Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia

Abstract

Cytoskeletal‐based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)‐labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3–6‐nanometer accuracy and 3.4‐ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin‐II and OSM‐3‐kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin‐II, a torque‐generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM‐3‐kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia‐dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.

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

“…To obtain a higher time resolution, we performed fast single-molecule imaging using only the more stable eGFP or paGFP, and we obtained a three to fivefold improvement of the time resolution compared to previous studies. In the future, the time resolution might be pushed further by using brighter fluorescent reporters such as mNeonGreen ( 48 ) or using other imaging methods such as Hessian structured illumination microscopy ( 25 ) with multiple copies of fluorescent proteins tagged to one motor. The three to fivefold improvement of the time resolution we obtained here is, however, sufficient to reveal that individual motors switch between IFT trains via a detach–diffuse–attach mechanism, directly exposing that diffusion is an intrinsic part of the motor turnaround process.…”

“…IFT trains are assembled in the ciliary base and transported to the ciliary tip by the cooperative action between two types of kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3 ( 5 , 9 , 16 , 17 ). Kinesin-II acts as the “import” motor to drive IFT trains through the base and transition zone toward the proximal segment, possibly avoiding obstacles on the microtubules by sidestepping ( 25 ). There, kinesin-II gradually hands over anterograde IFT trains to OSM-3, which drives the long-range transport toward the ciliary tip ( 18 ).…”

The structural basis of intraflagellar transport at a glance

“…It has been proposed that IFT-kinesin motors migrate towards the B-tubule by an intrinsic left-handed helical movement around the microtubules that is sterically blocked by the ODAs and thus results in a straight movement along the B-tubule (Stepp et al, 2017). Reduction of the motor sidestepping behavior in C. elegans did not result in traffic jams (Xie et al, 2020), suggesting that sidestepping is not involved in the positioning of kinesins on the B-tubule in Chlamydomonas or that a different IFT logistics co-evolved with the highly specialized axonemal structure of C. elegans cilia.…”

Mechanisms of Regulation in Intraflagellar Transport

“…On axonemes, however, no helical paths were observed, which could be explained by the motors hitting a steric barrier at the connection between A- and B-tubules and as a consequence follow a straight trajectory along a single PF. Sidestepping has also been observed in C. elegans chemosensory cilia [ 69 ]. It is, however, unclear whether tubule preference is unique to Chlamydomonas , or universal and similar in other organisms.…”