Abstract
Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.
Highlights
Motile cilia are microtubule-based cellular projections and their oriented beating generates fluid flows that are critical for development and homeostasis
For example, that the Heat Shock Protein (Hsp) co-chaperone Stip1/Hop displayed essentially total colocalization with the assembly factor Dnaaf5/Heatr2 (Fig. 1B), while by contrast, the outer dynein arm subunit Dnai2 clearly displayed only partial overlap with Dnaaf5 (Fig. 1C)
The protein is highly enriched in DynAPs, where it is restricted to the outer dynein arm (ODA) sub-DynAP (Fig. 6-8)
Summary
Motile cilia are microtubule-based cellular projections and their oriented beating generates fluid flows that are critical for development and homeostasis. Ciliary beating is driven by a complex set of axoneme-specific dynein motors that drive sliding of the axonemal microtubule doublets. Based on their relative positions within the axoneme, these motors are classified as either outer dynein arms (ODAs) or inner dynein arms (IDAs)(Fig. 1A, upper inset), the former driving ciliary beating generally, and the latter tuning the waveform (Kamiya and Yagi, 2014; King, 2018a). Mutations in genes encoding ODA or IDA subunits are the major cause of the motile ciliopathy syndrome known as primary ciliary dyskinesia (PCD; MIM 244400) This genetic disease results in repeated sinopulmonary disease, bronchiectasis, cardiac defects, situs anomalies, and infertility (Horani et al, 2016; Mitchison and Valente, 2017; Wallmeier et al, 2020). PCD can result from mutations in genes encoding any of several cytoplasmic proteins collectively known as Dynein Axonemal Assembly Factors (DNAAFs)(Desai et al, 2018; Fabczak and Osinka, 2019)
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