Biology subjects

Zhu, X.

Publications and source records attributed to Zhu, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Benchmarking TCR-pMHC structure prediction: a unified evaluation and CDR3-based functional insights

Interactions between T cell receptors (TCRs) and peptide-major histocompatibility complexes (pMHCs) are central to adaptive immunity. Recent advances in structure prediction tools have enabled atomic-level modeling of TCR-pMHC interactions. However, the lack of systematic evaluation forces practitioners to invest substantial resources in selecting appropriate tools. Here, we present a comprehensive benchmark of TCR-pMHC structure prediction with 70 previously unseen complexes and ten models spanning MSA-based, PLM-based, and docking-based approaches, revealing the superior modeling accuracy and docking quality of MSA-based methods, especially AlphaFold3. To further enhance the utility of AlphaFold3 predictions, we identify the pLDDT score of the TCR CDR3 region as an informative indicator of both structural correctness and functional relevance. Specifically, it enables up to 4.3% Top-1 success gain through reranking and captures mutation-induced affinity changes in over 80% of cases. Overall, our analysis would facilitate the practical usage of immune structure prediction models and guide the advancement of these models.

bioinformatics↗

bioRxiv2025

Upregulation of MAM by C99 disrupts ACSL4 activity and phospholipid homeostasis in Alzheimer disease.

The structure and function of cellular and intracellular membranes are critically governed by the fatty acid (FA) composition of phospholipids (PLs), which is dynamically regulated by a network of enzymes that fine-tune lipid species according to cellular demands. In this study, we identify a mechanism through which the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) modulates the activity of the acyl-CoA synthetase long-chain family member 4 (ACSL4), an enzyme that channels polyunsaturated fatty acids (PUFAs) into phosphatidylcholine (PC) via the Lands cycle. Through integrated biochemical, proteomic, and lipidomic analyses in both cellular and animal models, we demonstrate that MAM formation enhances ACSL4 activity, promoting arachidonic acid (AA) activation and its preferential incorporation into PC in concert with the MAM-localized lysophospholipid acyltransferase 4 (LPCAT4). Our findings further uncover an unexpected link between this pathway and the pathogenesis of Alzheimers disease (AD). We show that elevated levels of C99--the {beta}-secretase cleavage product of amyloid precursor protein (APP)--induce MAM remodeling through cholesterol clustering, which in turn activates ACSL4 and alters PC composition. This effect is mirrored in AD models as well as in fibroblasts, neurons, and immune cells derived from both familial and sporadic AD patients, all of which exhibit chronically increased C99 levels, heightened ACSL4 activity, and enrichment of PUFA-containing PC species, leading to lipid imbalance and membrane dysfunction. Together, these results establish MAMs as dynamic lipid-regulatory hubs that coordinate ACSL4-dependent membrane remodeling and highlight the contribution of MAM dysregulation to lipid abnormalities observed in AD.

molecular biology↗

bioRxiv2025

Single-Cell Multi-Omics Dissects Transcript Isoform and Immune Repertoire Dynamics in Human Immunosenescence

Immunosenescence, a major hallmark of systemic aging, refers to the progressive functional decline of the immune system. This decline not only compromises host defense and immunological memory but also fuels chronic inflammation and tissue degeneration (collectively known as inflammaging). While single-cell RNA sequencing (scRNA-seq) has revealed transcriptomic alterations in immune aging, analyses restricted to transcript abundance fail to capture deeper regulatory layers, such as transcript isoform diversity and the remodeling of immune receptor repertoires. To address this, we present the human peripheral immune single-cell multi-omics atlas that integrates gene expression, transcript isoforms diversity, and immune receptor repertoires. By combining single-cell full-length transcriptome sequencing (scCycloneSEQ), short-read scRNA-seq, and single-cell immune receptor sequencing (scTCR/BCR-seq), we systematically profiled peripheral blood mononuclear cells (PBMCs) from healthy young and elderly donors. Our analyses uncovered extensive age-related remodeling of immune cell composition, functional states, and TCR/BCR diversity. Notably, we identified in CD4 effector memory T cells exhibited widespread differential isoform usage (DIU), 3'UTR length variation, and a marked reshaping of cytotoxic T lymphocyte (CTL) clonotypes--all closely associated with aging-related inflammation and cellular senescence. This multi-omics atlas delineates key molecular features of immunosenescence and provides a high-resolution resource for deciphering the regulatory architecture underlying immune aging.

immunology↗

bioRxiv2025

Sensory neuronal mTORC1 signaling establishes neuroimmune memory that initiates allergic immunity

Environmental allergens are enriched in protease activity, a feature that directly activates cutaneous sensory neurons, triggering itch and Substance P release, which promotes Th2-skewing CD301b+ dendritic cell migration and initiates allergic immunity. However, allergens are typically encountered as repeated, subthreshold exposures, and how these cumulatively induce sensitization is unknown. We identify a sensory neuron-intrinsic mechanism of neuroimmune memory that enables this process. Initial activation by protease allergens induces sustained mTORC1 signaling and PGC-1-associated mitochondrial remodeling, establishing a metabolically primed state that amplifies neuroimmune responses upon allergen re-exposure. This heightened state, independent of adaptive immunity, drives enhanced itch, increased CD301b+ dendritic cell migration, and augmented Th2 differentiation. Disrupting neuronal mTORC1 signaling or mitochondrial stability abrogates this amplification while leaving primary responses intact. Importantly, this mechanism generalizes across distinct protease allergens, revealing mTORC1-driven metabolic reprogramming in sensory neurons as a form of innate neuroimmune memory underlying allergen cross-sensitization and polysensitization.

immunology↗

bioRxiv2025

Skull Bone Marrow Drainage and Its Associations with Inflammation, Sleep Quality, and Cognitive Performance

BackgroundRecent studies using human craniotomy samples and murine models have revealed channels that connect the inner skull cortex to the skull marrow cavities. While cerebrospinal fluid (CSF) tracer access to skull bone marrow has been described in CSF circulation disorders, evidence on regional patterns and clinical correlates in broader clinical cohorts is limited. MethodsThree-dimensional imaging was performed at baseline, 4.5, 15, and 39 hours post-gadolinium administration in 87 patients with neurological conditions. Signal changes in skull bone marrow near the superior sagittal sinus, lateral fissure, and cisterna magna were recorded. CSF drainage function was characterized as the percentage changes in the signal unit ratio within the skull bone marrow from baseline to time points post administration of gadolinium. ResultsThe tracer drained from CSF into the skull bone marrow, highlighting the drainage variations nearby different regions. Reduced drainage was associated with female sex, hypertension, diabetes, and elevated neutrophil levels (p < 0.05). Drainage near the superior sagittal sinus was inversely associated with sleep quality and positively associated with cognitive scores, and partially accounted for the association of sleep quality with cognitive function (indirect effect {Delta}{beta} = -0.092; bootstrap 95% CI -0.195 to -0.004; proportion mediated {approx} 38%). ConclusionThese results support the presence of skull bone marrow adjacent routes for CSF tracer movement and suggest that skull bone marrow tracer dynamics relate to systemic inflammation, sleep quality, and cognitive performance in this clinical cohort.

neuroscience↗

bioRxiv2025

A multi-tissue atlas of genetic regulatory effects in sheep

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,468 primary and 114,017 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

genomics↗

bioRxiv2025

Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

For viruses that replicate in the proximity of or bud at the endoplasmic reticulum (ER) associated membranes, proper processing of their glycoproteins is critical for successful infection. Rotavirus outer capsid protein VP7 is an ER-resident protein. However, its N-terminal signal peptide is removed by an unknown proteolytic mechanism. In this study, we leveraged tandem affinity purification followed by high-resolution mass spectrometry to profile host proteins that interact with VP7. We identified members of the signal peptidase complex (SPC) family as important host factors that facilitate rotavirus infection. CRISPR knockout or siRNA knockdown of distinct SPC subunits resulted in significant decrease in infectious rotavirus titers in a viral strain- and cell type-independent manner. While viral transcription, translation, and replication were not altered in the absence of SPC, we observed formation of abnormal viral particles by transmission electron microscopy (TEM) in SPCS1 knockout cells. Mechanistically, loss of SPC proteins led to inefficient cleavage of VP7 signal peptide and severely impaired the final steps of virion maturation and assembly. Additionally, we identified residue E256 within VP7 as a key site for SPC binding. An E to R mutation abolished VP7 interaction with SPC and subsequently led to reduced viral infectivity. Taken together, these findings define SPC as a novel regulator of VP7 maturation and rotavirus assembly and highlight its role as a novel cellular target for potentially broad-spectrum antiviral therapeutic development. Author summaryPrevious reports demonstrated that SPC is involved in the replication cycles of several members of the flavivirus family and human T-cell leukemia virus type 1 (HTLV-1). As an ER-resident outer capsid protein, rotavirus VP7 must undergo proper post-translational modifications, including the cleavage of its N-terminal signal peptide, to be functionally incorporated into mature virions. However, the processing mechanism remains unknown. For the first time, our study identifies SPC as an essential regulator of rotavirus assembly by mediating the cleavage of the VP7 signal peptide. Loss of SPC impairs VP7 signal peptide cleavage and maturation, thereby disrupting correct virion assembly and reducing infectious particle production. Using Alphafold3, we predicted the VP7 residue E256 to be at the binding interface with SPC complex. Experimentally, mutation of glutamic acid to arginine (E256R) substantially weakens this interaction and results in reduced viral propagation. Our findings unveil a novel post-translational checkpoint in rotavirus regulated by SPC and underscore the promise of SPC as a broad-spectrum antiviral target, especially for rotavirus, flavivirus, and HTLV-1, whose viral glycoproteins and structural proteins require SPC processing for proper maturation.

microbiology↗

bioRxiv2025

Single-component self-assembling protein nanoparticles displaying stabilized prefusion-closed hemagglutinin trimers for influenza vaccine development

Current influenza vaccines primarily target hemagglutinin (HA), the major surface glycoprotein and principal determinant of neutralizing antibody (NAb) responses. However, antigenic drift and shift, together with HAs intrinsic metastability and low-pH sensitivity, pose major challenges to the development of broadly protective influenza vaccines. Here, we stabilize HA in the prefusion-closed conformation through rationally selected amino acid substitutions. One approach targets a conserved residue across multiple influenza A subtypes (H1, H3, H5, and H7) and both influenza B lineages, providing a unified and broadly applicable framework for HA stabilization to support vaccine development. Using H1 and H3 as test cases, stabilized HA trimers were displayed on 24- and 60-mer single-component self-assembling protein nanoparticles (SApNPs) to enhance lymph node trafficking and immunogenicity. Compared with soluble trimers, HA-presenting SApNPs exhibited prolonged retention in lymph node follicles and elicited robust germinal center (GC) responses, key hallmarks of effective virus-like particle (VLP) vaccines. In mice, these SApNPs induced strong humoral immunity and conferred protection against homologous viral challenge. Furthermore, glycan engineering to enrich oligomannose content augments vaccine-induced NAb responses. Together, these findings provide mechanistic insights and establish design principles for next-generation HA-based influenza vaccines targeting both seasonal and pandemic strains. ONE-SENTENCE SUMMARYRational HA design and nanoparticle display guide next-generation strategies for influenza vaccine development

microbiology↗

bioRxiv2025

Population density triggers wing dimorphism via NlNompC-mediated insulin signaling in Nilaparvata lugens

The Nilaparvata lugens employs wing dimorphism as a key adaptive strategy to environmental heterogeneity. While hormonal pathways and transcription factors governing nutrient-induced wing plasticity are known, how population density triggers developmental divergence remains unresolved. Here, we mechanistically dissect density-dependent wing morph determination, revealing tactile inter-individual contact as the primary signal. High-density conditions amplify gentle-touch frequency, which is detected by mechanosensitive ion channels NlNompC and NlNMDARs. Genetic silencing or pharmacological inhibition of these channels abolished density-induced long-winged morphogenesis. The conserved role of NlNompC in gentle-touch sensation was validated by rescuing tactile perception deficits in Drosophila nompC mutants. Crucially, NlNompC integrates tactile cues into insulin/insulin-like growth factor signaling (IIS): crowding-driven mechanical stimuli upregulate NlIlp3, while suppressing NlInR2, to activate wing elongation programs. Epistasis experiments confirmed IIS as the key downstream pathway, with co-silencing NlInR2 or NlFoxO rescuing the short-winged phenotype induced by NlNompC knockdown or low population density, silencing NlIlp3 or NlInR1 rescuing the long-winged phenotype induced by high population density. Our work uncovers a mechano-endocrine axis linking tactile perception to developmental plasticity, bridging ecological cues with molecular pathways. These findings redefine density sensing beyond chemical modalities and offer actionable targets for disrupting pest dispersal strategies, advancing sustainable agriculture.

molecular biology↗

bioRxiv2025

Multi-platform framework for mapping somatic retrotransposition in human tissues

Mobile element insertions (MEI) shape the human genome in both germline and somatic tissues. While inherited MEIs are well characterized, mapping somatic MEIs (sMEI) in non-cancer tissues remains challenging due to their low allelic fraction and repetitive nature. We established an integrative framework for sMEI analysis leveraging modern sequencing technologies and analytical innovations. We first benchmarked sMEI detection and demonstrated advantages of long-read and MEI-targeted sequencing for ultra-low-frequency events using a mixture of well-established cell lines. We then showed that haplotype phasing and donor-specific assemblies refine sMEI detection, effectively distinguishing from germline and false signals in in-silico tumor-normal mixtures. We further developed a source-tracing strategy based on internal sequence variation, expanding the catalogue of active source elements beyond traditional transduction-based methods. Applying this framework to donor tissues, we identified 18 rare somatic L1 insertions, revealing structural and source diversity. Our work provides a foundational framework and biological insight into sMEIs.

genomics↗

bioRxiv2025

Macrophages display spatiotemporal specificity in intrahepatic cholangiocarcinoma and drive tumour progression via OSM and THBS1

Tumor-associated macrophages (TAMs) are abundant in intrahepatic cholangiocarcinoma (ICC) and correlate with poor prognosis, but their non-immunosuppressive roles remain unclear. This study aimed to define TAM transcriptional heterogeneity and map their interactions with ICC cells that promote malignancy beyond immune suppression. Single-cell and spatial transcriptome analysis with mouse and human ICC tissues revealed a TAM subpopulation, designated M{varphi}_VEGFA, that emerged as the major macrophage population from the intermediate stage of ICC development onward. Data from Gene Expression Omnibus (GEO) and spatial transcriptome indicate that M{varphi}_VEGFA was associated with poor prognosis and spatially localized to the ICC cells. Cell co-culture and cell-cell communication analyses revealed that M{varphi}_VEGFA promoted ICC cell proliferation via secreted OSM and downstream OSMR-STAT3-CCND1 axis, and enhanced ICC cell invasion through THBS1 and downstream CD47-mTOR/DBN1 signaling. Critically, multiplex immunofluorescence with human ICC tissue microarray indicated that OSM and THBS1 were co-elevated in human ICCs, and dual blockade of both targets inhibited ICC progression in mouse models. This study uncovers the direct and critical roles of M{varphi}_VEGFA in driving ICC progression with paracrine signaling and further highlights the potential of targeting macrophages in ICC treatment.

cancer biology↗

bioRxiv2025

Development of a High-Throughput Screening Method for Anti-NNV Drugs

Nervous necrosis virus (NNV) is an infectious pathogen, characterized by rapid infection and high mortality, commonly encountered in the aquaculture industry. Therefore, there is an urgent need to develop effective antiviral drugs against NNV, which is crucial for targeted treatment strategies, pathogen control, and loss reduction. Screening of effective antiviral active compounds, namely drug precursors, is key to developing highly efficient drugs against NNV. Reverse transcription-quantitative real-time PCR (RT-qPCR) is widely applied to screen and evaluate various substances to prevent and control virus infection. However, RT-qPCR is a cumbersome procedure and not suitable for high-throughput rapid screening. The aptamer TNA1c, which specifically binds to NNV-infected cells, was used to construct a target-driven activatable aptamer probe (TAA). Then, the TAA probe was applied to establish a high-throughput screening (TAA-HTS) method for efficient evaluation of substances against NNV infection. TAA-HTS technology achieved rapid, sensitive, and specific screening of bioactive substances with significant anti-NNV effects. As compared to commonly used analytical methods, such as RT-qPCR, TAA-HTS has the advantages of easy operation and high sensitivity and specificity. The findings of this study provide data support and a theoretical basis for the development of effective antivirus preparations. IMPORTANCEIn this study, a target-driven activatable aptamer probe was employed to establish a high-throughput screening method for anti-NNV drugs. This method enables rapid, sensitive, and specific screening of anti-NNV compounds while reducing background interference. Although RT-qPCR is currently the most widely used technique for antiviral drug screening and validation, the screening approach developed in this study significantly shortens screening time while achieving results consistent with those of RT-qPCR. This demonstrates that the screening method presented here has the potential to become a universal strategy for future drug screening against viral diseases. Moreover, this study not only provides a novel technical solution for NNV control but also demonstrates the potential of aptamer-based probes in high-throughput antiviral screening, serving as a model for combating viral pathogens in aquaculture and other fields.

pathology↗

bioRxiv2025

miR155 triplicated in Down syndrome regulates hippocampal GABAergic neurogenesis in Alzheimer's disease

MicroRNA dysregulation is implicated in neurodegenerative disorders, including Alzheimers disease (AD). The role of neuronal microRNA155 (miR155), elevated in both AD and Down syndrome (DS), remains unknown. We found that MIR155HG (miR155 host gene) colocalizes with APP (amyloid-beta precursor protein) in a neuron-specific, topologically-associated domain (TAD) within a regulatory network, in the obligate portion of chromosome 21 triplicated in DS which causes AD neuropathology and in most cases, dementia. We investigated miR155 role during neuron development and then validated these findings in an amyloidopathy model. In human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs), cortical neurons and cortical organoids, MIR155 deletion enhanced NSC proliferation, ventral patterning and GABAergic interneuron generation. However, MIR155 overexpression inhibited NSC marker expression and GABAergic interneuron generation. MIR155 upregulates its mRNA targets, NR2F1/2, key modulators of hippocampal GABAergic interneuron development. In an amyloidopathy mouse model, miR155 deletion induced the expansion of hippocampal NSCs and increased hippocampal GABAergic interneurons. These findings reveal previously unrecognized miR155 roles in NSC dynamics and GABAergic interneuron development which directionally diverge from extensively studied microglial miR155 in their beneficial vs negative impact on AD mouse models, suggesting that approaching miR155 therapeutically may require balancing the effects in neurons and microglia.

neuroscience↗

bioRxiv2025

Hyperactivated Glycolysis Drives Spatially-Patterned Kupffer Cell Depletion in MASLD

Metabolic dysfunction-associated steatotic liver disease (MASLD) progression is characterized by hepatic inflammation and cell death, yet the mechanisms underlying Kupffer cells (KCs) loss remain poorly understood. Here, we sought to elucidate the metabolic basis of KCs death during MASLD. Using metabolomics, immunostaining, and flow cytometry, we evaluated metabolic alterations and KCs death throughout early MASLD progression. We found that KCs death is an early hallmark of MASLD, exhibiting greater susceptibility and displaying a spatial distribution consistent with KCs zonation. Moreoever, KCs undergo progressive metabolic reprogramming toward enhanced glucose utilization during MASLD development, which is correlated with KCs death. In combination of biochemical agonist, isotope tracing and primary KCs culture, we further demonstrated that augmented glycolytic metabolism directly drives KCs death in vitro. Consistently, using Chil1-deficient mice, we further demonstrated that increased glucose utilization accelerates KCs death in vivo. Together, these findings establish a causal link between glycolytic activation and KCs loss during MASLD progression, highlighting glucose metabolic pathways as potential therapeutic targets to preserve KCs homeostasis and mitigate MASLD.

pathology↗

bioRxiv2025

Clonal dynamics shaped by diverse drug-tolerant persister states in melanoma resistance

Most advanced melanomas initially respond to targeted therapy but eventually relapse. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells that enter a reversible drug-refractory state. We developed MeRLin, a high-resolution lineage tracing platform integrating cellular barcoding, single-cell transcriptomics, RNA fluorescence in situ hybridization (FISH), and computational analyses to track clonal and transcriptional dynamics in patient-derived melanoma models during prolonged therapy. Clonal dynamics revealed that persister subpopulations first responded to treatment but persisted and expanded during minimal residual disease, ultimately leading to tumor recurrence. Pre-treatment melanoma populations diversified into four conserved persister states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics showed the organization of these adaptive programs and a complex signaling network of autocrine and paracrine interactions among persister subpopulations. Barcoded RNA-FISH enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone with SLC2A1 expression. MeRLin provides a robust framework for dissecting cancer heterogeneity and identifying vulnerabilities in persister populations.

cancer biology↗

bioRxiv2025

Novel Function of Arylalkalamine N-acetyltransfe- rase-1 in Modulating Host Coagulation and Blood Feeding in Aedes aegypti

During blood feeding, mosquitoes introduce substantial quantities of salivary proteins into their hosts to facilitate feeding. Arylalkylamine N-acetyltransferase-1 (aaNAT1) is a salivary protein prominently produced in the salivary glands of Aedes aegypti. Previous research has demonstrated that mosquitoes with AaaaNAT1 mutations fail to efficiently inhibit host blood coagulation; however, the precise regulatory mechanisms remain unknown. This study identified the primary metabolites and proteins interacting with AaaaNAT1 through integrated transcriptomic, proteomic, and metabolomic analyses. Combined with isothermal titration calorimetry (ITC), immunohistochemistry, ELISA, platelet function assays, and coagulation tests, we further established that AaaaNAT1 binds norepinephrine, reducing host platelet aggregation and thereby limiting activation of the host coagulation cascade and fibrinolytic system. Additionally, behavioral investigations showed that AaaaNAT1 knockdown in Aedes aegypti significantly prolonged the time required for female mosquitoes to locate a host and successfully obtain blood, while also markedly reducing blood consumption. This effect may stem from the inability of aaNAT1-depleted mosquitoes to effectively suppress host blood aggregation during feeding, as well as the downregulation of octopamine following AaaaNAT1 knockdown. Therefore, this study reveals a novel role for aaNAT1 distinct from its known functions in pigmentation and immunity, advancing our understanding of aaNAT1s role in insects.

molecular biology↗

bioRxiv2025

Dorsal Attention Network as a Convergent Hub of Diverse Non-pharmacological Interventions Preserving Functional Gradients and Cognition in Aging

Non-pharmacological interventions (NPIs) in aging neuroscience have largely focused on intervention-specific regional effects, with limited understanding of generalizable network-level mechanisms. Here, adopting a previously unexplored gradient-based perspective of functional brain organization, we analyzed an NPI dataset involving four interventions in older adults (training/control group: n = 112/59). NPIs led to strengthened intra-network functional integration and maintained macroscale gradient architecture. Virtual lesion analyses identified the dorsal attention network (DAN) as a key contributor to gradient maintenance. Critically, enhanced post-intervention DAN connectivity was associated with maintained gradient structure and improved global cognition. These findings establish a unifying framework in which the DAN acts as a convergent hub through which diverse NPIs preserve functional brain organization and attenuate cognitive aging.

neuroscience↗

bioRxiv2025

Coiled-coil-mediated phase separation of Spef1 for non-centrosomal microtubule organization and function

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 non-centrosomal CP-MT organization and function in the axoneme. Significance statementThe MT-associated protein Spef1 is a new essential player for the non-centrosomal CP-MT formation in motile cilia and flagella. This study reveals the unexpected LLPS feature of Spef1, leading to forming biomolecular condensates that enrich tubulins to facilitate non-centrosomal MT assembly. Spef1-CC contains a unique charge-distribution pattern, together with its dimerization capacity, contributing to multivalent interactions for initiating LLPS. The LLPS property of Spef1 is important for CP-MT formation and Spef1-mediated ciliary function. The formation of Spef1-LLPS condensates indicates that they work as MT nucleation centers and tubulin sources for the continuous growth of CP-MTs or repairing CP-MTs damaged during ciliary beating, and suggests that LLPS may be a common process for generating and organizing non-centrosomal CP-MTs in the axoneme.

cell biology↗