Central-pair microtubules (CP-MTs) are non-centrosomal MTs essential for planar beat pattern of cilia. The CP-MT formation requires the MT-associated protein Spef1, but the underlying molecular mechanism remains unclear. Here, we show that Spef1 undergoes liquid-liquid phase separation (LLPS) to facilitate non-centrosomal MT assembly by enriching tubulins. The LLPS of Spef1 is mediated by its C-terminal coiled-coil (CC) domain. Crystallography reveals that the Spef1-CC domain forms a parallel CC dimer with a unique charge distribution pattern on the surface. The dimerization capacity and charge distribution of Spef1-CC are both critical for controlling in vitro LLPS. Disruption of the dimerization capacity abolishes ciliary functions of Spef1. In contrast, a charge-changing mutant with attenuated LLPS still supports the CP-MT formation but results in cilia with abnormal beat pattern. Thus, the CC-mediated LLPS of Spef1 provides a mechanistic explanation for its prominent role in controlling CP-MT organization and function in the axoneme.

The central apparatus of motile cilia, consisting of central microtubules and various protein projections, is essential for dictating the ciliary movement. Although three proteins (FAP65, FAP147, and FAP70) have been localized to the C2a projection in Chlamydomonas reinhardtii , the full protein composition and functional roles of the vertebrate C2a remain inadequately defined. Here, we use three knockout mouse models corresponding to their respective homologs ( Ccdc108 , Mycbpap , and Cfap70 ) to systematically investigate their functions in vertebrates. Notably, all three knockout strains exhibit distinct phenotypes related to primary ciliary dyskinesia (PCD), including hydrocephalus and sinusitis. The ciliary incorporation of CCDC108, MYCBPAP, and CFAP70 is essential for one another’s stability, with the loss of any single component triggering C2a collapse, which destabilizes the central pair microtubules and ultimately alters the ciliary movement pattern. Furthermore, we significantly expand the vertebrate C2a proteome by identifying ARMC3 and MYCBP as additional C2a components. Collectively, our findings illuminate the proteomic composition and strict physiological requirements of the vertebrate C2a projection, providing new insights into the molecular pathogenesis of PCD.

In metazoan epithelia, arrays of motile cilia beat in a tissue-wide coordinated manner to drive unidirectional fluid flow. Whether radial spokes (RSs), which mediate mechano-signal transduction between the central pair and axonemal dynein arms, acquire metazoa-specific subunit(s) in evolution to facilitate this function remains poorly known. Here we report that Gα-GTPase activating protein family member RGS22 is a metazoa-specific component of RS1/2 complexes. Mouse Rgs22-deficient motile cilia exhibit ultrastructural defects, including deformation of the RS1 and disorganization of outer dynein arms. These abnormalities correlate with impaired planar polarity, manifested as unsynchronized ciliary beating and disrupted rotational polarity of basal bodies across the ependyma. The motility deficits further lead to progressive ciliary loss and shortening, resulting in postnatal hydrocephalus. Together, our data identify RGS22 an animal-lineage RS1/2 component required for normal RS architecture and associated with tissue-level ciliary motility and coordination phenotypes.

Rabies virus (RABV) causes severe neuroinflammation after entering the central nervous system, posing a serious threat to mammals. The NLRP3 inflammasome is a key sensor of RNA virus infection, yet how RABV activates NLRP3 remains unclear. We found that the attenuated RABV strain BNSP induced IL-1β secretion and that its M protein is essential for NLRP3 activation by binding to NLRP3 and facilitating speck formation. Mechanistically, threonine 148 of BNSP-M promotes HDAC6-mediated deacetylation of TRIM33 at lysine 966, leading to TRIM33 nuclear export. Cytoplasmic TRIM33 then activates NLRP3 via K63-linked polyubiquitination of NLRP3 at lysine 650. A recombinant mutant virus, BNSP-M-T148A, generated by reverse genetics, showed markedly reduced NLRP3 inflammasome activation both in vitro and in vivo. Intracranial infection of HDAC6-knockout mice with BNSP or BNSP-M-T148A resulted in weaker NLRP3 responses than in wild-type mice. Therefore, we have revealed a novel mechanism by which proteins transcribed by rabies virus interact with host proteins to induce NLRP3 inflammasome response after infection and verifies HDAC6 as a promising therapeutic target for alleviating rabies-induced neuroinflammation, thus providing novel theoretical insights into virus-host protein interaction and anti-rabies intervention strategies.

To maintain protein homeostasis, which is essential for health, animals have developed complex protective mechanisms against various acute and chronic stresses. However, the coordination of responses to these protein stresses, especially their age-dependent changes, is not well understood. HSF-1 is a key regulator of protein homeostasis. Our study identifies PBS-7, a proteasome subunit, as its crucial regulator. In aged C. elegans, decreased PBS-7 binding reduces proteasome-mediated degradation of HSF-1. The increase in HSF-1 enhances responses to chronic stresses, like accumulating protein aggregates, by upregulating heat shock proteins (HSPs) and autophagy genes. Meanwhile, the upregulated HSPs suppress the activation of HSF-1 upon acute stress, such as heat shock. Our findings reveal a mechanism that coordinates responses to acute and chronic protein stresses and highlights an adaptation prioritising protection against increasing protein aggregates in ageing.

Peste des Petits Ruminants (PPR), a highly contagious disease of domestic and wild small ruminants, is characterized by severe morbidity and mortality. PPRV, the causative agent, is a morbillivirus in the family Paramyxoviridae. The virus poses a significant barrier to sustainable agricultural development in the developing world. Currently, no effective therapeutics agent for PPRV infection is available. Ginsenoside Rb3, the major bioactive constituent in the plants of ginseng, was reported to exert a wide range of pharmacologic and immunologic effects. However, it is unclear whether Ginsenoside Rb3 can act as an antiviral against PPRV infection. Here, we show that Ginsenoside Rb3 exhibits significant antiviral activity against PPRV in cell culture models. The mechanism of action of Ginsenoside Rb3 against PPRV is mainly attributed to its ability to inhibit PPRV-mediated autophagy, thus leading to promotion of interferon responses. In summary, our study establishes Ginsenoside Rb3 as a novel antiviral agent effective against PPRV, sheds light on its mode of action, and reveals a novel immunomodulatory strategy that may prove essential for combating both current and future viral outbreaks.

Peste des Petits Ruminants (PPR), a highly contagious disease of domestic and wild small ruminants, is characterized by severe morbidity and mortality. PPRV, the causative agent, is a morbillivirus in the family Paramyxoviridae. The virus poses a significant barrier to sustainable agricultural development in the developing world. Currently, no effective therapeutics agent for PPRV infection is available. Ginsenoside Rh1, a protopanaxadiol ginsenoside, the major pharmacological ingredient in the plants of ginseng, was reported to inhibit the replication of a broad range of human viruses. However, it is unclear whether Ginsenoside Rh1 can act as an antiviral against PPRV infection. Here, we demonstrate that Ginsenoside Rh1 exhibits significant antiviral activity against PPRV in cell culture models. The mechanism of action of Ginsenoside Rh1 against PPRV is mainly attributed to its ability to block PPRV mediated autophagy, thus leading to stimulation of interferon responses and inhibition of inflammatory responses. In summary, our study establishes Ginsenoside Rh1 as a novel antiviral agent effective against PPRV and potentially other related morbilliviruses, sheds light on its mode of action, and reveals a novel autophagy-dependent dual immunomodulatory strategy that may prove essential for combating both current and future viral outbreaks.

Vesicular stomatitis virus (VSV), a member of the Vesiculovirus genus within the Rhabdoviridae family, is a widespread pathogen affecting all hoofed livestock species, leading to reduced animal growth and productivity. To date, no effective therapeutic treatment for VSV infection has been developed. Natural medicinal compounds with immunomodulatory properties represent a promising supportive strategy for infection control. Ginsenoside Rh1, a primary bioactive component of ginseng plants, has been reported to possess broad pharmacological and immunoregulatory activities. Nevertheless, its potential antiviral effects against VSV remain unexplored. In this study, we demonstrate that Ginsenoside Rh1 exhibits considerable antiviral activity against VSV in cellular models. Mechanistically, its antiviral effect is primarily mediated through the inhibition of VSV-induced autophagy, thereby enhancing interferon-mediated antiviral responses. Collectively, our findings identify Ginsenoside Rh1 as a novel antiviral agent active against VSV and potentially related vesiculoviruses, clarify its mechanism of action, and highlight an autophagy-dependent immunomodulatory approach that could be critical for confronting existing and emerging RNA viral infections.

As animals evolved from external to internal fertilization, sperm flagella, once transiently propelling sperm in water to reach nearby eggs, developed to beat for days in the viscous female reproductive tract. How flagella are remodeled accordingly remains unclear. Unlike externally fertilizing zebrafish and sea urchins, mammalian flagella feature a barrel between radial spokes (RSs) RS1 and RS2. Here, we show that this RS1-RS2 barrel (RRB) is a unique T-complex protein-1 ring complex (TRiC) that folds locally translated polypeptides to sustain flagellar motility. Cryo-electron microscopy (cryo-EM) reveals a flagellum-specific TRiC structure. An in situ cryo-electron tomography (cryo-ET) map of flagellar axonemes captures the RRB TRiC in an active, substrate-receptive state, with additional densities suggestive of folding substrates and cofactors. Mammalian flagella contain components of translation machineries and locally synthesize proteins. Cross-linking mass spectrometry identifies candidate locally translated axonemal proteins and folding substrates. Furthermore, a TRiC ATPase inhibitor markedly represses mouse sperm motility. Our findings provide insights into flagellar remodeling in internally fertilizing species.

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.

Motile cilia are critical for diverse cellular activities, affecting the survival and development of most eukaryotic organisms. Central microtubules (MTs), which are located in the lumen of ciliary axonemes, are non-centrosomal MTs that are crucial for motile cilia beating. However, the formation mechanism of central MTs remains elusive. Here, by using a Drosophila model, we identify Ccdc13 as a novel regulator for the assembly of central MTs. We show that Ccdc13 localizes along the central MTs and is essential for its formation in sperm flagella, with its deletion consequently affecting the sperm motility and the fertility of male flies. Mechanistically, we demonstrated that Ccdc13 directly interacts with Spef1, acting upstream of Spef1 to regulate central MT elongation. Remarkably, we demonstrated that the role of Ccdc13 in ciliary central MT formation is conserved in mammals. Ccdc13 deficiency in mice leads to the loss of central MTs in motile ependymal cilia, resulting in abnormal cilia motility and hydrocephalus. Our results mark the discovery of Ccdc13 as a novel regulator for ciliary central MT assembly and reveal that the Ccdc13-Spef1 complex is an evolutionarily conserved module that is critical for central MT formation in motile cilia of both flies and mammals.

Motile cilia are evolutionarily conserved protrusions critical for motility and homeostasis. Their rhythmic movements require the central pair microtubules (CP-MTs). While the initial CP-MT assembly in mammals is mediated by WDR47 and microtubule minus-end-binding CAMSAPs, the mechanism by which CP-MTs are stabilized remains unclear. Here, we demonstrate that WDR47 coordinates JHY and SPEF1 to maintain the stability of mammalian CP-MTs. By generating a proximity interactome of WDR47, we identify a group of CP-MT-associated proteins, including SPEF1 and JHY. WDR47 enriches JHY and SPEF1 to the central lumen and tip of nascent cilia, whereas SPEF1 recruits WDR47 and JHY to CP-MTs through direct interactions. Jhy deficiency in mice preferentially disrupts distal CP-MTs, resulting in rotatory ciliary beats. Phylogenetic analyses suggest conserved functions of WDR47 and SPEF1 in protozoa and metazoans, as well as a role for JHY in animals with radial or bilateral body symmetry. We propose that JHY emerges to further reinforce CP-MTs, enabling the transition from switchable to fixed ciliary waveforms in metazoan evolution.

Peste-des-petits-ruminants (PPR) is an acute, contact infectious disease caused by peste-des-petits-ruminants virus (PPRV) infection. PPRV mainly infects goats, sheep and other small ruminants, posing a serious threat to the development of the livestock industry and the safety of wildlife. In this study, a competitive ELISA (c-ELISA) method for detecting neutralizing antibodies against PPRV was developed using the extracellular domain of haemagglutinin (tH) protein and a monoclonal antibody against tH protein. The results showed that the specificity and sensitivity of c-ELISA were 99.38% and 100%, respectively. The agreement rate of the c-ELISA and viral neutralization test (VNT) was 96.65%, suggesting that the c-ELISA could detect neutralizing antibody against PPRV and could be used for protective immunity evaluation.

Cilia are membrane-covered hair-like organelles built on specialized centrioles and conserved throughout eukaryotic evolution. They are either motile or immotile, serving respectively as versatile signaling antennae or elegant beating nanomachines. Accordingly, their dysfunctions cause a wide variety of developmental and degenerative disorders, which in human are syndromes termed ciliopathies. Motile cilia in mammals reside in epithelial cells. Their rapid, rhythmic beating facilitates reproduction, left-right patterning, and organ homeostasis by propelling directional gamete transport, nodal flow, cerebrospinal fluid circulation, and mucus clearance. They merge mostly as multicilia, with up to hundreds per cell. Multiciliated cells need not only to break the tight cellular control on centriole biogenesis and ensure accurate assemblies of numerous structural components for their formations, but to properly organize and polarize them for their functions as well. This review mainly focuses on the cell biology of mammalian motile cilia, with the mouse as the model organism.

Radial spokes in motile cilia and flagella regulate rhythmic ciliary motility, which is essential for rapid cell movement and extracellular fluid flow. In humans, defective radial spokes can cause a genetic disorder known as primary ciliary dyskinesia (PCD), resulting in respiratory defects and infertility. Despite their critical role in ciliary motility, the molecular composition and related physiological functions remain to be elucidated. Here, we identify EF-hand calcium-binding domain protein 10 (EFCAB10) and adenylate kinase (AK8) as radial spoke proteins and describe their roles in mouse motile cilia. Using Efcab10-/- and Ak8-/- mice, we show that loss of either protein can affect ciliary motility and lead to PCD-related phenotypes in mice. Interestingly, ciliary AK8 is completely absent in Efcab10-/- cilia, but the loss of AK8 has no effect on ciliary EFCAB10. Further biochemical analyses reveal that EFCAB10 interacts with AK8 and RSPH3B, which fastens AK8 to the radial spoke. Overall, our findings demonstrate the essential role of EFCAB10 as a radial spoke protein in maintaining the integrity of the radial spoke and provide valuable insights into the molecular basis of related ciliopathies.

AbstractEpendymal multicilia position at one-side on the cell surface and beat synchronously across tissue to propel the flow of cerebrospinal fluid. Loss of ependymal cilia often causes hydrocephalus. However, molecules contributing to their maintenance remain yet fully revealed. Cytosolic carboxypeptidase (CCP) family are erasers of polyglutamylation, a conserved posttranslational modification of ciliary-axoneme microtubules. CCPs possess a unique domain (N-domain) N-terminal to their carboxypeptidase (CP) domain with unclear function. Here, we show that a novel mutant mouse ofAgbl5, the gene encoding CCP5, with deletion of its N-terminus and partial CP domain (designatedAgbl5M1/M1), developed lethal hydrocephalus due to degeneration of ependymal multicilia. Interestingly, multiciliogenesis was not impaired inAgbl5M1/M1ependyma. The initially formed multicilia beat at a normal frequency, but in intercellularly diverse directions, indicative of aberrant tissue-level coordination. Moreover, actin networks are severely disrupted and basal body patches are improperly displaced in mutant cells, suggesting impaired cell polarity. In contrast,Agbl5mutants with disruption solely in the CP domain of CCP5 (Agbl5M2/M2) do not develop hydrocephalus despite increased glutamylation levels in ependymal cilia as similarly seen inAgbl5M1/M1. This study revealed an unappreciated role of CCP5, particularly its N-domain, in ependymal multicilia stability associated with their polarization and coordination.

Cilia, essential organelles for cell motility and signaling, comprise an axoneme extended from the basal body (BB). The assembly process of BBs and axonemes during ciliogenesis, however, remains largely unknown due to the lack of structural information. Here, we leverage in-situ cryo-electron tomography to capture within mouse ependymal cells the dynamic processes of BB biogenesis and multiciliogenesis at various stages. This approach enables 3D visualization of the complete motile machinery, revealing the continuous microtubule-based scaffold from BBs to axonemes at sub-nanometer resolution with unprecedented structural details. Furthermore, we elucidate along BBs and cilia heterogeneous landscapes of microtubule-binding proteins underlying the establishment of structural periodicity and diverse subregions. Notably, the chronological binding patterns of microtubule-inner proteins (e.g., CEP41) correlate with the progressive assembly of ciliary beating machinery. We also resolve a substructure that borders the BB and the axoneme. Our findings provide key insights into intricate orchestrations during ciliogenesis.

The shell-and-tube phase change heat accumulator has extensive application in industrial production due to its small temperature change and high heat storage density. A novel structure with annular fin branches was designed to enhance the shell-and-tube phase change heat accumulator’s heat transfer performance. The phase change heat transfer performance in the shell-and-tube heat accumulator with branch annular fins was studied by using FLUENT software. By analyzing the evolution of the PCM liquid phase, the distribution of temperature and velocity, the variation of melting rate, and the uniformity of the temperature distribution and so on, the heat exchange property of the branch annular fin during melting was analyzed. The new type structure of annular fin with branches preserves the radial heat transfer depth of ordinary annular fins, while increasing the longitudinal heat transfer depth and heat transfer area. Compared with heat accumulator with ordinary annular fins, the time that the PCM completes melting is shortened by 27.13%, the uniformity of melting rate is increased by 34.72% and the uniformity of temperature distribution is increased by 1.55%. Furthermore, the heat transfer performance of phase change heat accumulator with the shell-and-tube structure can be significantly enhanced by shortening the main fin’s length or increasing the branch angle of the main fins. The study result of this paper has great guiding significance for the research and development of the shell-and-tube heat accumulator with fins.

Peste des petits ruminants virus (PPRV) is currently the only member of the Morbillivirus caprinae species within the genus Morbillivirus of the family Paramyoxviridae. PPRV causes a highly contagious disease in small ruminants, especially goats and sheep. Succinylation is a newly identified and conserved modification and plays an important role in host cell response to pathogen infection. However, the extent and function of succinylation in Vero cells during PPRV infection remains unknown. In this study, a global profile of the succinylome in Vero cells infected with PPRV Nigeria 75/1 vaccine strain (PPRVvac) was performed by dimethylation labeling-based quantitative proteomics analysis. A total of 2633 succinylation sites derived from 823 proteins were quantified. The comparative analysis of differentially succinylated sites revealed that 228 down-regulated succinylation sites on 139 proteins and 44 up-regulated succinylation sites on 38 proteins were significantly modified in response to PPRVvac infection, seven succinylation motifs were identified. GO classification indicated that the differentially succinylated proteins (DSuPs) mainly participated in cellular respiration, biosynthetic process and transmembrane transporter activity. KEGG pathway analysis indicated that DSuPs were related to protein processing in the endoplasmic reticulum. Protein-protein interaction networks of the identified proteins provided further evidence that various ATP synthase subunits and carbon metabolism were modulated by succinylation, while the overlapped proteins between succinylation and acetylation are involved in glyoxylate and dicarboxylate metabolism. The findings of the present study provide the first report of the succinylome in Vero cells infected with PPRVvac and provided a foundation for investigating the role of succinylation alone and its overlap with acetylation in response to PPRVvac.