Center for Nonlinear Dynamics » Cargo Transport in vivo

Inside living cells organelles and other cell constituents are constantly moving to reach where they are needed and establish internal cellular organization. This transport is accomplished by molecular motor proteins that haul the cargoes through stepping along the network of intracellular filaments, microtubules and actin filaments. In vitro work with artificial cargoes had immensely contributed to understanding the function of individual isolated microtubule motors of opposite polarity (kinesin and dynein). However, in contrast to the unidirectional artificial cargos, those in living cells spend energy to constantly move back and forth while their net direction is precisely controlled by the cell. The particulars of how opposite polarity motors work together and how their function is regulated in vivo remain for the most part enigmatic at many levels of detail.

We study opposite polarity motor regulation both in vivo and using endogenous purified cargoes. For our studies in vivo we use the transport of endogenous lipid droplets in the Drosophila embryo as a model system. The lipid droplets are transported bidirectionally by kinesin 1 and cytoplasmic dynein. Their distribution is developmentally regulated so that their net transport switches from plus-end directed to minus-end directed.This well defined directionality, together with the advanced genetics possible in the fly system, enables us to use biophysical tools as described below to study transport in the living embryos.

High resolution DIC microscopy time-lapse sequence showing lipid droplets in a Drosophila embryo transported by molecular motors in a bidirectional fashion. (the moving lipid droplets are ~ 500 nm in diameter)

Molecular motors use the energy released from ATP hydrolysis to generate force to haul the cargos, so measuring that force amounts to directly probing their function. We use an advanced optical trap system to measure the forces generated by motors to haul individual lipid droplets in the embryos. Measuring the force enables us to count the number of motors moving a cargo as it moves along the microtubule ( read more about the significance of motor numbers ).

An optical trap (tweezers) is a focused laser beam that captures the lipid droplets. Motors pulling a droplet out of the trap center experience an ever-increasing backwards force that eventually stalls the motors. The maximum distance the motors were able to pull the droplet is proportional to the motors’ stall force.

A histogram of stall forces for plus-end directed lipid droplets shows peaks at commensurate values of force. The three peaks are due to lipid droplets hauled by 1, 2 and 3 motors respectively. Measuring the force enables us to count the number of motors pulling a cargo as it moves along the microtubule.