Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains

Abstract

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.

IFT-A Structure Reveals Carriages for Membrane Protein Transport into Cilia

“…To date, the major reported target of ICK and CDPK1 is kinesin-2 (Chaya et al, 2014; Liang et al, 2014), which is proposed to dissociate from the IFT train upon phosphorylation (Liang et al, 2014). Kinesin-2 dissociation may initiate the major conformational change that IFT trains undergo as they convert to retrograde trains (Chien et al, 2017; Jordan et al, 2018; Stepanek and Pigino, 2016; Wingfield et al, 2021; Zhang et al, 2021).…”

Mechanisms of Regulation in Intraflagellar Transport

“…Another aspect that requires a closer inspection are the binding and unbinding kinetics of IFT motors to and from IFT trains ( Figure 5 ). A recent single-molecule study in C. elegans has revealed that the interaction of IFT motors with IFT trains is highly dynamic [ 142 ]. Heterotrimeric kinesin-II motors were shown to stochastically detach from anterograde trains, switching from a directed to a diffusive state (likely autoinhibited).…”

Classifying directed and diffusive transport in short, noisy single-molecule trajectories with wMSD

“…s %" ), diffusion coefficients (𝐷: 0.01 -1.8 µm $ . s %" ), integration times (∆𝑡: 30, 60, 100 ms) and localization precision (𝑃𝐿: 0, 30, 60, 90 nm), reflecting typical values we have obtained in single-molecule trajectories of ciliary components in the chemosensory cilia of C. elegans (20)(21)(22). For each data set, we simulated 20 XY-trajectories of 200 steps (see parameters in Supplemental Table 1).…”

“…Furthermore, the location of the turnaround, whether the turnaround contains a pause or not, what the duration of this pause is, and whether the motor is diffusing during the pause can be extracted. We have applied this wMSDc analysis approach to a complete data set as mentioned above (20).…”

“…In our previous work on intracellular transport in the chemosensory phasmid cilia of Caenorhabditis elegans (17)(18)(19) we have frequently observed switches between diffusion and directed motion. Switches can be immediate or can involve pauses of variable duration between transport in opposite directions (20). The complex motion of ciliary components in C. elegans cilia reveals a wide heterogeneity not only in terms of motion diversity (directed motion and diffusion / sub-diffusion) but also in terms of locationdependent velocity along the length of the cilium (21)(22)(23).…”