bars, 1 μm.

Figure 2

We compared lipid droplet distribution in embryos laid by wild-type flies (Oregon-R) or by flies transheterozygous for two weak alleles of Dhc64C, the gene for the heavy chain of cytoplasmic dynein (Gepner et al. 1996). Fixed embryos were stained for lipid droplets as described in Welte et al. 1998. In both genotypes, droplets accumulate in the center, towards the plus end of microtubules, in early cycle 14 (not shown); in Dhc64C mutants, they fail to redistribute towards the minus ends (the periphery) during gastrulation. Bar, 200 μm.

Figure 3

Droplet stalling force measurements, showing the percentage of droplets stalled as a function of applied force, for droplets moving towards microtubule minus ends, in wild-type or Dhc64C mutant backgrounds. Because droplets need to be clearly moving to attempt to stop them, these measurements represent the behavior of droplets moving long distances (see text). To avoid bias, force measurements were made in a blind fashion, with the genotype of the embryo being measured unknown to the person making the force measurement. Each data point is derived from measurements on six to seven embryos, with ∼30 minus-moving droplets tested per embryo.

Figure 4

(A) Track of the motion of a representative lipid droplet, with both plus and minus end runs of greatly varying lengths. Decreasing ordinate values correspond to minus end motion. One example each of a long minus end and plus end run is labeled. Dotted boxes labeled B and C indicate the regions that are magnified in B and C. (B) Portion of A showing two pauses. (C) Portion of A showing typical sharp reversals (no obvious pause involved). One example each of a short minus end and plus end run is labeled.

Figure 7

Mean travel speed as a function of run distance for minus end motion. The average speed of short (35–100 nm) or long (500–1,000 nm) runs is shown, for droplets in wild-type embryos from phase II and phase III and in Dhc64C mutant embryos from phase II. The error bars are the standard error of the average.