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.

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.

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.

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.

[Effects of DA-6 and EDDS on Growth and Cd Uptake by Solanum nigrum L. and on the Soil Bacterial Community Structure].

The effects of the plant growth regulator DA-6 and chelator EDDS on the growth, Cd absorption, and soil bacterial community structure of Solanum nigrum L. on Cd-polluted soil were examined through a pot experiment, so as to evaluate the practical application effects of different remediation treatments. The results showed that the addition of DA-6 alone and the combined application of DA-6 and EDDS could promote the growth of S. nigrum L., and the shoot fresh weight increased by 24.58% and 19.57%, respectively, compared with that in the control. At the same time, the chlorophyll content and antioxidant enzyme activity of S. nigrum L. leaves also increased, whereas the content of malondialdehyde decreased. The application of DA-6 and EDDS also significantly increased the content of soil available Cd and promoted the absorption of Cd by S. nigrum L. Compared with that in the control, the extraction amount of Cd from S. nigrum L. shoots was significantly increased by 39.42% (DA-6), 30.17% (EDDS), and 51.04% (DA-6+EDDS), respectively. It was also found that the application of EDDS alone significantly reduced the ACE index of the soil bacterial community and significantly increased the Simpson index, and the abundance of the Lysobacter genus in the soil was significantly reduced. After the combination of DA-6 and EDDS, the diversity and taxonomic composition of the soil bacterial community were improved.

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.

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.

Cilia locally synthesize proteins to sustain their ultrastructure and functions.

Cilia are microtubule-based hair-like organelles propelling locomotion and extracellular liquid flow or sensing environmental stimuli. As cilia are diffusion barrier-gated subcellular compartments, their protein components are thought to come from the cell body through intraflagellar transport or diffusion. Here we show that cilia locally synthesize proteins to maintain their structure and functions. Multicilia of mouse ependymal cells are abundant in ribosomal proteins, translation initiation factors, and RNA, including 18 S rRNA and tubulin mRNA. The cilia actively generate nascent peptides, including those of tubulin. mRNA-binding protein Fmrp localizes in ciliary central lumen and appears to function in mRNA delivery into the cilia. Its depletion by RNAi impairs ciliary local translation and induces multicilia degeneration. Expression of exogenous Fmrp, but not an isoform tethered to mitochondria, rescues the degeneration defects. Therefore, local translation defects in cilia might contribute to the pathology of ciliopathies and other diseases such as Fragile X syndrome.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

The effects of water control on the survival and growth of Alternanthera philoxeroides in the vegetative reproduction and seedling stages.

Alternanthera philoxeroides (Martius) is an infamous invasive alien plant that is widely distributed in aquatic and terrestrial habitats. To investigate the vegetative reproduction, growth, survival strategy, and the function of leaves in fragment of A. philoxeroides under different water conditions, two water control experiments were conducted with different leaf treatments: (1) water control with stolon fragments, and (2) water control with plants. The water control was subjected to five levels: I 30% soil water content, II 70% soil water content, III 97% soil water content, IV water depth of 5 cm, and V water depth of 10 cm in combination with the two leaf treatments, fragments with two leaves and fragments without leaves. Based on the results, A. philoxeroides produced a significantly higher stem length, node number, leaf number, stem biomass, leaf biomass, and total biomass in the 97% soil water content and in treatments with leaves. Additionally, the stem mass ratio increased and the root mass ratio decreased with the increase of the water content. In Exp. 1, the survival rate was the highest in the 97% water content and was 0 in the 30% water content. Therefore, the leaves of stolon fragments contribute to the vegetative reproduction and growth of A. philoxeroides. In response to different water conditions, A. philoxeroides adopts different strategies according to the resource reserves by itself, which are conducive to its survival and widespread occurrence.

The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment.

Background: Hormones and immune imbalance are critical factors in polycystic ovary syndrome (PCOS). The alternation of immune microenvironment of oocytes may play a significant role in infertility of PCOS patients. Objective: This study explores the role of follicular fluid microenvironment change in inflammatory pathways activation of granulosa cells (GCs) in PCOS women infertility. Methods: We enrolled 27 PCOS patients and 30 controls aged 22 to 38 years who underwent IVF and collected their luteinized granulosa cells (LGCs). Meanwhile, a granulosa-like tumor cell line (KGN) as a cell-model assisted this study. Key inflammatory markers in human ovarian GCs and follicular fluid were detected by RT-qPCR, Western blotting, or ELISA. The KGN cells were treated with follicle supernatant mixed with normal medium to simulate the microenvironment of GCs in PCOS patients, and the inflammation indicators were observed. The assembly of NLRP3 inflammasomes was detected by immunofluorescence techniques. Dihydroethidium assay and EdU proliferation assay were used to detect ROS and cell proliferation by flow cytometry. Results: Compared with normal controls (n = 19), IL-1ß (P = 0.0005) and IL-18 (P = 0.021) in the follicular fluid of PCOS patients (n = 20) were significantly increased. The NF-κB pathway was activated, and NLRP3 inflammasome was formatted in ovarian GCs of PCOS patients. We also found that inflammation of KGN cells was activated with LPS irritation or stimulated by follicular fluid from PCOS patients. Finally, we found that intracellular inflammation process damaged mitochondrial structure and function, which induced oxidative stress, affected cellular metabolism, and impaired cell proliferation. Conclusion: Inflammatory microenvironment alteration in the follicular fluid of PCOS patients leads to activated inflammatory pathway in GCs, serving as a crucial factor that causes adverse symptoms in patients. This study provides a novel mechanism in the inflammatory process of PCOS.

Phase separation of EML4-ALK in firing downstream signaling and promoting lung tumorigenesis.

EML4-ALK fusion, observed in about 3%-7% of human lung adenocarcinoma, is one of the most important oncogenic drivers in initiating lung tumorigenesis. However, it still remains largely unknown about how EML4-ALK fusion exactly fires downstream signaling and drives lung cancer formation. We here find that EML4-ALK variant 1 (exon 1-13 of EML4 fused to exon 20-29 of ALK) forms condensates via phase separation in the cytoplasm of various human cancer cell lines. Using two genetically engineered mouse models (GEMMs), we find that EML4-ALK variant 1 can drive lung tumorigenesis and these murine tumors, as well as primary tumor-derived organoids, clearly show the condensates of EML4-ALK protein, further supporting the findings from in vitro study. Mutation of multiple aromatic residues in EML4 region significantly impairs the phase separation of EML4-ALK and dampens the activation of the downstream signaling pathways, especially the STAT3 phosphorylation. Importantly, it also significantly decreases cancer malignant transformation and tumor formation. These data together highlight an important role of phase separation in orchestrating EML4-ALK signaling and promoting tumorigenesis, which might provide new clues for the development of clinical therapeutic strategies in treating lung cancer patients with the EML4-ALK fusion.

The decrease of intraflagellar transport impairs sensory perception and metabolism in ageing.

Sensory perception and metabolic homeostasis are known to deteriorate with ageing, impairing the health of aged animals, while mechanisms underlying their deterioration remain poorly understood. The potential interplay between the declining sensory perception and the impaired metabolism during ageing is also barely explored. Here, we report that the intraflagellar transport (IFT) in the cilia of sensory neurons is impaired in the aged nematode Caenorhabditis elegans due to a daf-19/RFX-modulated decrease of IFT components. We find that the reduced IFT in sensory cilia thus impairs sensory perception with ageing. Moreover, we demonstrate that whereas the IFT-dependent decrease of sensory perception in aged worms has a mild impact on the insulin/IGF-1 signalling, it remarkably suppresses AMP-activated protein kinase (AMPK) signalling across tissues. We show that upregulating daf-19/RFX effectively enhances IFT, sensory perception, AMPK activity and autophagy, promoting metabolic homeostasis and longevity. Our study determines an ageing pathway causing IFT decay and sensory perception deterioration, which in turn disrupts metabolism and healthy ageing.

Fibrogranular materials function as organizers to ensure the fidelity of multiciliary assembly.

Multicilia are delicate motile machineries, and how they are accurately assembled is poorly understood. Here, we show that fibrogranular materials (FGMs), large arrays of electron-dense granules specific to multiciliated cells, are essential for their ultrastructural fidelity. Pcm1 forms the granular units that further network into widespread FGMs, which are abundant in spherical FGM cores. FGM cores selectively concentrate multiple important centriole-related proteins as clients, including Cep131 that specifically decorates a foot region of ciliary central pair (CP) microtubules. FGMs also tightly contact deuterosome-procentriole complexes. Disruption of FGMs in mouse cells undergoing multiciliogenesis by Pcm1 RNAi markedly deregulates centriolar targeting of FGM clients, elongates CP-foot, and alters deuterosome size, number, and distribution. Although the multicilia are produced in correct numbers, they display abnormal ultrastructure and motility. Our results suggest that FGMs organize deuterosomes and centriole-related proteins to facilitate the faithful assembly of basal bodies and multiciliary axonemes.

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.

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.

lncRNA H19 acts as a ceRNA to regulate the expression of CTGF by targeting miR-19b in polycystic ovary syndrome.

The etiology of polycystic ovary syndrome (PCOS) is complex and the pathogenesis is not fully understood. Some studies have shown that dysregulation of ovarian granulosa cells may be related to abnormal follicles and excessive androgen in women with PCOS. Our team has also confirmed the high expression status of H19 in PCOS patients in the early stage. However, the relationship between H19 and miR-19b in the development of PCOS is still unknown. Therefore, we used bioinformatics to predict the binding sites of human H19 and miR-19b, and of miR-19b and CTGF genes. After the silencing and overexpression of H19, real-time polymerase chain reaction (PCR) was used to detect the expressions of H19, miR-19b, and CTGF. Western blotting was used to detect CTGF protein. Proliferation of KGN cells after H19 silencing was detected by CCK8. Flow cytometry was used to detect the apoptosis of KGN cells after H19 silencing. After the overexpression of H19, it was found that the expression of miR-19b gene decreased and the expression of CTGF increased, whereas silencing of H19 did the opposite. In addition, H19 could promote cell proliferation and decrease cell apoptosis. Finally, luciferase reporter assays showed that the 3'-end sequences of lncRNA H19 and CTGF contained the binding site of miR-19b. In conclusion, our study indicated that lncRNA H19 acted as a ceRNA to bind to miR-19b via a "sponge" to regulate the effect of CTGF on KGN cells, which may play a vital role in PCOS.

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.

Habitat preferences rather than morphological traits affect the recovery process of Collembola (Arthropoda, Hexapoda) on a bare saline-alkaline land.

The Songnen Plain of China was once an important grassland used for sheep grazing, but it has largely been degraded to bare saline-alkaline land (BSAL). BSAL consists of plant-free areas characterized by high soil pH values (up to 10) and salt and alkali (e.g., Na+ and Ca2+) contents, as well as low soil organic matter and water contents; thus, very few soil faunal species can survive on BSAL. The recovery of degraded ecosystems provides a great opportunity to investigate the reconstruction of belowground soil faunal communities. Collembola are a class of widespread and abundant soil fauna that can colonize this harsh environment. Habitat changes on BSAL promote aboveground revegetation, which greatly facilitates the recovery of Collembola. A soil transfer experiment on the BSAL of the Songnen Plain was conducted to study the effects of habitat and Collembola morphological traits on the recovery process of Collembola. Defaunated and with-fauna soil blocks were transferred among three habitats: BSAL, reclaimed arable land, and naturally revegetated grassland. The recovered Collembola in the transferred soil blocks were compared two, seven, and 12 weeks after the start of the experiment. The results showed that (1) the majority of the Collembola, regardless of their morphological traits, recovered in the defaunated soil blocks within 2 weeks; (2) generalists and habitat-preferring species recovered faster than specialists; (3) the average total abundance, species richness, and community composition of Collembola recovered to the natural levels in 2 weeks; and (4) 12 weeks after replacement, the highest average total abundance and species richness of Collembola were found in the arable land. Our results indicate that the majority of Collembola in this study, regardless of their dispersal type, which is related to their morphological traits, are fast dispersers, and their recovery speeds are mainly affected by habitat preferences. We suggest that the reclamation of BSAL to arable land rather than its natural recovery to grassland aids in the recovery of Collembola in degraded grassland systems.