Shulin packages axonemal outer dynein arms for ciliary targeting

doi:10.1126/science.abe0526

GSTDTAP > 气候变化

DOI	10.1126/science.abe0526
	Shulin packages axonemal outer dynein arms for ciliary targeting
	Girish R. Mali; Ferdos Abid Ali; Clinton K. Lau; Farida Begum; Jérôme Boulanger; Jonathan D. Howe; Zhuo A. Chen; Juri Rappsilber; Mark Skehel; Andrew P. Carter
	2021-02-26
发表期刊	Science
出版年	2021
英文摘要	Motile cilia and flagella are vital cellular organelles with functions that include setting up the left-right body axis, clearing airways of mucus, and driving single-cell movements. Cilia beating is powered by arrays of dynein motors, the key force generators being the outer dynein arm (ODA) complexes. Using the protozoan Tetrahymena , Mali et al. identified a factor, which they name Shulin, that binds newly synthesized ODAs. Cryo–electron microscopy revealed how Shulin locks the dynein motors together by shutting off motor activity and facilitating delivery of ODAs from the cytoplasm to their final position in the cilia. Science , this issue p. [910][1] ### 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. ![Figure][2] Shulin binds and closes ODAs in the cell body and travels with them to their final destination in cilia. Shulin engages outer dynein arms (ODA), fully preassembled by dynein axonemal assembly factors (DNAAFs), in the cell body. It binds and tightly packages ODAs into a closed inhibited state for their unimpeded transport into cilia by intraflagellar transport (IFT). Shulin disengages from ODAs once they are incorporated at their final ciliary location. The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo–electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia. [1]: /lookup/doi/10.1126/science.abe0526 [2]: pending:yes
领域	气候变化 ; 资源环境
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文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/315936
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Girish R. Mali,Ferdos Abid Ali,Clinton K. Lau,et al. Shulin packages axonemal outer dynein arms for ciliary targeting[J]. Science,2021.
APA	Girish R. Mali.,Ferdos Abid Ali.,Clinton K. Lau.,Farida Begum.,Jérôme Boulanger.,...&Andrew P. Carter.(2021).Shulin packages axonemal outer dynein arms for ciliary targeting.Science.
MLA	Girish R. Mali,et al."Shulin packages axonemal outer dynein arms for ciliary targeting".Science (2021).