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HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

ABSTRACT: Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

SUBMITTER: Diggle CP

PROVIDER: S-EPMC4168999 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

Diggle Christine P CP Moore Daniel J DJ Mali Girish G zur Lage Petra P Ait-Lounis Aouatef A Schmidts Miriam M Shoemark Amelia A Garcia Munoz Amaya A Halachev Mihail R MR Gautier Philippe P Yeyati Patricia L PL Bonthron David T DT Carr Ian M IM Hayward Bruce B Markham Alexander F AF Hope Jilly E JE von Kriegsheim Alex A Mitchison Hannah M HM Jackson Ian J IJ Durand Bénédicte B Reith Walter W Sheridan Eamonn E Jarman Andrew P AP Mill Pleasantine P

PLoS genetics 20140918 9

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidat ...[more]

PMID: 25232951

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Project description:Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by approximately 80% for the flhA mutant and approximately 50% for the fliC mutant, showing that only part of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

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HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

Publications

HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

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