Explorer Acute Versus Chronic Loss of Mammalian Azi 1 / Cep 131 Results in Distinct Ciliary Phenotypes

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). Surprisingly, Azi1 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. Citation: Hall EA, Keighren M, Ford MJ, Davey T, Jarman AP, et al. (2013) Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes. PLoS Genet 9(12): e1003928. doi:10.1371/journal.pgen. 1003928 Editor: Susan K. Dutcher, Washington University School of Medicine, United States of America Received June 26, 2013; Accepted September 14, 2013; Published December 26, 2013 Copyright: 2013 Hall et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Core funding for this study was provided by the Medical Research Council of Great Britain (http://www.mrc.ac.uk). Grants provided support for LBS from the MRC (G1100354) and APJ from the Wellcome Trust (077266). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ian.jackson@igmm.ed.ac.uk (IJJ); pleasantine.mill@igmm.ed.ac.uk (PM)