Lipid Droplets Purified from Drosophila Embryos as an Endogenous Handle for Precise Motor Transport Measurements

Abstract

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.

As the fat flies: The dynamic lipid droplets of Drosophila embryos

“…Droplets isolated from rat liver are quite motile in vitro [122], although motion is dominated by kinesin-1 and not obviously bidirectional. Droplets purified from Drosophila embryos have kinesin-1, cytoplasmic dynein, and dynactin attached to them and can exhibit long-range unidirectional motility [58]. They also show frequent short-range (a few hundred nanometers) back-and-forth motion, but not yet the sustained bidirectional runs of 1 μm or more observed in vivo [58].…”

“…Droplets purified from Drosophila embryos have kinesin-1, cytoplasmic dynein, and dynactin attached to them and can exhibit long-range unidirectional motility [58]. They also show frequent short-range (a few hundred nanometers) back-and-forth motion, but not yet the sustained bidirectional runs of 1 μm or more observed in vivo [58]. Thus, robust bidirectional in-vitro motility has not yet been achieved, but even with the partial reconstitution possible so far, one can ask how impairing various known transport regulators ( e.g.…”

“…This complete lack of motion, in the plus- as well as the minus-end direction, likely reflects motor matchmaking [206], the little understood interdependence of the opposing motors on bidirectionally moving cargoes (see below), but might potentially indicate that kinesin-1's role is indirect, e.g ., being responsible for earlier assembly of the motor machinery during oogenesis, and that another motor powers droplet motion. However, both Khc and Klc can be detected by western analysis in purified droplet preparations [76, 85], and association of Khc and droplets was observed after immunostaining of purified droplets [58]. In addition, acute kinesin-1 inhibition via antibody injection into the embryo disrupts droplet motion within minutes [76], and Khc alleles that impair, but do not abolish kinesin-1 function support droplet motion, but with altered characteristics [76].…”

“…2D). On the one hand, dynein intermediate chain is present on droplets, as judged by immunostaining of squash preparations of embryos [77] or purified droplets [58] as well as by western analysis of purified droplets [87]. On the other hand, mutations in dynein heavy chain as well as in the known dynein regulators BicD and Dynactin alter specific parameters of droplet motion [77, 83, 84]; in addition, severe disruptions of dynein heavy chain and of BicD also prevent net minus end transport in Phase III, resulting in abnormally transparent embryos [77, 83].…”

“…Dynactin has been detected on purified droplets by immunostaining [58]. In embryos expressing a dominant-negative form of p150 Glued , minus-end motion is somewhat compromised, but plus-end motion is much more severely affected [84].…”