LAUSR

Significance Primary cilia are important organelles that exist in almost all eukaryotic cells. Intraflagellar transport (IFT) is a motor-protein–driven bidirectional intracellular transport mechanism in cilia. Previous studies have shown that motors in Caenorhabditis elegans chemosensory cilia undergo rapid turnarounds to effectively work together in driving orderly IFT. The mechanism of motor turnarounds has, however, remained unclear. Here, using a combination of advanced fluorescence imaging and single-molecule analysis, we directly show that the individual turnarounds are due to motors switching between opposite-direction IFT trains. Furthermore, we show that switching events consist of motors detaching from a train, diffusing to another one followed by attachment. This directly demonstrates that motors switch trains by diffusion, which clarifies the mechanism of motor turnarounds. Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach–diffuse–attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.