Unravelling 3D Cargo Transport Dynamics at the Microtubule Network

Motor-driven cargo transport helps deliver several types of vesicles to the proper location of function. Motor proteins, such as dynein and kinesin, move vesicles through the complex 3D microtubule network. Motors encounter different roadblocks on their way such as microtubule intersections, patches of microtubule associated proteins, or other vesicles. The mechanisms that allow motors to overcome these roadblocks remain unclear.We have developed a correlative imaging approach that combines single particle tracking with super-resolution microscopy (Balint, Verdeny, Sandoval, Lakadamyali, PNAS 2013). Using this method, we showed that vesicles pause at tight microtubule intersections, likely because the intersecting microtubule constitutes a roadblock. Our initial imaging approach was limited to 2D single particle tracking making it difficult to determine the precise mechanisms that motors use to overcome these roadblocks.We overcame several technical challenges to extend the correlative imaging method to 3D single particle tracking, which allowed us to visualize 3D motion of vesicles in the context of the microtubule network. We show that vesicles move in two different modes along individual microtubules. Typically, they follow the microtubule long axis in a straight line although in 30% of cases they can also move in 3D around the microtubule. This second mode of motion is correlated to events such as switching from one microtubule to another at microtubule intersections and passing other vesicles on the same microtubule. Moreover, vesicles perform longer runs when moving in 3D around the microtubule. Thus, this mode of motion seems to be a more efficient way of transport and it likely plays a role in avoiding roadblocks. Overall, these results provide new insights into the mechanisms that motor proteins may use to overcome obstacles when navigating through the microtubule network.