SSNA1 organizes the distal luminal centriolar network and promotes ciliogenesis without microtubule association

Abstract

The distal lumen of the centriole plays a critical role in ciliogenesis, yet the molecular composition, spatial organization, and targeting hierarchy of distal luminal proteins remain poorly understood. In this study, we identify Sjogrens syndrome nuclear autoantigen 1 (SSNA1) as a bona fide centriolar protein and a key regulator of ciliogenesis in mammalian cells. Using our newly developed knockout (KO)-validated antibody, we show that, contrary to previous reports, SSNA1 does not reside in the nucleus, midbody, or ciliary axoneme. Instead, super-resolution imaging combined with expansion microscopy (ExM) reveals that SSNA1 localizes to the distal lumen of centrioles and the basal bodies of both primary and motile cilia, where it is arranged in a ring-like configuration with 9-fold symmetry and apart from centriolar microtubules. Molecular dissection using tag-free SSNA1, its oligomerization-deficient mutants, and microtubule co-pelleting assays further demonstrates that SSNA1, previously described as a microtubule nucleator, stabilizer, and branching factor, does not bind microtubules in vitro. Interactor screening and KO analysis unveil a hierarchical targeting network involving a C2CD3-SSNA1-LRRCC1 axis in the distal lumen. Functional characterization indicates that although dispensable for cell division, overall centriole organization and duplication, SSNA1 promotes cilia assembly by facilitating CP110 removal. Our findings redefine the physiological role of SSNA1 as part of the distal luminal module contributing to ciliogenesis and provide new insights into the molecular architecture and functional relevance of the distal centriolar lumen. SignificanceCentrioles template the assembly of cilia and flagella, whose dysfunction causes human diseases known as ciliopathies. Key centriolar structures - the cartwheel in the proximal lumen, the inner scaffold in the central lumen, and the distal and subdistal appendages outside the distal lumen - have been extensively characterized. However, the molecular organization and functional relevance of the distal lumen remain largely unexplored. Here, we identify SSNA1 as a novel centriolar component at the distal lumen and an essential regulator of ciliogenesis. Our work challenges the long-held view of SSNA1 as a microtubule-associated protein and provides new insights into the architecture, interaction network, targeting hierarchy, and function of the distal centriolar lumen, exemplifying how it modulates centriolar organization and drives ciliogenesis.