Poc1 bridges basal body inner junctions to promote triplet microtubule integrity and connections.

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

The microtubule quartet protein SNAP1 in Trypanosoma brucei facilitates flagellum and cell division plane positioning by promoting basal body segregation.

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.

Basal body organization and cell geometry during the cell cycle in Tetrahymena thermophila.

Tetrahymena thermophila possesses arrays of motile cilia that promote fluid flow for cell motility. These consist of intricately organized basal bodies (BBs) that nucleate and position cilia at the cell cortex. Tetrahymena cell geometry and spatial organization of BBs play important roles in cell size, swimming, feeding, and division. How cell geometry and BB organization are established and maintained remains poorly understood, and prior studies have been limited due to difficulties in accurate BB identification and small sample size. We therefore developed an automated image processing pipeline that segments single cells, distinguishes unique BB populations, assigns BBs into distinct ciliary rows, and distinguishes new from mature BBs. We identified unique features to describe the variation of cell shape and BB spatial organization in unsynchronized single-cell images. The results reveal asymmetries in BB distribution and ingression of the cytokinetic furrow within the cell. Moreover, we establish novel spatial and temporal waves in new BB assembly through the cell cycle. Finally, we used measurements from single cells across the cell cycle to construct a generative model that allows synthesis of movies depicting single cells progressing through the cell cycle. Our approach is expected to be of particular value for characterizing Tetrahymena mutants.

FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation.

Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.

The apical ciliary adhesion complex is established at the basal foot of motile cilia and depends on the microtubule network.

The Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network. The results of this study reveal that the apical CA complex forms at the distal end of the basal foot and depends on microtubules. Our data also raise the possibility that CAs may have additional roles in the regulation of the organization of the microtubule network of MCCs.

Basal bodies bend in response to ciliary forces.

Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.

Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT).

Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P7 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation.

The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell-cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.

Plasmodium SAS4: basal body component of male cell which is dispensable for parasite transmission.

The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella, and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. Plasmodium, a divergent unicellular eukaryote and causative agent of malaria, displays an atypical, closed mitosis with an MTOC (or centriolar plaque), reminiscent of an acentriolar MTOC, embedded in the nuclear membrane. Mitosis during male gamete formation is accompanied by flagella formation. There are two MTOCs in male gametocytes: the acentriolar nuclear envelope MTOC for the mitotic spindle and an outer centriolar MTOC (the basal body) that organises flagella assembly in the cytoplasm. We show the coordinated location, association and assembly of SAS4 with the BB component, kinesin-8B, but no association with the kinetochore protein, NDC80, indicating that SAS4 is part of the BB and outer centriolar MTOC in the cytoplasm. Deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for either male gamete formation or parasite transmission.

TFK1, a basal body transition fibre protein that is essential for cytokinesis in Trypanosoma brucei.

In Trypanosoma brucei, transition fibres (TFs) form a nine-bladed pattern-like structure connecting the base of the flagellum to the flagellar pocket membrane. Despite the characterization of two TF proteins, CEP164C and T. brucei (Tb)RP2, little is known about the organization of these fibres. Here, we report the identification and characterization of the first kinetoplastid-specific TF protein, named TFK1 (Tb927.6.1180). Bioinformatics and functional domain analysis identified three distinct domains in TFK1 - an N-terminal domain of an unpredicted function, a coiled-coil domain involved in TFK1-TFK1 interaction and a C-terminal intrinsically disordered region potentially involved in protein interaction. Cellular immunolocalization showed that TFK1 is a newly identified basal body maturation marker. Furthermore, using ultrastructure expansion and immuno-electron microscopies we localized CEP164C and TbRP2 at the TF, and TFK1 on the distal appendage matrix of the TF. Importantly, RNAi-mediated knockdown of TFK1 in bloodstream form cells induced misplacement of basal bodies, a defect in the furrow or fold generation, and eventually cell death. We hypothesize that TFK1 is a basal body positioning-specific actor and a key regulator of cytokinesis in the bloodstream form Trypanosoma brucei.

Roles of the actin cytoskeleton in ciliogenesis.

Primary cilia play a key role in the ability of cells to respond to extracellular stimuli, such as signaling molecules and environmental cues. These sensory organelles are crucial to the development of many organ systems, and defects in primary ciliogenesis lead to multisystemic genetic disorders, known as ciliopathies. Here, we review recent advances in the understanding of several key aspects of the regulation of ciliogenesis. Primary ciliogenesis is thought to take different pathways depending on cell type, and some recent studies shed new light on the cell-type-specific mechanisms regulating ciliogenesis at the apical surface in polarized epithelial cells, which are particularly relevant for many ciliopathies. Furthermore, recent findings have demonstrated the importance of actin cytoskeleton dynamics in positively and negatively regulating multiple stages of ciliogenesis, including the vesicular trafficking of ciliary components and the positioning and docking of the basal body. Finally, studies on the formation of motile cilia in multiciliated epithelial cells have revealed requirements for actin remodeling in this process too, as well as showing evidence of an additional alternative ciliogenesis pathway.

Male infertility-associated Ccdc108 regulates multiciliogenesis via the intraflagellar transport machinery.

Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for various biological processes. Dysfunction of these cilia causes human diseases such as sinopulmonary disease and infertility. Here, we show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFT-B complex, and the ciliation requirement for Ift74 overlaps with Ccdc108 in MCCs. Both Ccdc108 and IFT-B proteins localize to migrating centrioles, basal bodies, and cilia in MCCs. Importantly, Ccdc108 governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Together, our studies indicate that Ccdc108 and IFT-B complex components cooperate in multiciliogenesis.

Lrrcc1 and Ccdc61 are conserved effectors of multiciliated cell function.

Ciliated epithelia perform essential functions in animals across evolution, ranging from locomotion of marine organisms to mucociliary clearance of airways in mammals. These epithelia are composed of multiciliated cells (MCCs) harboring myriads of motile cilia, which rest on modified centrioles called basal bodies (BBs), and beat coordinately to generate directed fluid flows. Thus, BB biogenesis and organization is central to MCC function. In basal eukaryotes, the coiled-coil domain proteins Lrrcc1 and Ccdc61 have previously been shown to be required for proper BB construction and function. Here, we used the Xenopus embryonic ciliated epidermis to characterize Lrrcc1 and Ccdc61 in vertebrate MCCs. We found that they both encode BB components, localized proximally at the junction with striated rootlets. Knocking down either gene caused defects in BB docking, spacing and polarization. Moreover, their depletion impaired the apical cytoskeleton and altered ciliary beating. Consequently, cilia-powered fluid flow was greatly reduced in morphant tadpoles, which displayed enhanced mortality when exposed to pathogenic bacteria. This work illustrates how integration across organizational scales make elementary BB components essential for the emergence of the physiological function of ciliated epithelia.

The Role of Centrosome Distal Appendage Proteins (DAPs) in Nephronophthisis and Ciliogenesis.

The primary cilium is found in most mammalian cells and plays a functional role in tissue homeostasis and organ development by modulating key signaling pathways. Ciliopathies are a group of genetically heterogeneous disorders resulting from defects in cilia development and function. Patients with ciliopathic disorders exhibit a range of phenotypes that include nephronophthisis (NPHP), a progressive tubulointerstitial kidney disease that commonly results in end-stage renal disease (ESRD). In recent years, distal appendages (DAPs), which radially project from the distal end of the mother centriole, have been shown to play a vital role in primary ciliary vesicle docking and the initiation of ciliogenesis. Mutations in the genes encoding these proteins can result in either a complete loss of the primary cilium, abnormal ciliary formation, or defective ciliary signaling. DAPs deficiency in humans or mice commonly results in NPHP. In this review, we outline recent advances in our understanding of the molecular functions of DAPs and how they participate in nephronophthisis development.

Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment.

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

The human sperm basal body is a complex centrosome important for embryo preimplantation development.

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Hook-basal-body assembly state dictates substrate specificity of the flagellar type-III secretion system.

The assembly of the bacterial flagellum is orchestrated by the secretion of distinct early and late secretion substrates via the flagellar-specific type-III secretion system (fT3SS). However, how the fT3SS is able to distinguish between the different (early and late) substrate classes during flagellar assembly remains poorly understood. In this study, we investigated the substrate selectivity and specificity of the fT3SS of Salmonella enterica at different assembly stages. For this, we developed an experimental setup that allowed us to synchronize hook-basal-body assembly and to monitor early and late substrate secretion of fT3SSs operating in either early or late secretion mode, respectively. Our results demonstrate that the fT3SS features a remarkable specificity for only the substrates required at the respective assembly stage. No crosstalk of substrates was observed for fT3SSs operating in the opposing secretion mode. We further found that a substantial fraction of fT3SS surprisingly remained in early secretion mode. Our results thus suggest that the secretion substrate specificity switch of the fT3SS is unidirectional and irreversible. The developed secretion substrate reporter system further provides a platform for future investigations of the underlying molecular mechanisms of the elusive substrate recognition of the T3SS.

Planar polarization of cilia in the zebrafish floor-plate involves Par3-mediated posterior localization of highly motile basal bodies.

Epithelial cilia, whether motile or primary, often display an off-center planar localization within the apical cell surface. This form of planar cell polarity (PCP) involves the asymmetric positioning of the ciliary basal body (BB). Using the monociliated epithelium of the embryonic zebrafish floor-plate, we investigated the dynamics and mechanisms of BB polarization by live imaging. BBs were highly motile, making back-and-forth movements along the antero-posterior (AP) axis and contacting both the anterior and posterior membranes. Contacts exclusively occurred at junctional Par3 patches and were often preceded by membrane digitations extending towards the BB, suggesting focused cortical pulling forces. Accordingly, BBs and Par3 patches were linked by dynamic microtubules. Later, BBs became less motile and eventually settled at posterior apical junctions enriched in Par3. BB posterior positioning followed Par3 posterior enrichment and was impaired upon Par3 depletion or disorganization of Par3 patches. In the PCP mutant vangl2, BBs were still motile but displayed poorly oriented membrane contacts that correlated with Par3 patch fragmentation and lateral spreading. Thus, we propose an unexpected function for posterior Par3 enrichment in controlling BB positioning downstream of the PCP pathway.

The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus.

Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.