Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

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.

mRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis

“…1, E–G ). This is similar to the subcompartmentalization observed in other RNP granules, including the germ granules in the Drosophila ovary, stress granules, P granules, nucleoli, and the dynein axonemal particles in human and Xenopus multiciliated cells ( Boisvert et al, 2007 ; Jain et al, 2016 ; Lee et al, 2020a Preprint ; Little et al, 2015 ; Trcek et al, 2015 ; Wang et al, 2014 ). We noted that kl-granule formation is unlikely to be dependent upon any one mRNA constituent, as RNAi-mediated knockdown of kl-3 , kl-5 , kl-2 , or Dhc98D ( bam-gal4>UAS-kl-3 TRiP.HMC03546 or bam-gal4>UAS-kl-5 TRiP.HMC03747 or bam-gal4>UAS-kl-2 GC8807 or bam-gal4>UAS-Dhc98D TRiP.HMC06494 ) did not perturb granule formation despite efficient knockdown ( Fig.…”

Emerging Genotype-Phenotype Relationships in Primary Ciliary Dyskinesia

“…In addition to the proteins which make up the structural components of the cilia, there is a group of pre-assembly factors that are crucial to proper cilia assembly but reside in the cytoplasm and not in the cilia itself [ 12 ]. This large group is thought to play key roles in assembly of components in the cilia prior to their transport into a newly growing cilium.…”

Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

“…We found that many of the Jam3-containing endosomes were also apical-sorting endosomes co-labeled with EEA1 (Mander’s coefficients, M1 = 0.28 and M2 = 0.34; displaced images: M1 (five-pixel shift) = 0.12 and M2 (five-pixel shift) = 0.13; M1 vs. M1 (five-pixel shift) p = 0.03 and M2 vs. M2 (five-pixel shift) p = 0.01; see section “Materials and Methods”) ( Figures 3E–E”” and Supplementary Figure 2 ) and co-localization with Rab11 was almost not found ( Supplementary Figure 2 ). Recently, liquid-like organelles have been related to cilia development ( Lee et al, 2020 ); we tested whether Jam3 appeared in those organelles where Daap1 or Dnai1 was concentrated and found no co-staining in MCCs ( Supplementary Figure 2 ). To further locate Jam3 at the membrane, we did a co-staining of Jam3 with Vangl1, a planar cell polarity-related protein localized in adherent junctions.…”