ABSTRACT
Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.
Subject(s)
Axonemal Dyneins/metabolism , Axoneme/metabolism , Biological Transport, Active/genetics , Cell Movement/genetics , Cilia/metabolism , Embryo, Mammalian/metabolism , Microtubules/metabolism , Animals , Axonemal Dyneins/genetics , Axoneme/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cilia/genetics , Embryo, Mammalian/physiology , Embryo, Mammalian/ultrastructure , Ependyma/embryology , Ependyma/metabolism , Ependyma/physiology , Fluorescent Antibody Technique , Genotype , Immunoprecipitation , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Microtubules/genetics , Mutation , Phenotype , Trachea/embryology , Trachea/metabolism , Trachea/physiology , Trachea/ultrastructureABSTRACT
Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
Subject(s)
Adenine Nucleotides/metabolism , Asthenozoospermia/genetics , Cytoskeletal Proteins/deficiency , Situs Inversus/genetics , Adolescent , Adult , Animals , Asthenozoospermia/pathology , Axoneme/ultrastructure , CRISPR-Cas Systems/genetics , Cilia/metabolism , Cilia/ultrastructure , Cytoskeletal Proteins/genetics , DNA Mutational Analysis , Disease Models, Animal , Epididymis/pathology , Female , Flagella/metabolism , Flagella/ultrastructure , Humans , Loss of Function Mutation , Male , Mice , Mice, Knockout , Middle Aged , Planarians/cytology , Planarians/genetics , Planarians/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/pathology , Situs Inversus/diagnostic imaging , Situs Inversus/pathology , Sperm Motility/genetics , Tomography, X-Ray Computed , Exome SequencingABSTRACT
Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.
