Cytoplasmic HEATR2 is required for the pre-assembly of axonemal dynein machinery necessary for motility.

<p>(A) Immunofluorescence for axonemal dynein heavy chain 5 (DNAH5: green) on respiratory cells from patients with <i>HEATR2</i> mutations compared to non-related control cells, shows loss of type 1 and type 2 DNAH5-positive staining although axonemes are still present (acetylated tubulin:red). Nuclei are stained with DAPI. (Scale bar: 10 µm). (B) Extracts prepared from control human terminally differentiated respiratory airway cultures (40 days ALI, Epithelyx) were subjected to immunoprecipitation (IP) with antibodies to HEATR2 (Proteintech) or control rabbit immunoglobulin G (GFP). Resulting immunocomplexes (IP: right) as well as dilutions of original extract (INPUT: left) were subjected to immunoblot analysis with antibodies to HEATR2 (Proteintech) or DNAI2 (Abnova). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s008" target="_blank">Figure S8</a>). (C) No staining of axonemal dynein light intermediate chain 1 (DNALI1: green) is observed in patients with <i>HEATR2</i> mutations. No signal above background is detected in patient cells, in contrast to strong axonemal localization in non-related control cells. Nuclei are stained with DAPI. (Scale bar: 10 µm). (D) The DNALI1 orthologue in fly, <i>CG6971::mVenus</i> (green), fails to localize to ciliary axonemes (Ci: cilia, marked with square bracket) of Ch neurons (magenta: 22C10/Futsch) in <i>CG31320</i> knock-down larvae. (E) Schematic of dynamic role of HEATR2 in developing airway epithelial MMCs. Progenitor cells exit the cell cycle to commit to the MMC lineage with primary cilia. These cells express low levels of RFX3 (light red nuclei). Other upstream factors governing multiciliogenesis (MCN, MYB) induce centriole amplification as well as expression of FOXJ1 (bright green nuclei), required for centriole docking. High FOXJ1 and RFX3 drive a cilia motility transcriptional cascade leading to high expression of HEATR2 as well as expression of axonemal dynein components. HEATR2 is involved in the pre-assembly and/or delivery of future dynein arms to the apical cilia base. In fully mature MMCs, once inner and outer arm dynein complexes are delivered and incorporated into motile ciliary axonemes, relative levels of HEATR2 as well as RFX3 and FOXJ1 are reduced. This conserved regulatory motility module is required to drive high levels of HEATR2 when axonemal dyneins are being assembled and trafficked.