Distal Appendages Research Articles

Proliferation associated 2G4 is required for the ciliation of vertebrate motile cilia

Motile cilia are critical structures that regulate early embryonic development and tissue homeostasis through synchronized ciliary motility. The formation of motile cilia is dependent on precisely controlled sequential processes including the generation, migration, and docking of centrioles/basal bodies as well as ciliary growth. Using the published proteomics data from various organisms, we identified proliferation-associated 2G4 as a novel regulator of ciliogenesis. Loss-of-function studies using Xenopus laevis as a model system reveal that Pa2G4 is essential for proper ciliogenesis and synchronized movement of cilia in multiciliated cells (MCCs) and the gastrocoel roof plate (GRP). Pa2G4 morphant MCCs exhibit defective basal body docking to the surface as a result of compromised Rac1 activity, apical actin network formation, and immature distal appendage generation. Interestingly, the regions that include the RNA-binding domain and the C-terminus of Pa2G4 are necessary for ciliogenesis in both MCCs and GRP cells. Our findings may provide insights into motile cilia-related genetic diseases such as Primary Ciliary Dyskinesia.

Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes.

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Morphology and feeding biology of larvae of the South African endemic water beetle genus Prosthetops Waterhouse, 1879 (Coleoptera: Hydraenidae)

The larvae of three species of the South African endemic water beetle genus Prosthetops Waterhouse, 1879 are described and illustrated for the first time. The second and third instar larvae of Prosthetops nitens (Péringuey, 1892), as well as third instar larva of P. megacephalus (Boheman, 1851) and P. wolfbergensis Bilton, 2013, are treated here. These three species share a combination of morphological and chaetotaxic characters that can be used for positive identification of larvae of this genus: head capsule with pores FC1 present, second antennomeres with two well-developed distal sensory appendages, maxillary seta Cdo1 very reduced, thoracic subprimary setae Dd’ absent, subprimary setae Dc’ minute and inserted on the boundary between pretergal and tergal areas, urogomphi moderately separated at the base, anal lobe with well-developed dorsal, lateral and ventral plates, and a lack of anal hooks. Some insights on the gut-contents of the larva of P. wolfbergensis are offered and an unusual urogomphal malformation observed in a larva of P. nitens is also described and illustrated. These are the first described larvae of the subfamily Prosthetopinae, and their morphology is compared to that of other known hydraenid larvae.

SMYD3 Controls Ciliogenesis by Regulating Distinct Centrosomal Proteins and Intraflagellar Transport Trafficking.

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

P14 Midface Toddler Excoriation Syndrome (MiTES): a new mimic of non-accidental injury

Abstract Background Midface Toddler Excoriation Syndrome (MiTES) is a rare genetic condition first described in 2017. Commencing in the first year of life, it is characterised by intense mid-­facial itch with persistent scratching and skin breakdown. Symptoms improve later in childhood, leaving atrophic scarring. Disbelief that infants can damage themselves in this way can lead to an erroneous and damaging misdiagnosis of non-accidental injury (NAI). Cases A boy now aged 11 years presented at 6 months with recurrent scratching of the face, leading to ulceration of the nasal bridge, sides of the nose, infraorbital areas and central forehead. Extensive investigations were unhelpful and NAI was suspected with significant psychosocial sequelae for the family. When his sister, now aged 4 years, presented with strikingly similar lesions MiTES was suspected and confirmed by genetic testing which showed homozygosity for 18 alanine repeats in the PRDM12 gene. Testing of sibling and parents is planned. Both children are now asymptomatic but have significant facial scarring. Discussion Biallelic variants in PRDM12 usually cause hereditary sensory and motor neuropathy type 8 with widespread damage to distal appendages due to pain insensitivity. The specific variant found here and in all reported MiTES cases causes highly localised lesions with rarely any extra-facial ­involvement. Due to its novelty and rarity, MiTES may be misdiagnosed and safeguarding concerns raised, leading to distress for affected families and breakdown of parent-­provider relationships. This case, which for the first time provides longitudinal data on affected siblings, highlights the need for early recognition and diagnosis.

Nuclei and tracts in the epithalamus of crocodiles.

The epithalamus, an area of the dorsal diencephalon found in all vertebrates, consists of the habenula, the subhabenular nuclei, and associated tracts. The habenula is itself divisible into two parts-a medial and a lateral nucleus differing in theirinputs, outputs, and cellular morphology. The medial component is related to the limbic system and serotonergic raphe, while the lateral nucleus is more interconnected with the basal ganglia and midbrain dopamine systems. These findings, which come from experiments mainly done on mammals, serve as a basis for comparison with other vertebrates. However, similar studies in other amniotes, such as reptiles, are few. To fill this gap in knowledge, two species of crocodiles were examined utilizing a variety of histological methods in various planes of section. The following results were obtained. First, the habenula was divided into medial and lateral parts based on its cytoarchitecture. Neurons in the medial habenula were small, were closely packed, and had a limited dendritic arbor characterized by unusual distal dendritic appendages, whereas neurons in the lateral habenula were larger, were more loosely packed, and had longer dendritic processes that were commonly beaded. Second, the stria medullaris, the major input to the habenula, was identified by its immunoreactivity to parvalbumin. Third, the fasciculus retroflexus (habenulointerpeduncular tract), the primary output of the habenula, was visualized by staining with acetylcholinesterase. Fourth, nuclei associated with the habenula, the subhabenular nuclei, have been identified and characterized. These features provide a means to recognize the major nuclei and tracts in the epithalamus in crocodiles and are likely applicable to other reptiles.

PO-02-229 AUTOMATED LOCALIZATION OF ATRIAL ECTOPIC BEATS USING 12-LEAD ECG MEAN SPATIO-TEMPORAL VECTOR POSITION: IMPORTANCE OF INCORPORATING PATIENT-SPECIFIC ANATOMIC MODELS

Localizing the origin of atrial ectopic activity from the 12-lead ECG can be challenging, particularly in patients with atrial pathology. To identify the origin of paced atrial ectopic beats (simulating atrial ectopy) using software that quantifies the mean spatio-temporal vector position of cardiac depolarization projected onto generic and patient-specific atrial models. 12-lead ECG data were collected during sinus rhythm (SR) and during regular pacing from the high and low right atrium (HRA/LRA), proximal and distal coronary sinus (CS), pulmonary veins (LSPV, LIPV, RSPV, RIPV), and left atrial appendage (LAA). The origin and trajectory of the atrial signals were calculated with software that analyzes P-waves from the 12-lead ECG and automatically constructs a net vector of electrical activity projected onto a standard generic model of human atria (CineECG; Nieuwerbrug, The Netherlands). The model atria were split into 9 segments, and the software accuracy was scored based on localization of the paced atrial signal origin to the correct segment. To see if patient-specific anatomy improved localization, we also incorporated atrial anatomy from cardiac MRIs acquired from a subset of 12 patients and compared localization to the generic model. We enrolled 19 patients (mean age 70±9, 4 female) with paroxysmal (63%) or persistent (37%) AF and left atrial (LA) enlargement (mean LA volume 58 cc/m2) undergoing pulmonary vein isolation for treating AF. Using the generic atrial model, the software correctly localized 105/142 (74%) beats to the correct atrial segment (Figure). Accuracy was better for RA compared to LA signals (90% vs. 62%, p < 0.05). Localization worked best in the coronary sinus (19/19, 100%) and worst in the LPVs (14/33, 42%). Patient-specific models improved localization of PV signals, particularly left-sided ones (generic vs. patient-specific: RPVs 70% vs 85%, p = 0.36, LPVs 42% vs 76%, p = 0.03). Accuracy improved and was similar for RA vs. LA localization when using patient-specific models (90% vs. 82%, p = 0.48). The novel software algorithm can automatically localize paced signals in both the left and right atria despite significant atrial pathology. Patient-specific anatomic models improve localization. This software holds promise for automatically localizing and classifying atrial arrhythmias and guiding therapy, including catheter ablation, based on standard 12-lead ECG data.

Control of centrosome distal appendages assembly and disassembly

Centrosomes are microtubule organizing centers involved in chromosome segregation, spindle orientation, cell motility and cilia formation. In recent years, they have also emerged as key modulators of asymmetric cell division. Centrosomes are composed of two centrioles that initiate duplication in S phase. The conservative nature of centriole duplication means that the two centrioles of a G1 cell are of different ages. They are also structurally different as only the older centriole carries appendages, an assembly of a subset of proteins primarily required for cilia formation. In a growing tissue, the non-motile, primary cilium acts as a mechano- and sensory organelle that influences cell behavior via modulation of signaling pathways. Here, we discuss the most recent findings about distal appendage composition and function, as well as cell cycle-specific regulation and their implications in various diseases.

Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.

Single-molecule localization microscopy of expanded mammalian centrioles unravels the ultrastructural constitution of distal appendages.

Distal appendages (DAPs) are vital in ciliogenesis, mediating membrane docking and ciliary gating. Although the nine-fold symmetric constitution of DAP proteins has been investigated via superresolution microscopy analyses, the root structure of DAPs remains elusive owing to insufficient spatial optical resolution. Here, we proposed the in situ drift correction for optimized two-color imaging incorporating single-molecule localization microscopy with sample expansion approaches to achieve effective localization precision down to sub-5 nm. The robust workflow enables us to systematically perform the ultrastructural imaging study of protein mapping within cells, not limited to the proximity of coverslips. Notably, our findings unraveled the ultrastructural arrangement of the core DAP proteins (CEP83, CEP89, SCLT1, FBF1, and CEP164) and their spatial connection to microtubule triplets. Surprisingly, we found that C2CD3 was localized near a tubule of triplets guiding the DAP formation from microtubules to outer components of blades with nine-fold symmetry. We further identified the dual-layered distribution of ODF2. Intriguingly, the distal-layered ODF2 displayed a high spatial correlation with other DAP proteins, acting as a structural coordinator at the DAP root. Moreover, our results showed that the N- and C-terminus of ODF2 jointly encircled the microtubule triplets. Together, our studies suggest the unprecedented architectural framework of DAPs and provide a pragmatic roadmap for protein mapping with optical resolution at a molecular level.

Three-dimensional single-molecule super-resolution imaging of proteins involved in primary cilia formation.

Primary cilia are organelles that protrude from the cell membrane of most human cells where they perform critical sensory and signaling functions. Defects and mutations in proteins in the primary cilium cause a range of diseases and syndromes collectively termed ciliopathies. Intracellular primary cilium formation begins with the recruitment of Golgi-derived pre-ciliary vesicles to the distal appendages of the mother centriole, but the molecular mechanisms behind the recruitment of the distal appendage proteins are not fully understood. The proteins Rab34 and MyosinVa were found through structured illumination microscopy (SIM) to be closely associated with these pre-ciliary vesicles during this step. However, because of the sub-diffraction size and three-dimensional (3D) structures of the centriole and of the involved proteins, higher resolution multi-target 3D microscopy is required to resolve their organization. To address this limitation, 3D two-color direct stochastic optical reconstruction microscopy (dSTORM) was implemented, providing a single-molecule localization precision of roughly 20 nm in all dimensions. A two-channel 4f emissions system was constructed using a 2-μm axial range double helix phase mask for single-molecule localization in 3D through point spread function engineering. This system allowed for 3D single-molecule super-resolution imaging of Rab34 and MyosinVa in two different color-channels together with imaging of the centriolar protein FOP in both color-channels for context and precise channel registration. The resulting 3D super-resolved reconstructions revealed the nanoscale structures formed by these proteins at the pre-ciliary vesicles, the structural relation between Rab34 and MyosinVa, and their position in relation to the mother centriole.

Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding

Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

Tissue-specific requirement of sodium channel and clathrin linker 1 (Sclt1) for ciliogenesis during limb development.

Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.

The central scaffold protein CEP350 coordinates centriole length, stability, and maturation.

The centriole is the microtubule-based backbone that ensures integrity, function, and cell cycle-dependent duplication of centrosomes. Mostly unclear mechanisms control structural integrity of centrioles. Here, we show that the centrosome protein CEP350 functions as scaffold that coordinates distal-end properties of centrioles such as length, stability, and formation of distal and subdistal appendages. CEP350 fulfills these diverse functions by ensuring centriolar localization of WDR90, recruiting the proteins CEP78 and OFD1 to the distal end of centrioles and promoting the assembly of subdistal appendages that have a role in removing the daughter-specific protein Centrobin. The CEP350-FOP complex in association with CEP78 or OFD1 controls centriole microtubule length. Centrobin safeguards centriole distal end stability, especially in the compromised CEP350-/- cells, while the CEP350-FOP-WDR90 axis secures centriole integrity. This study identifies CEP350 as a guardian of the distal-end region of centrioles without having an impact on the proximal PCM part.

This Week in The Journal

Jixiang Zhang, Jazzmine M. Junigan, Ronnie Trinh, Annemieke Kavelaars, Cobi J. Heijnen, et al. (see pages [7862–7874][1]) Chemotherapy-induced peripheral neuropathy (CIPN) increases pain sensitivity and can produce spontaneous pain in the distal appendages of many cancer patients. The symptoms

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.

The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Taxonomic applications of the esophageal flapper valve in Bairdoppilata and Glyptobairdia (Bairdiidae, Ostracoda), with comments on anatomy, ontogeny, and geography.

The esophageal valve follows a uniform design in species of Bairdoppilata and Glyptobairdia, having a thin, wedge-shaped plate with 1016 small teeth along the posterior edge. This structure may be added to the list of diagnostic anatomical traits, including the scissors-like antennal claws and complex distal appendages of the hemipenis, for identification of living species of these genera. These soft anatomical characters are invariably associated with bairdoppilatan supplemental dentition on the carapace, which supports the reliability of that trait for identification of fossils. This report evaluates the consistency of the esophageal plate in nine living species of Bairdoppilata. Photographs of the Oligocene type species, B. martyni, are provided for the first time. Geographic populations of the common Caribbean species, B. cushmani, display some heterogeneity in carapace size and proportions. B. hirsutella, n. sp., is proposed for a population previously misidentified as B. hirsuta. Supplemental information and illustrations are provided for Glyptobairdia coronata and G. trinodosa.

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.