Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Kinesin family members and cytoplasmic dynein are motor proteins that move along the microtubules in cells to transport their cargo¹. Most kinesins move toward the plus end, whereas dynein moves toward the minus end of a microtubule. The functions and mechanisms of cargo transport in the neuronal axon have been investigated extensively. Because of their length, axons require stable long-distance transport to maintain the health of neurons. Defects in the transport of mitochondria, autophagosomes, and vesicles containing amyloid-β protein precursor (APP) have been reported as causes of neurodegenerative diseases^2,3. Numerous in vitro investigations have revealed the mechanisms underlying coordinated transport by motor proteins, and various studies using purified motor proteins and microtubules have uncovered how motor molecules move along microtubules^4,5. Typically, multiple motors are involved in a single cargo⁶. However, there are some models of how the opposite motors determine the direction of cargo transport. One is the "association/dissociation model"; in this, only one-directional motors are associated with the cargo during the one-directional transport. In the second, the "coordination model", both motors are attached to the same cargo, and only one side of the motor is activated. In the third "tug-of-war model", the force balance between kinesins and dyneins determines the direction of transport^7,8,9. In addition, several reports have suggested that the balance and number of activated motor proteins influence the velocity of cargo transport in vitro^8,10.

An unanswered question is how these activities of kinesins or dyneins are regulated in living cells. Previous reports have shown acetylation of microtubules in axons, and neurotrophic factors enhance axonal transport^11,12. Also, various types of cargo and cargo adapters function as motor activators in corresponding ways^13,14. Many are associated with the membrane of transport vesicles, and their functions are regulated by signals such as post-translational modifications¹⁵. Therefore, observing transport direction and velocity in living cells offers valuable insight into the molecular regulation of cargo transport in in vitro experiments. The observation of axonal transport allows distinguishing between kinesin- and dynein-based transport. Because axons harbor plus-end unidirectional microtubules¹⁶, cargo is transported anterogradely (i.e., soma to axon terminal) by kinesins and retrogradely (i.e., axon terminal to soma) by dyneins.

In the present study, a method to observe and analyze axonal transport in primary cultured neurons is described. As an example, the procedure for observing the axonal transport of membrane proteins-APP, calsyntenin-1/alcadein α (Alcα), and calsyntenin-3/alcadein β (Alcβ)-containing vesicles-is described. It is known that the anterograde transport of APP-containing vesicles is considerably faster than that of Alcα-containing vesicles, although both are transported by kinesin-1^17,18,19. In previous reports, several methods have been used to measure velocity. The most variable step is the handling of pauses during transport. In live cells, transport is sometimes hindered by obstacles along the microtubules; however, motors can bypass a region with or without a pause²⁰. The calculation of velocity over a longer observation time may be affected by the pause, which can result in a slower speed estimation. Here, a method is described using movement over a segmented (200 ms) period to exclude the effect of the physical pause of motors. Finally, an open-access (Windows only) software program called "KYMOMAKER"²¹ is introduced. Kymographs are used widely to visualize vesicular transport and are useful for visualizing the transport direction of each cargo without requiring a movie. The software generates kymographs from time-lapse movies by applying a one-dimensional Watershed algorithm multiple times during the rotation of images. This enables the resulting kymograph to show fine structures efficiently and easily. Furthermore, KYMOMAKER automatically detects and highlights trails according to their direction and enables the creation of easy-to-understand diagrams.