A kinesin in command of primary ciliogenesis.

Primary cilia sense extracellular cues and in response transmit signals required for development and tissue homeostasis. A new study by Kobayashi et al. (2011) reports that the kinesin Kif24 controls the formation of primary cilia by restricting the nucleation of cilia at centrioles.

A semi-automated machine learning-aided approach to quantitative analysis of centrosomes and microtubule organization.

Microtubules (MTs) promote important cellular functions including migration, intracellular trafficking, and chromosome segregation. The centrosome, comprised of two centrioles surrounded by the pericentriolar material (PCM), is the cell's central MT-organizing center. Centrosomes in cancer cells are commonly numerically amplified. However, the question of how the amplification of centrosomes alters MT organization capacity is not well studied. We developed a quantitative image-processing and machine learning-aided approach for the semi-automated analysis of MT organization. We designed a convolutional neural network-based approach for detecting centrosomes, and an automated pipeline for analyzing MT organization around centrosomes, encapsulated in a semi-automatic graphical tool. Using this tool, we find that breast cancer cells with supernumerary centrosomes not only have more PCM protein per centrosome, which gradually increases with increasing centriole numbers, but also exhibit expansion in PCM size. Furthermore, cells with amplified centrosomes have more growing MT ends, higher MT density and altered spatial distribution of MTs around amplified centrosomes. Thus, the semi-automated approach developed here enables rapid and quantitative analyses revealing important facets of centrosomal aberrations.

Ciliate cortical organization and dynamics for cell motility: Comparing ciliates and vertebrates.

The generation of efficient fluid flow is crucial for organismal development and homeostasis, sexual reproduction, and motility. Multi-ciliated cells possess fields of motile cilia that beat in synchrony to propel fluid. Ciliary arrays are remarkably conserved in their organization and function. Ciliates have polarized multi-ciliary arrays (MCAs) to promote fluid flow for cell motility. The ciliate cortex is decorated with hundreds of basal bodies (BB) forming linear rows along the cell's anterior-posterior axis. BBs scaffold and position cilia to form the organized ciliary array. Nascent BBs assemble at the base of BBs. As nascent BBs mature, they integrate into the cortical BB and cytoskeletal network and nucleate their own cilium. The organization of MCAs is balanced between cortical stability and cortical dynamism. The cortical cytoskeletal network both establishes and maintains a stable organization of the MCA in the face of mechanical forces exerted by ciliary beating. At the same time, MCA organization is plastic, such that it remodels for optimal ciliary mobility during development and in response to environmental conditions. Such plasticity promotes effective feeding and ecological behavior required for these organisms. Together, these properties allow an organism to effectively sense, adapt to, and move through its environment.

Deubiquitylase USP9X maintains centriolar satellite integrity by stabilizing pericentriolar material 1 protein.

Centriolar satellites are small cytoplasmic granules that play important roles in regulating the formation of centrosomes and primary cilia. Ubiquitylation of satellite proteins, including the core satellite scaffold protein pericentriolar material 1 (PCM1), regulates centriolar satellite integrity. Currently, deubiquitylases that control centriolar satellite integrity have not been identified. In this study, we find that the deubiquitylase USP9X binds PCM1, and antagonizes PCM1 ubiquitylation to protect it from proteasomal degradation. Knockdown of USP9X in human cell lines reduces PCM1 protein levels, disrupts centriolar satellite particles and causes localization of satellite proteins, such as CEP290, to centrosomes. Interestingly, knockdown of mindbomb 1 (MIB1), a ubiquitin ligase that promotes PCM1 ubiquitylation and degradation, in USP9X-depleted cells largely restores PCM1 protein levels and corrects defects caused by the loss of USP9X. Overall, our study reveals that USP9X is a constituent of centriolar satellites and functions to maintain centriolar satellite integrity by stabilizing PCM1.

Microtubule glycylation promotes attachment of basal bodies to the cell cortex.

Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.

miR-219 regulates neural progenitors by dampening apical Par protein-dependent Hedgehog signaling.

The transition of dividing neuroepithelial progenitors to differentiated neurons and glia is essential for the formation of a functional nervous system. Sonic hedgehog (Shh) is a mitogen for spinal cord progenitors, but how cells become insensitive to the proliferative effects of Shh is not well understood. Because Shh reception occurs at primary cilia, which are positioned within the apical membrane of neuroepithelial progenitors, we hypothesized that loss of apical characteristics reduces the Shh signaling response, causing cell cycle exit and differentiation. We tested this hypothesis using genetic and pharmacological manipulation, gene expression analysis and time-lapse imaging of zebrafish embryos. Blocking the function of miR-219, a microRNA that downregulates apical Par polarity proteins and promotes progenitor differentiation, elevated Shh signaling. Inhibition of Shh signaling reversed the effects of miR-219 depletion and forced expression of Shh phenocopied miR-219 deficiency. Time-lapse imaging revealed that knockdown of miR-219 function accelerates the growth of primary cilia, revealing a possible mechanistic link between miR-219-mediated regulation of apical Par proteins and Shh signaling. Thus, miR-219 appears to decrease progenitor cell sensitivity to Shh signaling, thereby driving these cells towards differentiation.

Choosing sides--asymmetric centriole and basal body assembly.

Centrioles and basal bodies (CBBs) are microtubule-rich cylindrical structures that nucleate and organize centrosomes and cilia, respectively. Despite their apparent ninefold rotational symmetry, the nine sets of triplet microtubules in CBBs possess asymmetries in their morphology and in the structures that associate with them. These asymmetries define the position of nascent CBB assembly, the orientation of ciliary beating, the orientation of spindle poles and the maintenance of cellular geometry. For some of these functions, the orientation of CBBs is first established during new CBB biogenesis when the daughter structure is positioned adjacent to the mother. The mother CBB organizes the surrounding environment that nascent CBBs are born into, thereby providing a nest for the new CBB to develop. Protists, including ciliates and algae, highlight the importance of this environment with the formation of asymmetrically placed scaffolds onto which new basal bodies assemble and are positioned. Recent studies illuminate the positioning of nascent centrioles relative to a modular pericentriolar material (PCM) environment and suggest that, like ciliates, centrosomes organize an immediate environment surrounding centrioles for their biogenesis and positioning. In this Commentary, I will explore the positioning of nascent CBB assembly as the first event in building cellular asymmetries and describe how the environment surrounding both basal bodies and centrioles may define asymmetric assembly.

IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy.

Jeune asphyxiating thoracic dystrophy, an autosomal recessive chondrodysplasia, often leads to death in infancy because of a severely constricted thoracic cage and respiratory insufficiency; retinal degeneration, cystic renal disease and polydactyly may be complicating features. We show that IFT80 mutations underlie a subset of Jeune asphyxiating thoracic dystrophy cases, establishing the first association of a defective intraflagellar transport (IFT) protein with human disease. Knockdown of ift80 in zebrafish resulted in cystic kidneys, and knockdown in Tetrahymena thermophila produced shortened or absent cilia.

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

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

Differential subcellular localization of ASPM RNA and protein.

RNAs encoding some centrosomal components are trafficked to the organelle during mitosis. Some RNAs, including ASPM , localize to the centrosome co-translationally. However, the relative position of these RNAs and their protein after trafficking to centrosomes remained unclear. We find that mislocalization of ASPM RNA from the centrosome does not affect the localization of ASPM protein. Further, ASPM RNA and ASPM protein reside in two physically close yet distinct subcellular spaces, with ASPM RNA on the astral side of the centrosome and ASPM protein on the spindle side. This suggests subtly distinct locations of ASPM RNA translation and ASPM protein function.

The Unkempt RNA binding protein reveals a local translation program in centriole overduplication.

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication.

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography.

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

Label-free proteomic comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Poc1 is a basal body inner junction protein that promotes triplet microtubule integrity and interconnections.

Basal bodies (BBs) are conserved eukaryotic structures that organize motile and primary cilia. The BB is comprised of nine, cylindrically arranged, triplet microtubules (TMTs) that are connected to each other by inter-TMT linkages which maintain BB structure. During ciliary beating, forces transmitted to the BB must be resisted to prevent BB disassembly. Poc1 is a conserved BB protein important for BBs to resist ciliary forces. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 binding in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. Moreover, we identify a molecular response to ciliary forces via a molecular remodeling of the inner scaffold, as determined by differences in Fam161A localization. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.

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

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

Label-free proteomic comparison reveals ciliary and non-ciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila . A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia ­ structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.

Centrioles want to move out and make cilia.

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.

New Tetrahymena basal body protein components identify basal body domain structure.

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.

Trisomy 21 increases microtubules and disrupts centriolar satellite localization.

Trisomy 21, the source of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. We investigate how PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT accumulates around the centrosome as a cluster of enlarged cytoplasmic puncta that localize along microtubules (MTs) and at MT ends. Cytoplasmic PCNT puncta impact the density, stability, and localization of the MT trafficking network required for primary cilia. The PCNT puncta appear to sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins PCM1, CEP131, and CEP290, important for ciliogenesis, accumulate at enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta and pericentrosomal crowding, reestablish a normal density of MTs around the centrosome, and restore ciliogenesis to wild-type levels. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the MT network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.