SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.

Cilia contain complex assemblies of different microtubules. These microtubules are essential for the organization and motility of the cilium and provide tracks for intraciliary protein transport. At the base of a cilium is the basal body, a modified centriole consisting of a circular arrangement of nine triplet microtubules (TMTs). The A and B tubules of TMTs transition into the doublet microtubules (DMTs) of the axoneme. In motile cilia, these axonemal DMTs anchor thousands of dynein motors and regulatory complexes that together generate ciliary motility. DMTs also often surround a central apparatus (CA), an additional structure of two heavily patterned and interconnected microtubules, C1 and C2. DMTs themselves often transition into singlet microtubules (SMTs) near the ciliary tip, with the length of this singlet zone varying between species and cell types (1). For example, the singlet zone in immotile (primary) cilia often exceeds the length of the DMTs from which they originate (2, 3), whereas the motile flagellum of Trypanosoma brucei does not contain a singlet zone at all (4, 5). In cilia that do have a singlet zone, the transition from DMT to SMT either involves the abrupt termination or selective loss of the distal region of the B tubule or the splitting of A and B tubules into 2 separate microtubules, each with 13 protofilaments (6). B tubule loss seems to predominate in mammalian primary cilia (2, 3), whereas splitting has been observed in the motile flagella of rodent and human spermatozoa (7–9).The lumenal surfaces of the TMTs, DMTs, and SMTs of motile cilia are patterned by microtubule inner proteins (MIPs). The identities of some of these MIPs have become known through cryo-electron microscopy (cryo-EM) studies of DMTs from the model organism Chlamydomonas reinhardtii (10). We recently revealed that many of these MIPs are conserved in the DMTs of bovine respiratory cilia (11). In addition to near-universal MIPs, a subset of MIPs is specific to mammals. A possible function of MIPs is to fine-tune the physical properties of microtubules—for example, to modulate stability or elasticity (12). Because the required properties of ciliary microtubules may differ depending on the morphology of cilia, their function, and the environment in which they operate, this raises the question of whether different cilium types within an organism have different MIP repertoires. In humans, for example, the motile cilia of tracheal epithelia are an order of magnitude shorter than the motile flagella of spermatozoa (6 μm compared with 60 μm) (13, 14). The cilia of these two cell types also have different functions: respiratory cilia beat in metachronal waves to expel inhaled pathogens and particles, whereas sperm flagella evolved to propel spermatozoa through the female reproductive tract. Cell-specific specialization of axonemal structure within an organism is supported by the recent discovery that human sperm and airway cells have distinct compositions of axonemal dynein (15). Differences in the composition of axonemes between cell types may explain the tissue-specific effects on ciliary motility of mutations in some axonemal components (reviewed by Sironen et al. (16)). Resolving ciliary microtubule composition therefore has important implications for our ability to understand the phenotypic manifestation of human ciliopathies, which are associated with varying levels of infertility, respiratory disease, left–right body asymmetry, and heterotaxy (17).Compared with DMTs, the identities of MIPs in other ciliary microtubules are less well understood. Recent structures of C. reinhardtii CA microtubules (18, 19) have shown that they have a mutually exclusive set compared with DMTs. It may be expected that the MIPs of the TMTs and SMTs are identical to those of the DMTs with which they are contiguous. However, cryo-electron tomography (cryo-ET) studies of mammalian axonemes indicate that these microtubules have different patterns of MIPs (6, 20). We have previously used cryo-ET to show that the lumens of SMTs of human spermatozoa contain a structure called TAILS (tail axoneme intralumenal spiral) (6). This structure forms a noncontinuous left-handed spiral with an 8-nm pitch that breaks at the microtubule seam. TAILS forms not only in SMTs but up to 300 nm into the distal DMT (6). However, the resolution provided by cryo-ET was insufficient to identify the component or components responsible for TAILS.To address the question of how MIP repertoires differ among ciliary microtubules within an organism, we have used single-particle cryo-EM to determine the structures of human ciliary microtubules from two different cell types: the SMTs of spermatozoa and the DMTs of ciliated epithelial cells of the respiratory tract. Comparison of the structures of human and bovine respiratory DMTs (11) identified six additional MIPs, including SPACA9, which forms striations in the B tubule of human airway cilia. A cryo-EM structure of human sperm SMTs demonstrated that SPACA9 is also responsible for TAILS. Striations resembling those formed by SPACA9 are visible in tomographic reconstructions of other microtubules, leading us to speculate that SPACA9 could be a promiscuous binder of mammalian ciliary microtubules.