Correlating Cargo Orientation with Molecular Motor Activity during Axonal Transport

Axons of neurons present a unique challenge for intracellular transport: with a diameter of roughly one micrometer and length that can range up to a meter, transport machinery in the axon must be able to move cargo processively with high speed while moving largely unidirectionally. Motile cargoes must also be able to bypass static organelles which can be hundreds of nanometers in size, taking up significant portions of the cross section of the axon. Defects in this transport are found in a host of neurodegenerative disorders, including Alzheimer's and Parkinson's Diseases. One strategy that cells use to maintain healthy axonal transport is to provide each cargo with several copies of different molecular motors. However, details of the regulation required for appropriate teamwork between the motors are lacking. The motion that results from the actions of multiple motors moving a single cargo inside the cell can be complex, and conventional imaging approaches are limited to measuring the position of cargo along the length of the axon. We present an experimental approach to measure an additional parameter - cargo orientation. By constructing a dual-polarization dark field microscope, we achieve a high throughput readout of position and orientation of gold nanorod-containing endosomes in primary neurons with millisecond resolution. This allows us to relate particular translational-orientational behaviors specifically to teams of either kinesins or dyneins. We observed that changes in cargo velocity correlate with changes in orientation particularly at transitions between paused and moving states. Furthermore, we find that cargoes with similar translational dynamics can have very different orientational behavior and individual cargoes, though of identical origin, differ in global orientational dynamics.