Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling.

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome-mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2-GTP enters cilia by binding to IFT-B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT-B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP-locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine-tune cilia-dependent signaling for normal embryonic development and organismic homeostasis.

Self-construction of actin networks through phase separation-induced abLIM1 condensates.

The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg2+. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.

Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM.

The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 ß-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS-CP interactions and mechanosignal transduction.

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components.

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

Cep57 and Cep57l1 function redundantly to recruit the Cep63-Cep152 complex for centriole biogenesis.

The Cep63-Cep152 complex located at the mother centriole recruits Plk4 to initiate centriole biogenesis. How the complex is targeted to mother centrioles, however, is unclear. In this study, we show that Cep57 and its paralog, Cep57l1, colocalize with Cep63 and Cep152 at the proximal end of mother centrioles in both cycling cells and multiciliated cells undergoing centriole amplification. Both Cep57 and Cep57l1 bind to the centrosomal targeting region of Cep63. The depletion of both proteins, but not either one, blocks loading of the Cep63-Cep152 complex to mother centrioles and consequently prevents centriole duplication. We propose that Cep57 and Cep57l1 function redundantly to ensure recruitment of the Cep63-Cep152 complex to the mother centrioles for procentriole formation.

Parental centrioles are dispensable for deuterosome formation and function during basal body amplification.

Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to arise either spontaneously for massive de novo centriole biogenesis or in a daughter centriole-dependent manner as shuttles to carry away procentrioles assembled at the centriole. Here, we show that both parental centrioles are dispensable for deuterosome formation. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm are initially procentriole-free. They emerge at widely dispersed positions in the cytoplasm and then enlarge, concomitant with their increased ability to form procentrioles. More importantly, deuterosomes still form efficiently in mEPCs whose daughter centriole or even both parental centrioles are eliminated through shRNA-mediated depletion or drug inhibition of Plk4, a kinase essential to centriole biogenesis in both cycling cells and multiciliated cells. Therefore, deuterosomes can be assembled autonomously to mediate de novo centriole amplification in multiciliated cells.

Characterisation of centriole biogenesis during multiciliation in planarians.

BACKGROUND INFORMATION: Dense multicilia in protozoa and metazoa generate a strong force important for locomotion and extracellular fluid flow. During ciliogenesis, multiciliated cells produce hundreds of centrioles to serve as basal bodies through various pathways including deuterosome-dependent (DD), hyper-activated mother centriole-dependent (MCD) and basal bodydependent (BBD) pathways. The centrosome-free planarian Schmidtea mediterranea is widely used for regeneration studies because its neoblasts are capable of regenerating any body part after injury. However, it is currently unclear how the flatworms generate massive centrioles for multiciliated cells in the pharynx and body epidermis when their cells are initially centriole-free. RESULTS: In this study, we investigate the progress of centriole amplification during the pharynx regeneration. We observe that the planarian pharyngeal epithelial cells generate their centrioles asynchronously through a de novo pathway. Most of the de novo centrioles are formed individually, whereas the remaining ones are assembled in pairs, possibly by sharing a cartwheel, or in small clusters lacking a nucleation center. Further RNAi experiments show that the known key factors of centriole duplication, including Cep152, Plk4 and Sas6, are crucial for the centriole amplification. CONCLUSIONS AND SIGNIFICANCE: Our study demonstrates the distinct process of massive centriole biogenesis in S. mediterranea and helps to understand the diversity of centriole biogenesis during evolution.

Rsph9 is critical for ciliary radial spoke assembly and central pair microtubule stability.

BACKGROUND INFORMATION: In the "9+2"-type motile cilia, radial spokes (RSs) protruded from the nine peripheral microtubule doublets surround and interact with the central pair (CP) apparatus to regulate ciliary beat. RSPH9 is the human homologue of the essential protozoan RS head protein Rsp9. Its mutations in human primary ciliary dyskinesia patients, however, cause CP loss in a small portion of airway cilia without affecting the ciliary localization of other head proteins. RESULTS: We characterized mouse Rsph9 and investigated its function in ependymal motile cilia. Rsph9 was specifically expressed in mouse tissues containing motile cilia and upregulated during multiciliation. Its ciliary localization complied with its putative role as an RS subunit. Depletion of Rsph9 by RNAi in mouse ependymal cilia resulted in a near complete CP loss and altered the ciliary beat pattern from planar to rotational. Multiple RS proteins, including those in the head, were also markedly downregulated in the Rsph9-depleted cilia. CONCLUSION: Rsph9 is essential for both the RS head assembly and the CP maintenance in mammalian ependymal cilia. SIGNIFICANCE: Our results help to understand the assembly and functions of mammalian RS and pathology of RS-related ciliopathy.

A cross-sectional study of blood selenium concentration and cognitive function in elderly Americans: National Health and Nutrition Examination Survey 2011-2014.

BACKGROUND: Cognitive decline can develop into mild cognitive impairment, a high-risk factor in the progression of Alzheimer's disease. The antioxidant micronutrient selenium may have some effect on preventing cognitive decline, but the association between whole blood selenium concentration and cognitive function remains controversial. AIM: To investigate the association between whole blood selenium concentration and cognitive function score in elderly Americans. SUBJECTS AND METHODS: Data was obtained from the national health and nutrition survey between 2011 and 2014. A general linear model was used to adjust for possible risk factors to analyse the association between blood selenium concentration and cognitive function. RESULTS: 2068 participants were included in our study, and the average blood selenium concentration was high at 195.08 µg/L. The risk of lower cognitive scores was higher in people with lower blood selenium concentration (p < 0.05). The lower cognition may also be associated with one or more of the following characteristics: older, male, had a low poverty-income ratio, low education level, and consumed less alcohol. Related conditions such as stroke, diabetes and high blood pressure may also affect cognitive scores. CONCLUSIONS: Higher blood selenium is associated with higher cognitive scores in elderly Americans.

Long non-coding RNA H19 is associated with polycystic ovary syndrome in Chinese women: a preliminary study.

Polycystic ovary syndrome (PCOS) represents a serious reproductive and endocrine condition and is associated with high incidence rates. H19 is a compelling long noncoding RNA (lncRNA) which carries out a range of biological functions. However, prior to this study, little was known as to whether there was an association between lncRNA H19 and PCOS. In the current study, we used quantitative real-time polymerase chain reaction (qRT-PCR) to determine lncRNA H19 expression levels in peripheral blood leukocytes from patients with PCOS and compared this data with that derived from normal controls. We also screened data for potential relationships between lncRNA H19 and a range of endocrine variables in PCOS. The expression of lncRNA H19 was significantly higher in cases of PCOS than in controls. Individuals exhibiting higher expression levels of lncRNA H19 were associated with a significantly higher risk of PCOS than those with lower expression levels. Moreover, lncRNA H19 expression was positively correlated with fasting plasma glucose levels; this was the case with both raw data, and after adjustment for age and BMI in the PCOS group. However, lncRNA H19 expression showed no significant correlation with total testosterone or insulin resistance in either PCOS cases or the controls. In conclusion, we demonstrate the first evidence to indicate that lncRNA H19 is associated with PCOS, suggesting that elevated lncRNA H19 levels are a risk factor for PCOS. For susceptible individuals, lncRNA H19 may represent a useful biomarker of the early stages of endocrine and metabolic disorders in PCOS.

White spot syndrome virus IE1 and WSV056 modulate the G1/S transition by binding to the host retinoblastoma protein.

DNA viruses often target cellular proteins to modulate host cell cycles and facilitate viral genome replication. However, whether proliferation of white spot syndrome virus (WSSV) requires regulation of the host cell cycle remains unclear. In the present study, we show that two WSSV paralogs, IE1 and WSV056, can interact with Litopenaeus vannamei retinoblastoma (Rb)-like protein (lv-RBL) through the conserved LxCxE motif. Further investigation revealed that IE1 and WSV056 could also bind to Drosophila retinoblastoma family protein 1 (RBF1) in a manner similar to how they bind to lv-RBL. Using the Drosophila RBF-E2F pathway as a model system, we demonstrated that both IE1 and WSV056 could sequester RBF1 from Drosophila E2F transcription factor 1 (E2F1) and subsequently activate E2F1 to stimulate the G1/S transition. Our findings provide the first evidence that WSSV may regulate cell cycle progression by targeting the Rb-E2F pathway.

Branched F-actin as a negative regulator of cilia formation.

Cilia dysfunction leads to developmental defects and also a spectrum of human diseases termed ciliopathies. The actin cytoskeleton is a highly dynamic network and involved in many important biological processes, such as cell migration and membrane trafficking. Recently, actin dynamics has been shown to play a critical role in ciliogenesis. This review summarizes these results and provides insight into possible mechanisms.

A polarized multicomponent foundation upholds ciliary central microtubules.

Cilia's back-and-forth beat pattern requires a central pair (CP) of microtubules. However, the mechanism by which the CP is upheld above the transition zone (TZ) remains unclear. Here, we showed that a rod-like substructure marked by Cep131 and ciliary Centrin serves as a polarized CP-supporting foundation. This CP-foundation (CPF) was assembled independently of the CP during ciliogenesis in mouse ependymal cells. It protruded from the distal end of the basal body out of the TZ to enwrap the proximal end of the CP. Through proximity labeling, we identified 26 potential CPF components, among which Ccdc148 specifically localized at the proximal region of Centrin-decorated CPF and was complementary to the Cep131-enriched distal region. Cep131 deficiency abolished the CPF, resulting in CP penetration into the TZ. Consequently, cilia became prone to ultrastructural abnormality and paralysis, and Cep131-deficient mice were susceptible to late-onset hydrocephalus. In addition to Centrin, phylogenetic analysis also indicated conservations of Ccdc131 and Ccdc148 from protists to mammals, suggesting that the CPF is an evolutionarily conserved multicomponent CP-supporting platform in cilia.

Distinct roles of Kif6 and Kif9 in mammalian ciliary trafficking and motility.

Ciliary beat and intraflagellar transport depend on dynein and kinesin motors. The kinesin-9 family members Kif6 and Kif9 are implicated in motile cilia motilities across protists and mammals. How they function and whether they act redundantly, however, remain unclear. Here, we show that Kif6 and Kif9 play distinct roles in mammals. Kif6 forms puncta that move bidirectionally along axonemes, whereas Kif9 appears to oscillate regionally on the ciliary central apparatus. Consistently, only Kif6 displays microtubule-based motor activity in vitro, and its ciliary localization requires its ATPase activity. Kif6 deficiency in mice disrupts coordinated ciliary beat across ependymal tissues and impairs cerebrospinal fluid flow, resulting in severe hydrocephalus and high mortality. Kif9 deficiency causes mild hydrocephalus without obviously affecting the ciliary beat or the lifespan. Kif6-/- and Kif9-/- males are infertile but exhibit oligozoospermia with poor sperm motility and defective forward motion of sperms, respectively. These results suggest Kif6 as a motor for cargo transport and Kif9 as a central apparatus regulator.

Multi-scale structures of the mammalian radial spoke and divergence of axonemal complexes in ependymal cilia.

Radial spokes (RS) transmit mechanochemical signals between the central pair (CP) and axonemal dynein arms to coordinate ciliary motility. Atomic-resolution structures of metazoan RS and structures of axonemal complexes in ependymal cilia, whose rhythmic beating drives the circulation of cerebrospinal fluid, however, remain obscure. Here, we present near-atomic resolution cryo-EM structures of mouse RS head-neck complex in both monomer and dimer forms and reveal the intrinsic flexibility of the dimer. We also map the genetic mutations related to primary ciliary dyskinesia and asthenospermia on the head-neck complex. Moreover, we present the cryo-ET and sub-tomogram averaging map of mouse ependymal cilia and build the models for RS1-3, IDAs, and N-DRC. Contrary to the conserved RS structure, our cryo-ET map reveals the lack of IDA-b/c/e and the absence of Tektin filaments within the A-tubule of doublet microtubules in ependymal cilia compared with mammalian respiratory cilia and sperm flagella, further exemplifying the structural diversity of mammalian motile cilia. Our findings shed light on the stepwise mammalian RS assembly mechanism, the coordinated rigid and elastic RS-CP interaction modes beneficial for the regulation of asymmetric ciliary beating, and also facilitate understanding on the etiology of ciliary dyskinesia-related ciliopathies and on the ependymal cilia in the development of hydrocephalus.

EML4-ALK fusions drive lung adeno-to-squamous transition through JAK-STAT activation.

Human lung adenosquamous cell carcinoma (LUAS), containing both adenomatous and squamous pathologies, exhibits strong cancer plasticity. We find that ALK rearrangement is detectable in 5.1-7.5% of human LUAS, and transgenic expression of EML4-ALK drives lung adenocarcinoma (LUAD) formation initially and squamous transition at late stage. We identify club cells as the main cell-of-origin for squamous transition. Through recapitulating lineage transition in organoid system, we identify JAK-STAT signaling, activated by EML4-ALK phase separation, significantly promotes squamous transition. Integrative study with scRNA-seq and immunostaining identify a plastic cell subpopulation in ALK-rearranged human LUAD showing squamous biomarker expression. Moreover, those relapsed ALK-rearranged LUAD show notable upregulation of squamous biomarkers. Consistently, mouse squamous tumors or LUAD with squamous signature display certain resistance to ALK inhibitor, which can be overcome by combined JAK1/2 inhibitor treatment. This study uncovers strong plasticity of ALK-rearranged tumors in orchestrating phenotypic transition and drug resistance and proposes a potentially effective therapeutic strategy.

miR-375 is highly expressed and possibly transactivated by achaete-scute complex homolog 1 in small-cell lung cancer cells.

In this study, we identified five miRNAs highly expressed in the small-cell lung cancer (SCLC) cell line NCI-H209. Among them, the expression levels of miR-375 were dramatically elevated in all SCLC cell lines examined, coincident with the expression of the transcription factor achaete-scute complex homolog 1 (ASCL1). Moreover, miR-375 was upregulated and correlated with ASCL1 in the cell lines generated from mouse SCLC-like tumors as well. Dual-luciferase assays further showed that ASCL1 activated the expression of miR-375 by binding to the three E-box elements in the miR-375 promoter. These results imply a role of ASCL1 in SCLC via the upregulation of miR-375.

Rab-like small GTPases in the regulation of ciliary Bardet-Biedl syndrome (BBS) complex transport.

Primary cilia, microtubule-based hair-like structures protruding from most cells, contain membranes enriched in signaling molecules and function as sensory and regulatory organelles critical for development and tissue homeostasis. Intraflagellar transport (IFT), cilia-specific bidirectional transport, is required for the assembly, maintenance, and function of cilia. BBSome, the coat complex, acts as the adaptor between the IFT complex and membrane proteins and is therefore essential for establishing the specific compartmentalization of signaling molecules in the cilia. Recent findings have revealed that three ciliary Rab-like small GTPases, IFT27, IFT22, and Rabl2, play critical regulatory roles in ciliary BBSome transport. In this review, we provide an overview of these three Rab-like small GTPases and their relationship with BBSome.

Purification and Immunostaining of Mouse Ependymal Ciliary Shafts.

Cilia and flagella are microtubule-based hair-like organelles protruding from the surface of most eukaryotic cells, and play essential roles in cell locomotion, left-right asymmetry, embryo development, and tissue homeostasis. With isolated cilia and flagella, great progress has been made in understanding the composition, structure, and function of cilia. However, the current cilia/flagella isolation methods are deficient in the integrity or productivity of purified cilia when applied to mammalian motile cilia. Here, we describe a new protocol that isolates cilia shafts from mouse ependymal cells, by horizontal shear force and mild detergent. This method enables the production of virtually integral cilia with high yields and less cell body contamination. It is suitable for immunostaining, puromycin labeling assay, and proximity ligation assay of mammalian motile cilia. Graphical abstract.

Intertwined Wdr47-NTD dimer recognizes a basic-helical motif in Camsap proteins for proper central-pair microtubule formation.

Calmodulin-regulated spectrin-associated proteins (Camsaps) bind to the N-terminal domain of WD40-repeat 47 (Wdr47-NTD; featured with a LisH-CTLH motif) to properly generate axonemal central-pair microtubules (CP-MTs) for the planar beat pattern of mammalian motile multicilia. The underlying molecular mechanism, however, remains unclear. Here, we determine the structures of apo-Wdr47-NTD and Wdr47-NTD in complex with a characteristic Wdr47-binding region (WBR) from Camsap3. Wdr47-NTD forms an intertwined dimer with a special cross-over region (COR) in addition to the canonical LisH and globular α-helical core (GAC). The basic WBR peptide adopts an α-helical conformation and anchors to a tailored acidic pocket embedded in the COR. Mutations in this target-binding pocket disrupt the interaction between Wdr47-NTD and Camsap3. Impairing Wdr47-Camsap interactions markedly reduces rescue effects of Wdr47 on CP-MTs and ciliary beat of Wdr47-deficient ependymal cells. Thus, Wdr47-NTD functions by recognizing a specific basic helical motif in Camsap proteins via its non-canonical COR, a target-binding site in LisH-CTLH-containing domains.