Shulin packages axonemal outer dynein arms for ciliary targeting.

INTRODUCTION Motile cilia are slender, highly conserved cellular protrusions that move surrounding fluids. Their whip-like motion enables the swimming movement of many unicellular eukaryotes. In multicellular organisms, cilia beating orchestrates key developmental processes and plays a critical role in clearing mucus from the airways throughout life. Consequently, loss of cilia motility in humans leads to primary ciliary dyskinesia, a debilitating ciliopathy characterized by severe respiratory disease. Cilia movement is driven by molecular motors of the dynein family. These include outer dynein arms (ODAs) and inner dynein arms (IDAs), which are arrayed along a microtubule-based structure called the axoneme. During cilia formation, these large multisubunit machines are synthesized in the cell cytoplasm. They are put together by multiple assembly factors (DNAAFs) and then taken into the cilia by intraflagellar transport (IFT). The activity of the newly assembled dyneins needs to be controlled until they are delivered to their final location in the axoneme. However, the mechanisms underlying this process are unclear. RATIONALE We hypothesized that potential control factors would bind to newly assembled ODAs in the cytoplasm. We therefore removed cilia from the unicellular model organism Tetrahymena thermophila, purified recombinantly tagged ODAs from their cell bodies, and identified bound factors by mass spectrometry. A combination of genetic knockdowns, biochemical assays, and electron microscopy (EM) established roles for the factors in ciliary targeting and uncovered their molecular mechanisms. RESULTS We identified two factors, Q22YU3 and Q22MS1, previously unlinked to motile cilia, that specifically copurified with the ODAs from the cell body. Bioinformatic studies suggested that Q22YU3 has a human ortholog (C20ORF194) with no well-defined function, whereas Q22MS1 is Tetrahymena-specific. We generated genetic knockdowns of Q22YU3 and Q22MS1 in Tetrahymena. Both mutant strains displayed reduced swimming speeds caused by slower-beating cilia. Immunostaining experiments revealed that lack of either factor led to a reduction in dynein concentrations inside cilia. This indicated that both factors are important for dynein delivery and hence for normal cilia movement. ODAs in Tetrahymena contain three chains with dynein motor activity and can move microtubules in vitro. We found that both factors inhibited this ODA motor activity, with Q22YU3 showing the more severe effect. Negative-stain EM revealed that Q22YU3 compacts the three motors in ODAs from an open state to a tightly closed conformation, explaining its inhibitory function. We therefore named Q22YU3 “Shulin” from the Sanskrit meaning “one that holds the trident.” Cryo-EM revealed the architecture of the ODA complex and showed the molecular basis for Shulin’s inhibition. The three ODA motor domains are joined by a tail region that also contains the interaction sites for multiple dynein accessory chains. Shulin is a multidomain protein with an N terminus homologous to the FACT histone chaperone Spt16 and a C terminus resembling a bacterial guanosine triphosphatase. Our structure showed that Shulin predominantly binds the ODA tail, making multiple bridging contacts that cluster the three motor domains together. This locks them in a form with weak affinity for microtubules and hence inactivates them. Immunostaining showed that Shulin enters newly growing cilia but is lost when they mature. This suggests that Shulin keeps ODAs inactive during their transport but is released after they reach their docking site. CONCLUSION We have discovered two negative regulators of ciliary outer dynein arms. Whereas Q22MS1 modestly dampens dynein motor activity, Shulin completely shuts it down. Inhibiting dyneins in the cytoplasm and relieving inhibition once inside cilia is an efficient cellular mechanism to ensure proper engagement of molecular motors only in the correct cellular context.