Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium’s capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet–Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet–Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host–parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host–pathogen interface and offering insights into BBSome molecular mechanisms.Cilia, also called eukaryotic flagella, are emblematic organelles for which functional and structural similarities across diverse lineages indicate they have existed since the emergence of eukaryotes (1, 2). Although historically considered as machines for cell locomotion and movement of fluids across epithelia, cilia are now recognized as signaling platforms that sense and transduce environmental stimuli to drive cellular responses (3). As such, cilia constitute a critical cell–environment interface that is paramount for development and physiology of ciliated organisms. In vertebrates, cilium-dependent signaling orchestrates important developmental pathways, such as limb development and kidney morphogenesis, and is required for vision, hearing, and smell (4). In free-living protists, cilium signaling controls cell motility, mating, and response to extracellular cues (5).The cilium is anchored to the cell surface membrane and protrudes into the extracellular milieu. At the core of the organelle is a microtubule-based axoneme that originates at the basal body in the cytoplasm. The axoneme is encased by a ciliary membrane that is contiguous with the plasma membrane but constitutes a specialized domain with distinct protein and lipid composition (6). The base of the cilium is not entirely delimited by membrane, and the ciliary matrix (soluble fraction within the cilium) is, thus, topologically contiguous with the cytoplasm. Organellar identity is maintained by a diffusion barrier that bona fide ciliary proteins must traverse; traversal of this barrier in and out of the cilium is critical for cilium function (7).In keeping with their critical motility and sensory functions, defective cilia cause a wide range of inherited human diseases, termed ciliopathies, which exhibit diverse clinical manifestations and molecular etiologies (8). Bardet–Biedl Syndrome (BBS) is a ciliopathy that is mainly characterized by retinopathy, obesity, polydactyly, cognitive impairment, renal abnormalities, and hypogonadism; we currently know of 19 genes (bbs1–bbs19) that, when mutated, can cause BBS (9). Interestingly, eight bbs genes (bbs1, bbs2, bbs4, bbs5, bbs7, bbs8, bbs9, and bbs18) encode proteins that assemble into a complex termed the Bardet–Biedl Syndrome complex (BBSome) (10, 11). While cilium assembly is generally unaffected, BBSome mutants in vertebrates and protists exhibit sensing defects resulting from abnormal localization of select ciliary proteins (12–18). Although the BBSome is necessary for dynamic trafficking of these membrane-associated proteins through the ciliary compartment, its precise location and exact function remain enigmatic.Ciliated pathogens cause tremendous human suffering worldwide and limit economic development in some of the world’s poorest regions (19). Despite broad awareness of the cilium’s role in the pathology of inherited human diseases, the contribution of cilia and ciliary modules, such as the BBSome, to infection by eukaryotic parasites is mostly unknown (20). Parasite survival and virulence depend on successful interaction with the host environment, and this interaction is mediated, at least in part, by cilia and ciliary proteins (21). This paradigm applies to the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness in humans and Nagana in cattle. Sleeping sickness is endemic to sub-Saharan Africa, is almost always fatal if untreated, and remains one of the world’s most neglected diseases (22). T. brucei has a single flagellum, which emerges from the cytoplasm through the flagellar pocket at the posterior end of the cell (23). The flagellar pocket is a pronounced invagination of the plasma membrane that marks the boundary between the flagellar membrane and the rest of the plasma membrane. The trypanosome flagellar pocket is a key host–parasite portal, because it is the sole site of endocytosis, mediates uptake of growth factors, and is a necessary transit point for proteins en route to or from the cell surface (24).Given the role of the BBSome in controlling delivery of ciliary proteins important for interaction with the external environment, we examined BBSome functions in mammalian-infectious, bloodstream-stage T. brucei. We report that T. brucei BBS proteins assemble into a BBSome [T. brucei Bardet–Biedl Syndrome complex (TbBBSome)] that localizes to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. BBS gene knockouts (KOs) show the TbBBSome is dispensable for flagellum assembly and parasite viability but required for virulence in a mouse infection model. Quantitative proteomics and biochemical analysis suggest that the TbBBSome interacts with clathrin and ubiquitin to facilitate endocytic trafficking of select cell surface proteins and that defects in these processes underlie the virulence defect of BBSome mutants. Our combined studies offer insights into both parasite biology and the pathophysiology of human ciliopathies.     

Direct imaging of intraflagellar-transport turnarounds reveals that motors detach,diffuse, and reattach to opposite-direction trains

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach–diffuse–attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

Almost all eukaryotic cells contain one or multiple cilia, organelles that play crucial roles in sensory perception and signaling (1). The assembly and maintenance of cilia rely on bidirectional intraflagellar transport (IFT) along the cilium, which is mediated by kinesin-2 and IFT-dynein motors (2–8). In Chlamydomonas, a single motor, heterotrimeric kinesin-2 (9, 10), drives the anterograde transport of IFT trains, consisting of IFT-particle complexes and IFT motors to the ciliary tip. From the tip, kinesin-2 is passively recycled back to the ciliary base by diffusion, independent of retrograde IFT driven by IFT-dynein (11–15). In Caenorhabditis elegans chemosensory cilia, however, two different kinesin-2 motors cooperate to drive anterograde IFT, and they are actively recycled back to the base by IFT-dynein–driven retrograde IFT (5, 9, 16, 17). This marked difference in mode of kinesin returning from tip to base raises the question whether kinesin-2 motors in C. elegans are always associated to IFT trains or whether some motors are freely moving around in the cilia. If kinesin-2 motors are always associated with IFT trains, how can they rapidly jump between anterograde and retrograde trains, as inferred in previous studies (18, 19)?C. elegans chemosensory cilia consist of a cylindrical, microtubule-based axoneme surrounded by a specialized membrane (20). The axoneme emanates from the ciliary base and transition zone to a bipartite structure consisting of the proximal segment and the distal segment made of nine doublet and nine singlet microtubules, respectively (6, 21). IFT is required for proper ciliary biogenesis and maintenance, and IFT requires the cooperation of anterograde kinesin-2 motor proteins (5, 22) and retrograde IFT-dynein motors (8). The core machinery of IFT trains includes kinesin-2 and IFT-dynein motors and a stable IFT-train backbone composed of IFT-A and IFT-B complexes (11) linked by the BBSome (23) to which motors and cargoes can dock and undock (3, 24). IFT trains are assembled in the ciliary base and transported to the ciliary tip by the cooperative action between two types of kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3 (5, 9, 16, 17). Kinesin-II acts as the “import” motor to drive IFT trains through the base and transition zone toward the proximal segment, possibly avoiding obstacles on the microtubules by sidestepping (25). There, kinesin-II gradually hands over anterograde IFT trains to OSM-3, which drives the long-range transport toward the ciliary tip (18). At the tip, IFT trains are rapidly disassembled and reassembled (14, 26) and transported back to the ciliary base solely by IFT-dynein (8, 15, 27).Single-molecule imaging of kinesin-2 and IFT-dynein motors has revealed that motors undergo rapid turnarounds (directional switches) to achieve efficient motor cooperation (18, 19). The anterograde-to-retrograde (a-to-r) turnarounds of kinesin-II and r-to-a turnarounds of OSM-3 in the handover zone (located between 1 and 4 µm from the base) have been interpreted as kinesin-II switching from drivers of anterograde trains to passengers of retrograde trains and OSM-3 switching from passengers of retrograde trains to active drivers of anterograde trains, respectively (18). This handover mechanism is the origin of the efficiency of the cooperation between kinesin-II and OSM-3 because kinesin-II immediately starts to detach from anterograde trains after finishing their function as the import motor while OSM-3 starts to bind to anterograde trains to take over the long-range transport to the tip. Similarly, single-molecule imaging of IFT-dynein has revealed that IFT-dynein can undergo a-to-r turnarounds at all locations along the cilium (19), which has been interpreted as IFT-dynein switching from the deactivated passenger of anterograde trains to the active driver of retrograde trains. Taken together, these single-motor turnarounds are consistent with an overall picture of IFT, in which the IFT-train backbone provides a stable platform, moving from base to tip or back, to which individual motors can rapidly attach and detach during transport, driving effective and regular IFT (3, 18).So far, however, direct observations of this interpretation that IFT motors switch between opposite-direction trains to turn around have not been made, which leaves a possible alternative explanation, namely that turnarounds are partly due to freely moving motors not associated with IFT trains. To further zoom into the mechanism of IFT motor turnarounds, here we push the envelope of our single-molecule fluorescence imaging capabilities in living C. elegans. By applying alternating dual-color imaging, we directly show that during a turnaround, motors switch from one IFT train to another. By increasing our imaging time resolution (up to 30 ms per frame), we show that during the directional switch, motors use diffusion to hop from one train to another.     

Conditions for metachronal coordination in arrays of model cilia

On surfaces with many motile cilia, beats of the individual cilia coordinate to form metachronal waves. We present a theoretical framework that connects the dynamics of an individual cilium to the collective dynamics of a ciliary carpet via systematic coarse graining. We uncover the criteria that control the selection of frequency and wave vector of stable metachronal waves of the cilia and examine how they depend on the geometric and dynamical characteristics of a single cilium, as well as the geometric properties of the array. We perform agent-based numerical simulations of arrays of cilia with hydrodynamic interactions and find quantitative agreement with the predictions of the analytical framework. Our work sheds light on the question of how the collective properties of beating cilia can be determined using information about the individual units and, as such, exemplifies a bottom-up study of a rich active matter system.

Motile cilia are hair-like organelles that beat with a whip-like stroke that breaks time-reversal symmetry to create fluid flow or propel swimming microorganisms under low Reynolds number conditions (1–3). The beat is actuated by many dynein motors, which generate forces between microtubules that cause the cilium to bend in a robust cyclic manner with moderate fluctuations (4, 5). On surfaces with many cilia, the actuating organelles can coordinate with each other and collectively beat in the form of metachronal waves, where neighboring cilia beat sequentially (i.e., with a phase lag) rather than synchronously (6). The flows created from this coordinated beating are important for breaking symmetry in embryonic development (7, 8), creation of complex and dynamic flow patterns for the cerebrospinal fluid in the brain (9, 10), and providing access to nutrients (11). In microorganisms such as Paramecium and Volvox, the metachronal beating of cilia provides propulsion strategies in viscous environments (12, 13). It has been shown that depending on the parameters, beating ciliary carpets can exhibit globally ordered and turbulent flow patterns (14), which can be stable even with a moderate amount of quenched disorder (15), and that metachronal coordination optimizes the efficiency of fluid pumping (16, 17). Natural cilia have inspired various designs of artificial cilia (18–22), which may be used for pumping fluid (23, 24) and mixing (25), or fabrication of microswimmers (26).Hydrodynamic interactions have been shown to play a key role in coordinated beating of cilia (27, 28) and mediating cell polarity control (29). To achieve synchronization between two cilia via hydrodynamic interactions, it is necessary to break the permutation symmetry between them [e.g., by exploiting the dependence of the drag coefficient on the distance from a surface (30), flexibility of the anchoring of the cilia (31), nonuniform beat patterns (32, 33), or any combination of these effects (34)]. In addition to the hydrodynamic interactions, the basal coupling between cilia can also facilitate the coordination (35–37).How can we predict the collective behavior of arrays of many cilia coordinated by hydrodynamic interactions, and in particular, the properties of the emerging metachronal waves, from the single-cilium characteristics? Extensive numerical simulations using explicitly resolved beating filaments (16, 17, 27, 38–40) and simplified spherical rotors (13, 14, 41, 42) have demonstrated that metachronal coordination emerges from hydrodynamic interactions. However, insight into this complex many-body dynamical system at the level that has been achieved in studies of two cilia is still lacking. Here, we propose a theoretical framework for understanding the physical conditions for coordination of many independently beating cilia, which are arranged on a substrate in the form of a two-dimensional (2D) array immersed in a three-dimensional (3D) fluid. We uncover the physical conditions for the emergence of stable metachronal waves and predict the properties of the wave in terms of single-cilium geometric and dynamic characteristics.     

C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals

The primary cilium plays critical roles in vertebrate development and physiology, but the mechanisms underlying its biogenesis remain poorly understood. We investigated the molecular function of C2 calcium-dependent domain containing 3 (C2cd3), an essential regulator of primary cilium biogenesis. We show that C2cd3 is localized to the centriolar satellites in a microtubule- and Pcm1-dependent manner; however, C2cd3 is dispensable for centriolar satellite integrity. C2cd3 is also localized to the distal ends of both mother and daughter centrioles and is required for the recruitment of five centriolar distal appendage proteins: Sclt1, Ccdc41, Cep89, Fbf1, and Cep164. Furthermore, loss of C2cd3 results in failure in the recruitment of Ttbk2 to the ciliary basal body as well as the removal of Cp110 from the ciliary basal body, two critical steps in initiating ciliogenesis. C2cd3 is also required for recruiting the intraflagellar transport proteins Ift88 and Ift52 to the mother centriole. Consistent with a role in distal appendage assembly, C2cd3 is essential for ciliary vesicle docking to the mother centriole. Our results suggest that C2cd3 regulates cilium biogenesis by promoting the assembly of centriolar distal appendages critical for docking ciliary vesicles and recruiting other essential ciliogenic proteins.The primary cilium is a fundamentally important organelle, the loss of which in humans causes a broad spectrum of genetic disorders known as ciliopathies (1). In addition to sensory and motile functions, the primary cilium plays a central role in the signal transduction of the Hh, PDGF, Wnt, Hippo, and calcium signaling pathways (2–4). Recent genomic and proteomic approaches have identified a ciliome consisting of hundreds of proteins (5, 6). However, the molecular functions of most of these proteins remain elusive.The primary cilium originates from the basal body, a centriole-derived structure (1). A centriole comprises a core multiprotein complex surrounded by a cylinder of nine microtubule triplets. In addition, the oldest centriole, the mother centriole, possesses distal and subdistal appendages. Centrioles and surrounding pericentriolar material constitute the centrosomes and spindle poles in dividing cells (7). In quiescent cells, Golgi-derived ciliary vesicles dock at the distal end of the mother centriole (8). The mother centriole, now known as the basal body, migrates to the cell surface with the docked ciliary vesicle. The ciliary vesicle fuses with the plasma membrane, allowing the cilium to protrude from the cell surface.The distal appendages of the mother centriole, also known as transition fibers of the cilium, are protein complexes comprising at least five components (Ccdc41/Cep83, Cep89/Cep123, Sclt1, Fbf1, and Cep164) (9). Distal appendages are critical for the recruitment of Tau tubulin kinase 2 (Ttbk2), which appears to play a critical role in removing centrosomal protein of 110kD (Cp110, also known as Ccp110, Mouse Genome Informatics, www.informatics.jax.org/marker/MGI:2141942), an inhibitor of ciliogenesis, from the distal end of the mother centriole (10). Recent studies have also suggested that components of the distal appendages are essential for docking ciliary vesicles to the mother centriole (9, 11–14).The axoneme, the microtubule skeleton of the cilium, extends from the distal end of the mother centriole and requires intraflagellar transport (IFT), which was known to mediate cargo movement within the cilium (15). Mutations disrupting the functions of the IFT motors (kinesin II and cytoplasmic dynein) or the IFT cargo adaptor complexes (IFT-A and IFT-B) lead to defects in cilia biogenesis. Recent studies have revealed an essential role for distal appendage proteins in recruiting IFT proteins to the mother centriole, suggesting another mechanism by which centriolar distal appendages promote ciliogenesis (9, 12, 13).Centriolar satellites are electron-dense particles around the centrosome and basal body (16). Many centriolar satellite components have been identified, including Pcm1, the BBSome, a multiprotein complex comprising seven Bardet–Biedl syndrome-related proteins, as well as Cep290 and Ofd1 (17–20). Interestingly, the functions of various centriolar satellite proteins diverge. Pcm1 and the BBSome promote ciliary membrane biogenesis and the trafficking of ciliary membrane proteins (18, 21). Ofd1, on the other hand, appears to regulate ciliogenesis by recruiting components of the distal appendages and IFT particles, although a recent study appeared to suggest an additional negative role of Ofd1 in ciliogenesis (22, 23).Through the study of two loss-of-function mouse mutants, we have previously identified a novel C2 domain-containing protein, C2cd3, as an essential regulator of ciliogenesis and mouse embryonic development (24). In the current study, we demonstrate that C2cd3 is localized to centriolar satellites and that its localization is dependent on Pcm1 and dynein-mediated retrograde transport. C2cd3 is also localized to the distal ends of the mother and daughter centrioles and is required for the recruitment of five distal appendage proteins: Ccdc41, Sclt1, Cep89, Fbf1, and Cep164. Moreover, in the absence of C2cd3, Ttbk2 is not recruited to the distal end of the mother centriole, nor is Cp110 removed. In addition, the recruitment of Ift88 and Ift52, two IFT complex-B components, does not occur in C2cd3 mutants. Finally, we observed that the docking of the ciliary vesicles to the mother centriole is dependent on C2cd3. Our results suggest that C2cd3 regulates the initiation of ciliogenesis through centriolar maturation, ciliogenic protein recruitment and ciliary vesicle docking.     

Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons

Most central neurons in the mammalian brain possess an appendage called a primary cilium that projects from the soma into the extracellular space. The importance of these organelles is highlighted by the fact that primary cilia dysfunction is associated with numerous neuropathologies, including hyperphagia-induced obesity, hypogonadism, and learning and memory deficits. Neuronal cilia are enriched for signaling molecules, including certain G protein-coupled receptors (GPCRs), suggesting that neuronal cilia sense and respond to neuromodulators in the extracellular space. However, the impact of cilia on signaling to central neurons has never been demonstrated. Here, we show that the kisspeptin receptor (Kiss1r), a GPCR that is activated by kisspeptin to regulate the onset of puberty and adult reproductive function, is enriched in cilia projecting from mouse gonadotropin-releasing hormone (GnRH) neurons. Interestingly, GnRH neurons in adult animals are multiciliated and the percentage of GnRH neurons possessing multiple Kiss1r-positive cilia increases during postnatal development in a progression that correlates with sexual maturation. Remarkably, disruption of cilia selectively on GnRH neurons leads to a significant reduction in kisspeptin-mediated GnRH neuronal activity. To our knowledge, this result is the first demonstration of cilia disruption affecting central neuronal activity and highlights the importance of cilia for proper GPCR signaling.Primary cilia are typically solitary nonmotile appendages that project from nearly every cell type in the mammalian body (1). They are specialized sensory organelles that incorporate a myriad of extracellular stimuli into signal transduction pathways to modulate cell physiology (2–4). Consequently, ciliary dysfunction can result in numerous human diseases, termed ciliopathies, which impact many organ systems (5). Ciliopathies are associated with certain neuropathologies, including structural malformations, hyperphagia-induced obesity, intellectual disability, and hypogonadism, thereby highlighting the importance of cilia for proper CNS development and function (3).Most adult neurons in the mammalian brain possess a primary cilium that projects from its cell body. Specific signaling proteins are selectively targeted to and retained within neuronal cilia, which are restricted compartments and regulate entry and exit of proteins through multiple mechanisms (6, 7). These signaling proteins include type 3 adenylyl cyclase (AC3) (8), which converts ATP to cAMP, and the GPCRs, somatostatin receptor 3 (Sstr3) (9), serotonin receptor 6 (10, 11), melanin-concentrating hormone receptor 1 (Mchr1) (12, 13), dopamine receptor 1 (14), and neuropeptide Y receptors 2 and 5 (15). The functions of primary cilia are determined by the proteins that are enriched within them, thus, it is likely that neuronal cilia sense neuromodulators in the extracellular milieu and initiate signaling cascades. However, the precise roles of neuronal cilia remain unknown.Identification of signaling proteins that are selectively targeted to neuronal cilia is a critical step in elucidating the functions of these organelles. We previously identified ciliary localization sequences in the third intracellular loop and carboxy tail of ciliary GPCRs and used these sequences to predict novel ciliary GPCRs (12, 14). One of these, the kisspeptin receptor (Kiss1r, also known as GPR54), was considered a strong candidate ciliary GPCR given that loss of Kiss1r leads to hypogonadotropic hypogonadism in humans and mice (16, 17) and hypogonadotropic hypogonadism is a feature of the ciliopathy Bardet-Biedl syndrome (18). Kiss1r is expressed in a large proportion of gonadotropin-releasing hormone (GnRH) neurons (19), a population of hypothalamic neurons that are central effectors driving the neuroendocrine reproductive axis. Treatment of Kiss1r-expressing GnRH neurons with kisspeptin increases the firing rate of the GnRH neurons and augments GnRH secretion. GnRH stimulates luteinizing hormone and follicle-stimulating hormone secretion, which in turn initiates puberty and supports adult sexual function.Here, we show that Kiss1r is enriched in primary cilia on GnRH neurons in the mouse. Notably, GnRH neurons can possess multiple Kiss1r-positive cilia and the proportion of multiciliated GnRH neurons increases in parallel with sexual maturation. We also show that cilia are required for proper Kiss1r-mediated signaling on GnRH neurons. These results provide insight into the mechanism of Kiss1r signaling and demonstrate that loss of cilia on central neurons impairs neuronal signaling.     

Primary cilia are required in a unique subpopulation of neural progenitors

The apical domain of embryonic (radial glia) and adult (B1 cells) neural stem cells (NSCs) contains a primary cilium. This organelle has been suggested to function as an antenna for the detection of morphogens or growth factors. In particular, primary cilia are essential for Hedgehog (Hh) signaling, which plays key roles in brain development. Their unique location facing the ventricular lumen suggests that primary cilia in NSCs could play an important role in reception of signals within the cerebrospinal fluid. Surprisingly, ablation of primary cilia using conditional alleles for genes essential for intraflagellar transport [kinesin family member 3A (Kif3a) and intraflagellar transport 88 (Ift88)] and Cre drivers that are activated at early [Nestin; embryonic day 10.5 (E10.5)] and late [human glial fibrillary acidic protein (hGFAP); E13.5] stages of mouse neural development resulted in no apparent developmental defects. Neurogenesis in the ventricular–subventricular zone (V-SVZ) shortly after birth was also largely unaffected, except for a restricted ventral domain previously known to be regulated by Hh signaling. However, Kif3a and Ift88 genetic ablation also disrupts ependymal cilia, resulting in hydrocephalus by postnatal day 4. To directly study the role of B1 cells’ primary cilia without the confounding effects of hydrocephalus, we stereotaxically targeted elimination of Kif3a from a subpopulation of radial glia, which resulted in ablation of primary cilia in a subset of B1 cells. Again, this experiment resulted in decreased neurogenesis only in the ventral V-SVZ. Primary cilia ablation led to disruption of Hh signaling in this subdomain. We conclude that primary cilia are required in a specific Hh-regulated subregion of the postnatal V-SVZ.The primary cilium, a minute elongated organelle with a (9+0) microtubular cytoskeleton (axoneme) on the surface of most cells, is essential for signal transduction and particularly for Hedgehog (Hh) signaling (1–4). The primary cilium, therefore, has very important functions during vertebrate development (5, 6), including the development of the central nervous system (7–9). Primary cilia are required for the expansion of progenitor pool during cerebellar development (10, 11) and in the formation of neural stem cells (NSCs) and progenitors in the adult hippocampus (12–14). Moreover, it has been shown that primary cilia regulate dendritic refinement and synaptic integration of adult-born hippocampal neurons (15). Recent evidence also shows that Arl13b in primary cilia is essential for the early polarization of the neuroepithelium and the formation of radial glia (16). In addition, primary cilia and Arl13b regulate migration and placement of interneurons in the developing cerebral cortex (17, 18).The walls of the lateral ventricles retain an active germinal niche in the ventricular–subventricular zone (V-SVZ) that continues generating neurons and glial cells in the postnatal brain of many mammals (19). The astroglia-like NSCs (B1 cells) give rise to intermediate progenitor cells (C cells), which in turn generate neuroblasts (A cells) (20–22). These young neurons migrate along the rostral migratory stream to the olfactory bulb (OB). B1 cells retain epithelial characteristics, including an apical domain that contacts the lateral ventricle (23). This apical process contains a primary cilium and is surrounded by multiciliated ependymal (E1) cells in a pinwheel-like organization (23). Given their location and the important functions that primary cilia have in the processing of extracellular signals, B1 cells’ primary cilia could have key roles in the reception of ventricular signals for the regulation of adult neurogenesis (24, 25). However, the function of B1 cells’ primary cilia remains unknown. Genetically ablating primary cilia—by removing essential components of the intraflagellar transport (IFT) system (26)—inevitably eliminate the motile cilia of E1 cells, resulting in disruption of cerebrospinal fluid (CSF) flow and hydrocephalus. Because E1 cells and CSF are thought to play important roles in the regulation of B1 cell proliferation, it is not possible to dissociate non–cell-autonomous effects of disruption of ependymal cilia from direct effects of primary cilia removal in B1 cells.Here we used various approaches to genetically ablate primary cilia in NSCs at different developmental stages and in different locations. Surprisingly, we found that primary cilia removal during fetal development had strikingly little effect on the development of the telencephalon. During early postnatal life, primary cilia were also dispensable in most B1 cells, but were essential in a specific Hh-regulated subdomain of the V-SVZ. Our results suggest that primary cilia function is tightly linked to Hh signaling within a restricted domain of the postnatal neurogenic region.     

Primary cilia in satellite cells are the mechanical sensors for muscle hypertrophy

Skeletal muscle atrophy is commonly associated with aging, immobilization, muscle unloading, and congenital myopathies. Generation of mature muscle cells from skeletal muscle satellite cells (SCs) is pivotal in repairing muscle tissue. Exercise therapy promotes muscle hypertrophy and strength. Primary cilium is implicated as the mechanical sensor in some mammalian cells, but its role in skeletal muscle cells remains vague. To determine mechanical sensors for exercise-induced muscle hypertrophy, we established three SC-specific cilium dysfunctional mouse models—Myogenic factor 5 (Myf5)-Arf-like Protein 3 (Arl3)^−/−, Paired box protein Pax-7 (Pax7)-Intraflagellar transport protein 88 homolog (Ift88)^−/−, and Pax7-Arl3^−/−—by specifically deleting a ciliary protein ARL3 in MYF5-expressing SCs, or IFT88 in PAX7-expressing SCs, or ARL3 in PAX7-expressing SCs, respectively. We show that the Myf5-Arl3^−/− mice develop grossly the same as WT mice. Intriguingly, mechanical stimulation-induced muscle hypertrophy or myoblast differentiation is abrogated in Myf5-Arl3^−/− and Pax7-Arl3^−/− mice or primary isolated Myf5-Arl3^−/− and Pax7-Ift88^−/− myoblasts, likely due to defective cilia-mediated Hedgehog (Hh) signaling. Collectively, we demonstrate SC cilia serve as mechanical sensors and promote exercise-induced muscle hypertrophy via Hh signaling pathway.

Exercise is considered as the primary intervention to improve muscle strength and to counteract muscle atrophy. While physical exercise training is considered a suitable intervention to improve muscle strength and endurance in healthy individuals, some people are resistant to the beneficial effects of exercise (1–3). It has been debated whether exercise is beneficial or harmful for patients with myopathic disorders (4) and type 2 diabetes (5). This so-called “exercise resistance” is considered congenital, and one recently identified causative factor involved in exercise resistance is hepatokine selenoprotein P (2, 6).Primary cilia have a mechanosensory function in bone cells (7), renal cells (8), and airway smooth muscle cells exert a role in sensing oscillatory fluid flow and transducing extracellular mechano-chemical signals into intracellular biochemical responses (9). Intriguingly, low muscle tone is a clinical feature often present in congenital ciliopathies with unclear underlying mechanisms (10). Arf-like Protein 3 (ARL3) is a highly conserved ciliary protein across ciliated organisms. ARL3, a regulator of intraflagellar transport in primary cilia, has been reported involving with various ciliary signaling functions (11, 12) and maintaining cell division polarity (13). Arl3 mutations cause Joubert syndrome (14, 15). Arl3^−/− knockout does not affect cilia structure but compromises ciliary function (16).Cells utilize primary cilia to convert environmental cues, mechanical or chemical, into various cellular signaling essential for development (17–21). During skeletal muscle development, Hedgehog (Hh) signaling helps to initiate the myogenic program (22). In myoblast cells, Fu et al. (23) showed that primary cilia are assembled during the initial stages of myogenic differentiation but disappear as cells progress through myogenesis. The ablation of primary cilia suppresses Hh signaling and myogenic differentiation while enhancing proliferation. However, there are still significant gaps in our understanding of how exercise and mechanical signals activate the Hh signaling pathway. In the present study, we hypothesize that primary cilia in satellite cells (SCs) transduce mechanical stimulation through activation of Hh signaling and promote muscle hypertrophy induced by exercise.Hypertrophy of skeletal muscle is a complex biological process that involves multiple cell types, including SCs, fibro-adipogenic precursors, endothelial cells, fibroblasts, pericytes, and immune cells. Removing cilia from fibro-adipogenic precursors can reduce intramuscular adipogenesis and increase myofibril size during muscle healing (24). SCs play an essential role in muscle hypertrophy and exercise adaptation (25, 26), especially in young mice (27). Mechanical signals can interrupt SC suppression in a skeletal muscle loss model induced by ovariectomy. Diminished SC number and elevated adipogenic gene expression in muscle caused by ovariectomy are averted by mechanical stimulation (28). Experiments in vitro indicate that mechanical stimulation enhances the fusion of SCs (29). SCs are a heterogeneous population of stem cells and committed progenitors (30). Paired box protein Pax-7 (Pax7) is a traditional marker of SCs and acts at different levels in a nonhierarchical regulatory network controlling SC-mediated muscle hypertrophy (31). A major target gene of Pax7 is Myogenic factor 5 (Myf5), and loss of Pax7 significantly decreases Myf5 expression in myoblasts (32). However, Myf5 is present in Pax3/Pax7 double mutants, indicating Myf5 activation occurs independently of Pax3/Pax7 (33). Furthermore, 10% of Pax7-expressing satellite cells have never expressed Myf5 (30). Parise et al. (34) observed an approximately sixfold increase in the number of Myf5-expressing cells by 48 h following exercise, which remained elevated until at least 96 h after exercise. We established three mouse models of Myf5-Arl3^−/−, Pax7-Intraflagellar transport protein 88 homolog (Ift88^−/−), and Pax7-Arl3^−/− to investigate the SC during mechanical stimulation and exercise. In the present study, we provide exciting evidence that SC cilia act as the key mechanical sensor for exercise-induced hypertrophy.     

Highly motile nanoscale magnetic artificial cilia

Among the many complex bioactuators functioning at different scales, the organelle cilium represents a fundamental actuating unit in cellular biology. Producing motions at submicrometer scales, dominated by viscous forces, cilia drive a number of crucial bioprocesses in all vertebrate and many invertebrate organisms before and after their birth. Artificially mimicking motile cilia has been a long-standing challenge while inspiring the development of new materials and methods. The use of magnetic materials has been an effective approach for realizing microscopic artificial cilia; however, the physical and magnetic properties of the magnetic material constituents and fabrication processes utilized have almost exclusively only enabled the realization of highly motile artificial cilia with dimensions orders of magnitude larger than their biological counterparts. This has hindered the development and study of model systems and devices with inherent size-dependent aspects, as well as their application at submicrometer scales. In this work, we report a magnetic elastomer preparation process coupled with a tailored molding process for the successful fabrication of artificial cilia with submicrometer dimensions showing unprecedented deflection capabilities, enabling the design of artificial cilia with high motility and at sizes equal to those of their smallest biological counterparts. The reported work crosses the barrier of nanoscale motile cilia fabrication, paving the way for maximum control and manipulation of structures and processes at micro- and nanoscales.

Described as the “surprise organelle” of the first decade of the twenty-first century, the hair-like microscopic structure cilium remains as intriguing as at the time of its discovery (1, 2). Just a few micrometers long and some hundreds of nanometers thick, cilia perform a wide range of vital functions in our body as chemo- and mechanosensors (3, 4) and as actuators (2). Efforts made to artificially mimic their functions have demonstrated and further pointed toward a broad range of applications in major areas like microfluidics (5–12), micro- and nanorobotics (13), cell and particle manipulation (14–16), surface enhancements (17), and many more. Fabrication of artificial cilia to act as actuators has triggered the use and development of various materials responsive to external stimuli like electric fields, chemicals, light, and magnetic fields (18–20). Less implemented methods involving pneumatic (21), acoustic (22), piezo (23), and mechanical (24) actuation have also been reported. Toward exercising a high degree of control and actuation, the implementation of magnetic materials offers a promising approach, in particular due to the noninteracting nature of magnetic fields with biological fluids and cells (25–27). As magnetic biomimetic microactuators are remotely addressable and reversible in short response times, their use enjoys an overwhelming acceptance in the biomimicry of organelle–cilia and many other bioprocesses (28, 29).The magnetic materials investigated for artificial cilia so far are responsive hybrid composites known as magnetorheological elastomers (also referred to as magnetic elastomers) (30), which have magnetic particles embedded in an elastic polymer matrix. First developed for their field-dependent material properties, most importantly their tunable modulus (31), and to study the combined influence of magnetic field and elastic stresses in an elastomer (32), they quickly gained a lot of attention after a quasistatic model explaining their changing modulus was developed (33). Materials were further developed to enhance the field-dependent modulus by using magnetic particles with higher magnetization saturation values like the commonly used iron carbonyl particles. Magnetic artificial cilia with large sizes (i.e., lengths of hundreds of micrometers or more) have been successfully fabricated and demonstrated, but the fabrication of highly motile artificial cilia with submicrometer dimensions to faithfully mimic their biological counterparts has remained an open challenge due to a number of unresolved issues. First, magnetic particles most used in creating artificial cilia such as iron carbonyl and other magnetic particles have diameters of the order of a few micrometers (34), and the corresponding magnetic elastomers are therefore not suitable for fabricating artificial cilia with submicrometer dimensions. The magnetic artificial cilia we demonstrated earlier make use of these materials and therefore, have large diameters and lengths of tens and hundreds of micrometers or more, respectively (11, 35) (i.e., more than an order of magnitude larger than biological cilia). Conversely, magnetic elastomers with nanomagnetic particles have been less explored because of their much lower magnetization values and critical preparation processes. Complications in their preparation arise due to the presence of magnetic dipole moments and van der Waals forces existing between the nanoparticles, which heavily hamper their dispersion in an elastomer without agglomeration (36). Reduction in particle–particle interaction, and therefore, the particle agglomeration, is attained by coating them with a layer of polymer, but this limits the maximum particle concentration possible to attain in the elastomer (25). Consequently, artificial cilia fabricated with lower concentration of particles in an elastomer show a lower degree of response, making them less effective in use (25). Cilia-like nanostructures fabricated from the only magnetic elastomers with sufficiently high particle concentration and nanoscale homogeneity (37) have also shown a limited bending response (27, 38). Recently, highly responsive cilia with nanoscale dimensions fabricated from nanomagnetic particle chains have been reported (39); a drawback from this approach is that the cilia dimensions and properties are limited by the particles available. In this study, we report a material preparation process for preparing a magnetic elastomer that overcomes these issues and is suitable for fabricating highly responsive micro-/nanostructures. Another limiting factor that hampers creating small-scale cilia is the fabrication process for which conventional mold and release processes are often used, using molds made with photolithography (11, 35). This limits the downscaling and aspect ratio of the cilia. To resolve these limitations, we further report a robust micro-/nanomolding process to shape and successfully release the micro-/nanocilia structures from their template. The material preparation method coupled with the tailored fabrication process enables us to make a major step and realize artificial structures faithfully mimicking the highly motile cilia at sizes equivalent to the smallest sizes found in nature. The fabricated artificial cilia we report here have radii and lengths down to 200 nm and 6 μm, respectively, and show a maximum possible bending angle of 900 under an external magnetic field generated by a conventional magnet. This extremely large bending response is further exploited to demonstrate a complex 3600 rotary motion at large bending angles and at very high frequencies (up to 80 Hz) without loss of motility.     

Ciliopathy genes are required for apical secretion of Cochlin,an otolith crystallization factor

Here, we report that important regulators of cilia formation and ciliary compartment–directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.

Cilia are finger-like cell surface protrusions involved in an immense array of biological processes that range from limb patterning in the embryo to light detection in the eye. Numerous cilia genes are associated with human disorders collectively known as ciliopathies. While analyzing cilia function in vertebrate sensory organs, we focused on Cep290, a protein that localizes to centrosomes and the ciliary transition zone. Human CEP290 defects range from blindness to severe developmental defects causing perinatal lethality (1, 2). Otoliths are highly mineralized bodies that rest on the surface of sensory epithelia in the vestibular system of vertebrates. Otolith displacement that occurs in response to body movements stimulates mechanosensitive hair cells embedded in sensory epithelia, providing the organism with information about body position (3). The main inorganic component of the otolith is calcium carbonate (CaCO₃). Its crystal form (polymorph) varies across species and in different otolithic organs. Otolith calcium carbonate crystal form is controlled by proteinaceous matrix that remains poorly characterized and which is thought to control the balance between calcite, vaterite, and aragonite (4). In teleost fish, the utricular otolith mainly contains aragonite, the latter can be detected by Meigen’s Cobalt Nitrate stain (5–7). Defects in certain otolith matrix proteins lead to the appearance of other calcium carbonate polymorphic forms and abnormal crystal morphology (6). In humans and animals, this is likely to affect vestibular function and result in behavioral defects (8, 9).     

Intracellular and extracellular forces drive primary cilia movement

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin–Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.A living cell is a dynamic, nonequilibrium system dependent on chemical and mechanical communication with its environment. Communication is mediated in many mammalian cells by the primary cilium, a specialized antenna that typically extends from a cell’s apical surface. The primary cilium, with its specialized and segregated membrane compartment, has emerged as a key signaling center that transduces mechanical and chemical extracellular cues (1–4). Flow detection in the kidney epithelium promotes cell homeostasis, whereas defects in sensing or signaling can lead to polycystic kidney disease (5). Forces generated by fluid flow are thought to lead to Ca²⁺ influx through a transient receptor potential (TRP) family Ca²⁺ channel in the ciliary membrane, polycystin-2 (PC2) (6–9) (Fig. 1A). It has also been shown that ciliary mechanosensation regulates kidney epithelial cell size through the mammalian target-of-rapamycin pathway independent of Ca²⁺ transients (10). How do the mechanical properties of the cilium facilitate these cellular responses? Both bending and pivoting could trigger membrane channels. Previous studies have used fluid flow to estimate primary cilium bending stiffness based on deformation profiles (11–14), but the precise mechanical properties of the cilium remain unknown. It is likely that mechanosensation needs subtle coordination and calibration of the intracellular machinery involving adaptation and feedback mechanisms reacting to external stimuli, such as is the case in mammalian inner-ear cells, where active mechanical processes are crucial for hearing acuity (15, 16). We here applied micromanipulation and imaging methods to measure the mechanical properties of primary cilia, their cellular anchoring, and their fluctuations.Open in a separate windowFig. 1.Mechanical structure and anchoring of the primary cilium. (A) Schematic sketch of a primary cilium with PC2 Ca²⁺ channels and its intracellular anchoring. (B) Differential interference contrast (DIC) micrograph of a primary cilium deflected in buffer flow of ∼4.8 µm/s. (C) DIC micrograph of a primary cilium with an attached bead deflected by an optical trap.The core of the primary cilium is an axoneme made up of microtubules. Nine microtubule doublets extend from the cylindrical basal body formed by the cell’s mother centriole and provide the mechanical backbone of the cilium. Unlike beating cilia and flagella (9 + 2 cilia), primary cilia (9 + 0 cilia) lack a central microtubule doublet and cross-linking supports. As a consequence, the doublet microtubules can lose their ninefold symmetry as they extend away from the basal body. Electron micrographs have revealed so-called 9v arrangements in distal portions of cilia, in which the number of microtubule doublets varies from the usual nine down to a single doublet (17). These fundamental structural differences are expected to make the elastic properties of 9 + 0 cilia distinctly different from those of 9 + 2 cilia.Most 9 + 0 primary cilia are presumed to be passive sensors because they lack the dynein motors that drive beating of active cilia (18), with one exception: motile 9 + 0 cilia in the organizer node of early embryos possess dynein arms and drive fluid flow (19). Cilia in the epithelium lining renal ducts are thought to passively respond to flow in the ducts. Surprisingly, we found active ciliary fluctuations. Even in the absence of dyneins, internal activity in the cell can cause cilia to move, just as a skyscraper can sway because of both wind and an earthquake. The mechanical properties of the cilium and its anchoring inside the cell determine the response of the assembly to both external and internal forces. The distinct nonthermal component of cilium motion that we document here challenges the view that the cilium is a strictly immotile sensor and raises the possibility that endogenous cilium fluctuations may have a functional role.     

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA–DC has an ellipsoidal shape ∼24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.Cilia and flagella of eukaryotic cells are organelles that generate fluid flow on the cell surface and/or sense chemical or mechanical stimuli from the external environment (1). Cilia/flagella beating is driven by outer arm dynein (OAD) and inner arm dyneins. The arrangement of dyneins on the axoneme has an overall periodicity of 96 nm, within which OAD binds every 24 nm; this 24-nm periodicity is completely conserved in essentially all eukaryotic organisms with “9 + 2” axonemes (2, 3) and even occurs in insect sperm flagella containing multiple rows of doublet microtubules arranged in a spiral configuration (4, 5). OAD is best characterized in Chlamydomonas. It is a very large protein complex of ∼2 MDa, comprising 3 heavy chains, 2 intermediate chains, and 11 distinct light chains. Most of the subunits are conserved from protists to mammals (6). OAD is the most abundant and most powerful axonemal dynein, generating about two-thirds of the total propulsive force of ciliary beating (7, 8). Human diseases due to ciliary motility defects [termed primary ciliary dyskinesia (PCD)] are caused most commonly by defects in OAD assembly (9–11). The assembly process and the in situ structure of the OAD complex in the axoneme have been well studied (3, 12, 13). However, the mechanism underlying the periodic binding of OAD to the doublet is poorly understood.The outer dynein arm-docking complex (ODA-DC) has been identified as a complex that mediates OAD binding to the doublet (14, 15). In the flagella of Chlamydomonas mutants (e.g., outer-dynein-arm deficient oda6) retaining the ODA-DC but not OAD, the ODA-DC is observed by electron microscopy as a small projection linearly arrayed every 24 nm along the outer doublet (3, 14–17). It is composed of three subunits: DC1, ∼83 kDa, encoded by ODA3; DC2, ∼62 kDa, encoded by ODA1; and DC3, ∼21 kDa, encoded by ODA14 (18–20), which assemble in the cell cytoplasm and are transported into the flagella independently of OAD (16). DC1 and DC2 are coiled-coil proteins; they are essential for the OAD docking activity because mutants (oda1, oda3) lacking these proteins entirely lack OAD. In contrast, DC3 is a redox-sensitive Ca²⁺-binding protein and apparently is nonessential for OAD binding because a mutant (oda14) lacking it retains ∼40% of OADs (20, 21). Presumably, DC3 has an as yet unidentified regulatory function. DC2 is well conserved throughout evolution, and seems to have undergone duplication in humans (CCDC63 and CCDC114) (22, 23). Defects in CCDC114 are known to cause PCD and the patients lack outer dynein arms, suggesting that DC2 functions in OAD docking in humans also (22, 23). The ODA-DC binds OAD via at least three interactions: one between DC1 and the OAD intermediate chain IC1, one between DC1 and the other OAD intermediate chain IC2, and one between DC2 and the OAD light chain LC7b (24, 25). However, we still do not understand the architecture of the ODA-DC and the detailed manner of its binding to the doublet microtubules, which will be essential for understanding the molecular basis of OAD periodicity. In the present study, we analyzed the structure and microtubule-binding properties of the ODA-DC by using recombinant proteins as well as native proteins, and observed how it and OAD are incorporated in vivo into axonemes lacking one or both structures. We found that the ODA-DC itself is 24 nm in length and that it produces 24-nm periodicity by end-to-end association on the doublet microtubules. We propose that this periodicity determines the ordered arrangement of the OAD.     

Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole

Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centriole-targeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.The primary cilium (PC) functions as an antenna-like signaling organelle typically found on postmitotic cells (1–3). It consists of a microtubule-based axoneme enclosed within a ciliary membrane, and its assembly is triggered at the basal body (4, 5). The basal body, in turn, is derived from one of the two centrioles that make up the centrosome, specifically the mature (or “mother”) centriole (M centriole) (6–9). Long erroneously considered a vestigial organelle, the PC has emerged as a key structure for sensing extracellular stimuli and hence plays crucial roles in cellular responses to both mechanical and chemical cues. In vertebrates, PC function has been linked to the regulation of many important aspects of embryonic development as well as tissue homeostasis in adulthood (2, 10); moreover, defects in ciliary assembly or function have been linked to a large number of human diseases known as ciliopathies (3, 6, 11–13).PC structure and formation have long been studied at a morphological level (14, 15), but a molecular understanding of the regulation of PC assembly and resorption is only beginning to emerge (5, 7, 8, 16, 17). In cultured cells, PC formation generally occurs when cells exit the cell cycle to enter quiescence (Go), and, conversely, PCs are often resorbed when cells reenter the cell cycle. Thus, PC formation can readily be triggered by serum starvation of some cultured cells, including telomerase-immortalized retinal pigment epithelial (RPE-1) cells. Early steps in PC formation include the docking of membrane vesicles to centrioles (14, 18, 19), the removal of the capping protein CP110 from the distal part of the M centriole (20, 21), the recruitment of intraflagellar transport (IFT) protein complexes (22–24), the formation of a transition zone at the membrane (25, 26), and, finally, the outgrowth of the ciliary axoneme (27, 28).An important role in PC formation resides with specific components of the M centriole, termed distal appendages (14, 15). These appendages are considered critical for the early docking of Golgi-derived membrane vesicles and the subsequent anchorage of the basal body underneath the plasma membrane. Following the discovery of centrosomal protein 164kDa (Cep164), the first marker for distal appendages (29), several additional distal appendage proteins (DAPs) have recently been identified and functionally linked to ciliogenesis. These include centrosomal protein 83kDa (Cep83)/CCDC41, centrosomal protein 89kDa (Cep89)/CCDC123, SCLT1, and FBF1 (18, 19, 30, 31). The DAP Cep164 was discovered in a screen for components that are critical for PC formation (29). Subsequently, mutations in Cep164 were linked to ciliopathies, providing direct proof for the importance of this protein in human pathophysiology (32). At a mechanistic level, Cep164 was shown to be required at an early stage of PC formation, notably for the docking of membrane vesicles to the basal body (18). Moreover, two components of the vesicle transport machinery, the small GTPase Rab8 and its guanine–nucleotide exchange factor Rabin8, were identified as interaction partners of Cep164 (18). Despite this progress, the precise molecular functions of Cep164 remain to be fully understood.Importantly, protein kinases have also been implicated in ciliogenesis and in cilia-related diseases. These include Nek1 and Nek8, two members of the family of NIMA-related kinases (33, 34), and Tau Tubulin Kinase 2 (TTBK2), a member of the casein kinase 1 family (35–37).Here, we report that Cep164 and TTBK2 form a complex and that formation of this complex at M centrioles is essential for ciliogenesis. We show that the noncatalytic C-terminal domain of TTBK2 interacts with Cep164 and that formation of the complex critically depends on the WW domain within the N-terminal domain of Cep164. We also provide evidence that Cep164 is a likely physiological substrate of TTBK2. Use of chimeric TTBK2–Cep164 constructs in siRNA-rescue experiments leads us to conclude that a main function of Cep164 consists of the recruitment of TTBK2 to M centrioles. Once localized correctly, TTBK2 is then in a position to trigger PC formation through phosphorylation of appropriate substrates. Interestingly, our data also reveal a role for TTBK2 in the assembly of distal appendages. Overexpression of active kinase in fact enhances not only the association of DAPs with existing appendages but also triggers their occasional recruitment to daughter centrioles (D centrioles).     

Olfactory regulation by dopamine and DRD2 receptor in the nose

Olfactory behavior is important for animal survival, and olfactory dysfunction is a common feature of several diseases. Despite the identification of neural circuits and circulating hormones in olfactory regulation, the peripheral targets for olfactory modulation remain relatively unexplored. In analyzing the single-cell RNA sequencing data from mouse and human olfactory mucosa (OM), we found that the mature olfactory sensory neurons (OSNs) express high levels of dopamine D2 receptor (Drd2) rather than other dopamine receptor subtypes. The DRD2 receptor is expressed in the cilia and somata of mature OSNs, while nasal dopamine is mainly released from the sympathetic nerve terminals, which innervate the mouse OM. Intriguingly, genetic ablation of Drd2 in mature OSNs or intranasal application with DRD2 antagonist significantly increased the OSN response to odorants and enhanced the olfactory sensitivity in mice. Mechanistic studies indicated that dopamine, acting through DRD2 receptor, could inhibit odor-induced cAMP signaling of olfactory receptors. Interestingly, the local dopamine synthesis in mouse OM is down-regulated during starvation, which leads to hunger-induced olfactory enhancement. Moreover, pharmacological inhibition of local dopamine synthesis in mouse OM is sufficient to enhance olfactory abilities. Altogether, these results reveal nasal dopamine and DRD2 receptor as the potential peripheral targets for olfactory modulation.

Olfactory behavior is important for food seeking and animal survival. On the other hand, olfactory dysfunction is a common feature of several diseases such as psychiatric disorders, neurodegeneration, and COVID-19 (1–3). Interestingly, the olfactory ability can be regulated by feeding status and external environments (4, 5). Recent studies have made progress in identifying the neural circuits and circulating hormones in olfactory regulation (6–11). However, the peripheral targets modulating olfactory ability remain relatively unexplored (12).Dopamine (DA) is a monoamine neurotransmitter (13, 14), which plays important roles in a variety of brain functions. DA is released by dopaminergic neurons in the central nervous system. In addition, DA can be released by sympathetic nerves in the peripheral tissues including the olfactory mucosa (OM) (15–18). The sympathetic innervation of rodent OM originates predominantly from the superior cervical ganglion (SCG) (17). Tyrosine hydroxylase (TH) is the rate-limiting enzyme for DA synthesis (19). Intriguingly, the Th mRNA is locally translated in the sympathetic nerve axons, which facilitates local DA synthesis (20, 21).There are two types of DA receptors based on sequence homology and function: The excitatory D1-like receptors (DRD1 and DRD5) and inhibitory D2-like receptors (DRD2–DRD4) (22). Activation of DRD2, a Gα_i/o-coupled receptor, can reduce the intracellular levels of cyclic adenosine monophosphate (cAMP). Drd2 is associated with several neuropsychiatric diseases and is the target of some antipsychotic drugs (23–28). In the central nervous system including the olfactory bulb (OB), DA-DRD2 signaling plays important roles in regulating synaptic transmission and plasticity (29–33). However, the function and regulation of DA-DRD2 signaling in the peripheral tissues are relatively less understood.Here we show that DRD2 is expressed in the cilia and somata of mature olfactory sensory neurons (OSNs) in mice. We provide evidence that DA-DRD2 signaling has a tonic inhibition on OSN activity and olfactory function in mice. Intriguingly, hunger greatly reduces the N4-acetylcytidine (ac⁴C) modification of Th mRNA and local DA synthesis in mouse OM, which causes the olfactory enhancement during starvation. We further show that inhibition of local DA synthesis or DRD2 receptor in mouse OM recapitulates enhanced olfactory abilities during starvation. Collectively, these results reveal nasal DA and DRD2 receptor as the potential peripheral targets for olfactory regulation.