Mark Winey

The lumens of the highly stable microtubules that make up the core of basal bodies, cilia, and flagella are coated with a network of proteins known as MIPs, or microtubule inner proteins. MIPs are hypothesized to enhance the rigidity and stability of these microtubules, but how they assemble and contribute to cilia function is poorly understood. Here we describe a ciliate specific MIP, RIB22, in Tetrahymena thermophila. RIB22 is a calmodulin-like protein found in the A-tubule of doublet and triplet microtubules in cilia and basal bodies. Its localization is dependent on the conserved MIP RIB72. Here we use cryogenic electron tomography (cryoET) to examine RIB22 and its interacting partners in axonemes and basal bodies. RIB22 forms a ternary complex with the C-terminal EF-hand domain of RIB72A and another MIP, FAM166A. Tetrahymena strains lacking RIB22 or the EF-hand domain of RIB72A showed impaired cilia function. CryoET on axonemes from these strains demonstrated an interdependence of the three proteins for stabilization within the structure. Deletion of the RIB72A EF-hand domain resulted in the apparent loss of multiple MIPs in the region. These findings emphasize the intricacy of the MIP network and the importance of understanding MIPs’ functions during cilium assembly and regulation.

The cilium is a microtubule-based eukaryotic organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying cryo-electron tomography, we obtained structures of the inner junction in three regions of the cilium from Tetrahymena : the proximal, the central core of the basal body, and the axoneme. We identified several protein components in the basal body. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to specific regions, forming intricate local interaction networks in the inner junction and bolstering local structural stability. By examining the inner junction in a POC1 knockout mutant, we found the triplet microtubule was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to “infiltrate” into the mutant basal body. Our findings provide molecular insight into cilium assembly at the inner junctions, underscoring its precise spatial regulation.

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to “infiltrate” into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the two poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the spindle pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their spindle pole body earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances achieved with ciliate models. Studies on patterning and regeneration have been revived in the giant ciliate Stentor, facilitated by modern omics methods. Cryo-electron microscopy and tomography have revolutionized the structural study of complex macromolecules such as telomerase, ribozymes, and axonemes. DNA elimination, gene scrambling, and mating type determination have been deciphered, revealing interesting adaptations of processes that have parallels in other kingdoms of life. Studies of common eukaryotic processes, such as intracellular trafficking, meiosis, and histone modification, reveal conservation as well as unique adaptations in these organisms that are evolutionarily distant from other models. Continual improvement of genetic and molecular tools makes ciliates accessible models for all levels of education and research. Such advances open new avenues of research and highlight the importance of ciliate research.

Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from Tetrahymena. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an FAP115 knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

We performed a proteomic screen of cilia missing known microtubule inner proteins (MIPs) Rib72A and Rib72B to determine additional MIP candidates. One of these candidates, Fap115, was confirmed to be a structural and functional MIP using cell biology and cryo-electron tomography.

Summary: Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila . We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of α-tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a “molecular staple” stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas , we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the two poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body in Saccharomyces cerevisiae . Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the spindle pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their spindle pole body earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

Basal bodies (BBs) are microtubule-based organelles that act as a template for and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs, yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised and BB overproduction is exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia. This article has an associated First Person interview with the first author of the paper.