title: Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.
abstract: Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Emma A
authors.initials: EA
authors.lastname: Hall
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Margaret
authors.initials: M
authors.lastname: Keighren
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ford
authors.affiliation: Electron Microscopy Research Services, Medical School, Newcastle University, Newcastle, United Kingdom.
authors.forename: Tracey
authors.initials: T
authors.lastname: Davey
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Jarman
authors.affiliation: MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: Lee B
authors.initials: LB
authors.lastname: Smith
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Jackson
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.
authors.forename: Pleasantine
authors.initials: P
authors.lastname: Mill
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins; D002502:Centrioles / Q000235:genetics / Q000378:metabolism; D002923:Cilia / Q000235:genetics* / Q000473:pathology; D003598:Cytoskeletal Proteins; D005407:Flagella / Q000378:metabolism / Q000473:pathology; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007248:Infertility, Male / Q000209:etiology / Q000235:genetics*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008870:Microtubules / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering; D013082:Sperm Tail / Q000473:pathology*
chemical_list: D018797:Cell Cycle Proteins; C097241:Cep131 protein, mouse; D003598:Cytoskeletal Proteins; D011506:Proteins; D034741:RNA, Small Interfering