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.

Cilia, despite sharing a conserved axonemal structure, display diverse structural and molecular features, particularly at their tips. This study uncovers a mechanism that shapes the molecular features of fly mechanosensory cilia tips through localized active transport mediated by Kif19A, a kinesin-8 family member. Kif19A is predominantly expressed in peripheral neurons and is essential for enriching mechanosensory molecules at the ciliary tip, though it does not affect overall ciliary structure. In vitro studies show that Kif19A motors bind transiently to microtubules and exhibit non-processive movement individually, while multiple motors work together to drive plus end-directed movement of the cargoes, essential for precise localization and function of Kif19A in vivo. Building on the experimental observations, we propose a theoretical model in which nanoscale molecular polarity is established by local transport and binding sites that counteract rapid diffusion. This model highlights key principles by which molecular motors shape nanoscale cellular structures, thereby contributing to the diversity of cell architecture.

Polarimetric synthetic aperture radar (PolSAR) semantic segmentation remains challenging due to large intra‑class scattering variability, high inter‑class scattering similarity, and boundary ambiguity caused by speckle. Real‑valued networks discard polarimetric phase information, while existing complex‑valued methods often directly extend real‑valued architectures without coordinated design. To address these issues, this paper proposes Complex‑Valued HRU‑Net, a lightweight encoder‑decoder network. A three‑stage complex‑valued HRNet encoder preserves fine spatial details through parallel multi‑resolution streams, and a complex‑valued U‑Net decoder progressively restores high‑resolution semantic feature maps. At the second skip connection, a Complex‑Valued Cross‑Gated Attention Enhancement (CV‑CGAE) module suppresses speckle‑contaminated responses and selectively transfers boundary‑ and texture‑related cues via sequential enhancement, cross‑attention interaction, and gated recalibration. Experiments on three airborne and spaceborne PolSAR datasets—AIRSAR San Francisco, AIRSAR Flevoland, and RADARSAT‑2 San Francisco—show that the proposed method achieves mean intersection‑over‑union (mIoU) values of 97.21%, 98.25%, and 96.38%, respectively. In the 15‑class Flevoland scene, all per‑class IoU values exceed 94%. Parameter‑fair comparisons and phase randomization experiments confirm that the performance gains stem from explicit use of phase information, not merely increased model capacity. The network provides improved boundary delineation and post‑hoc physical interpretability for complex‑valued PolSAR segmentation.

Mitochondrial calcium fluxes serve as pivotal regulators of optimal organellar function and cellular viability, yet the spatiotemporal regulation of nanodomain Ca2+ transients at mitochondria-ER contact sites (MERCS) and their integration into adaptive mitochondrial stress signaling remain unresolved. In this study, we employed custom-built high temporal-spatial resolution GI/3D-SIM imaging techniques to achieve nanoscale resolution of calcium transients. We identify that MERCS-localized calcium oscillations gate retrograde stress signaling. Mechanistically, we demonstrate that augmented mitochondria-associated ER membrane (MAMs) connectivity unexpectedly attenuated global mitochondrial Ca2+ efflux, which triggering ATF5 shuttling-mediated transcriptional licensing and calcium-sensitive epigenetic reprogramming that synergistically activating stress-resilience programs. Quantitative protein expression and transcriptome analyses confirm that CsA-mediated calcium retention mimics MAMs induction preserves mitochondrial integrity and protecting cells from apoptosis in Aβ1-42-challenged neurons through synchronized UPRmt activation. Our findings reveal a novel mechanism by which MERCS decode proteotoxic stress into transcriptional and epigenetic adaptations, offering therapeutic potential for neurodegenerative diseases.

Phosphatidylinositol-4-phosphate (PI4P) is a low-abundance membrane lipid that plays crucial roles in lipid exchange and homeostasis, signal transduction, and vesicle trafficking. PI4KA, a type III phosphatidylinositol 4-kinase, catalyzes PI4P synthesis at the plasma membrane (PM). However, the mechanism by which cytoplasmic PI4KA is recruited to the PM to regulate PI4P levels in response to extracellular and intracellular signals remains unclear. We found that PI4KA is tightly associated with membranous organelles, including the endoplasmic reticulum (ER). In response to Ca2+, the membrane tethering protein extended synaptotagmin 1 (E-Syt1) recruits ER-localized PI4KA to ER-PM junctions, facilitating its PM recruitment and the assembly of the enzyme complex. In hippocampal neurons undergoing synaptic potentiation, neuronal activity-induced PM localization of PI4KA and PM PI4P synthesis also rely on E-Syt1 function. Thus, E-Syt1-mediated PI4KA localization to ER-PM junctions serves as a critical mechanism by which Ca2+ signaling regulates lipid metabolism at the PM.

In eukaryotic cells, organelles communicate through membrane contact sites - specialized regions where their membranes come into close apposition without fusing. Among these, contacts between the endoplasmic reticulum (ER) and the Golgi are crucial for lipid trafficking and polarized sorting of protein cargoes, yet their regulation and physiological roles remain poorly understood due to limited research tools. Here, we developed genetically encoded biosensors that selectively label ER-Golgi contact sites by building upon split GFP/YFP systems. These fluorescent probes reliably detect ER-Golgi contacts whose formation depends on Golgi-enriched phosphatidylinositol 4-phosphate and the lipid transfer activity of oxysterol-binding protein, and reveal the dynamic remodeling of these structures in live cells. Notably, the biosensors captured alterations in ER-Golgi contacts during cell division and ER stress, as well as their developmental loss in mammalian neurons. We propose these biosensors as powerful tools for investigating ER-Golgi interactions in response to physiological cues or pathological perturbations across diverse cell types.

Interpreting missense variants in highly conserved, paralog-rich gene families remains a major barrier to understanding protein function and advancing precision medicine. Here, we combine comprehensive mutagenesis, high-content live-cell imaging, and automated quantitative analysis to generate a complete functional atlas of all 2,683 single-nucleotide coding variants in human α-tubulin TUBA1A. Systematic profiling of microtubule assembly revealed distinct classes of mutations that disrupt folding, chaperone engagement, and protofilament geometry, defining structural constraints that govern tubulin function. This approach complements conservation-based predictors and enables functional reinterpretation of disease-associated variants. Molecular dynamics simulations further revealed how local perturbations in GTP (guanosine triphosphate) binding, dimer contacts, and lateral interfaces propagate to alter filament architecture. Integrating these datasets produced a predictive framework that generalizes across tubulin isotypes and species, enabling accurate inference of variant effects. This work establishes a scalable, high-resolution strategy for functional annotation of conserved proteins and provides mechanistic insight into cytoskeletal assembly and flexibility.

Abstract Deoxyguanosine triphosphatases (dGTPases) are nucleotide-depleting enzymes known to play a role in antiviral defense. While their enzymatic mechanism is established, the structural and functional diversity of dGTPases remains poorly understood. Here, we report a systematic analysis of dGTPase homologs across bacteria, archaea, and eukaryotes, revealing their widespread distribution and association with diverse immune-related domains. Through integrative bioinformatics and structural mining, we identify a class of bacterial dGTPases that assemble into stable octameric and higher-order oligomeric structures. Using cryo-electron microscopy, we resolve the octameric and 16-mer assemblies of a representative Vibrio dGTPase (Vdg) and further captured filamentous forms. Functional assays demonstrate that octamer formation is essential and sufficient for antiviral activity, while higher-order assemblies are dispensable. We also identify dAMP as an allosteric regulator, underscoring the functional versatility of dGTPases. Our findings provide insights into the modular architecture, oligomerization-driven activation, and immune function of bacterial dGTPases, and broaden our understanding of nucleotide depletion-based antiviral strategies.

The innate immune sensor AIM2 detects cytosolic DNA and initiates inflammatory responses, yet its activation mechanism remains incompletely understood. Here, we show that AIM2 undergoes liquid-liquid phase separation upon DNA binding, forming dynamic condensates both in vitro and in cells. These condensates serve as platforms for inflammasome and PANoptosome assembly, promoting immune activation across multiple pathways. Direct structural determination from condensates reveals the assembly of active-form ASC filaments. Mechanistically, liquid-phase condensation is governed by multivalent interactions involving different AIM2 domains, including previously uncharacterized regions and species-specific elements. In vitro and in vivo assays show that mutants specifically disrupting condensation impair immune complex assembly, cell death initiation, antimicrobial defense, and intestinal homeostasis. Moreover, AIM2-DNA condensates function as regulatory hubs targeted by host- and pathogen-derived factors to balance immune homeostasis or facilitate immune evasion. These findings establish liquid-phase condensation as a fundamental mechanism of AIM2 activation and a potential therapeutic target.

Abstract Cancer epidemiology shows consistent sex dimorphism across many non reproductive cancers: males typically have earlier onset, higher incidence, and worse outcomes. While these differences have been partly attributed to differential exposure to risk factors, they persist after adjustment, implicating intrinsic biological contributors. Changes in cancer behavior around the menopausal transition further implicate estrogenic environments in modulating core processes such as cell division and tumor survival. Here, we examine effects of high concentrations of estrogen on proliferation in multiple non reproductive cancer types, including bladder cancer. We find that estrogen induces tumor cell death through a mechanism independent of classical estrogen receptors (ERα, ERβ, and GPER). To identify mediators of this cytotoxicity, we conducted an unbiased whole genome CRISPR knockout screen. Notably, the ten most significant genes—KIFC1, TPX2, LIN37, KIF4A, KIF18B, KIF2C, WDR62, CLASP1, CLIP1, and VPS37C—are all required for spindle assembly and function. These results implicate spindle assembly as a key determinant of cellular sensitivity to estrogen. Kinetic studies showed that estrogen inhibits both microtubule polymerization and depolymerization. Biochemical analysis identifies estrogen binds αβ-tubulin dimers and acts as a negative catalyst of microtubule dynamics, impairing spindle assembly, disrupting mitosis, and triggering cell cycle-associated death pathways. Consistent with this model, overexpression of the microtubule polymerase Ch-TOG attenuates estrogen’s effects. Together, these data suggest that elevated estrogen levels can compromise spindle assembly and promote tumor cell death, providing a potential mechanism by which reproductive age females may be relatively protected from many non reproductive cancers and offering insight into the sex disparities observed in epidemiological studies. Citation Format: Pu Liang, Song Zeng, Wei Chen, Dong Li, Wei Feng, Xiaopeng Hu, Wensheng Wei, Xin Liang, Xi Wang. Estrogen induces cell death in non- reproductive cancer cells through disrupting mitotic spindle assembly [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4677.

Membrane remodeling is essential for numerous cellular functions. Although liquid-liquid phase separation (LLPS) of intrinsically disordered region (IDR)-rich proteins could drive dramatic membrane remodeling of artificial giant unilamellar vesicles, it remains elusive whether LLPS-mediated membrane-remodeling functions in live cells and what role it plays in specific bioprocesses. Here, we show that three IDR-rich integral transmembrane fusion proteins (MFPs), generated by chromosomal translocations, can lead to de novo remodeling of their located membranous organelles. Taking FUS-CREB3L2, prevalent in low-grade fibromyxoid sarcoma (LGFMS), as a proof of concept, we recorded super-resolution long-time imaging of endoplasmic reticulum (ER) remodeling dynamics as accumulating FUS-CREB3L2, meanwhile causing spontaneous ER stress to hijack the X-box-binding protein 1 (XBP1) pathway. We further reveal the underlying mechanisms of how FUS-CREB3L2 transduces its tumorigenic signals and aberrant LLPS effects from the ER membrane into the nucleus autonomously, which activates hundreds of LGFMS-specific genes de novo compared with CREB3L2, thus sufficiently reprogramming the cells into an LGFMS-like status.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism of piRNA-guided heterochromatin formation and its relationship to transcriptional termination remains elusive. Through RNA interference screening, we discovered Pcf11 and PNUTS as essential for piRNA-guided silencing in Drosophila germ line. Enforced tethering of Pcf11 leads to co-transcriptional repression and RNA polymerase II (RNA Pol II) stalling, and both are dependent on an cc-helical region of Pcf11 capable of forming condensates. An intrinsically disordered region can substitute for the cc-helical region of Pcf11 in its silencing capacity and support animal development, arguing for a causal relationship between phase separation and Pcf11's function. Pcf11 stalls RNA Pol II by preferentially forming condensates with the unphosphorylated Spt5, promoted by the PP1/PNUTS phosphatase during termination. We propose that Pcf11/Spt5 condensates control termination by decelerating polymerase elongation, a property exploited by piRNAs to silence transposons and initiate RNA-mediated heterochromatin formation.

Cell migration plays a central role in a wide range of physiological, developmental, and disease-related processes. Studies using single-cell models, such as Dictyostelium discoideum, have provided important insights into the molecular principles underlying this process. Migrating cells exhibit a polarized morphology, with actin-rich protrusions at the leading edge driving forward motion and an actomyosin network at the trailing edge enabling retraction. While actin polymerization and direct cytoskeletal regulators are essential, a complex network of signaling molecules also play a critical role in cell migration. Initially viewed as part of the directional sensing machinery in guided migration, this signaling network is now also recognized as an integral component of the motility module itself. Its spontaneous activity coordinates with cytoskeletal reorganization, enabling cell migration even in the absence of external cues. This review highlights key cytoskeletal and signaling molecules involved in leading-edge protrusion formation, with an emphasis on findings from Dictyostelium studies. We also discuss recent advances in understanding how these cytoskeletal and signaling molecules organize into excitable networks to regulate cell motility.

Cells dynamically adapt their migration modes to environmental conditions, but their response to sticky surfaces, where they risk becoming immobilized, remains unclear. In our study, we discovered that strong adhesion prompts substantial changes in Dictyostelium discoideum, leading to "inchworm migration," a novel subtype of amoeboid migration. This adaptation involves minimal contact between the cell and the surface, with the cell body standing upright and twisting, followed by rapid reattachment for directed movement. Concurrently, the cells shed migrasomes loaded with negatively charged molecules and adhesion receptors, controlling their adhesion traits to resume pseudopod migration. We identify the repurposing of cytokinesis machinery for migration mode shifting and selective membrane shedding as a crucial mechanism. Neutrophils also exhibit inchworm migration under strong adhesion, suggesting its broader application among amoeboid cells in adapting to high-adhesion environments. Our findings illuminate a programmed, adaptive response in amoeboid cells to navigate effectively through strongly adhesive terrains.

Lattice light-sheet microscopy provides a crucial observation window into intra- and intercellular physiology of living specimens but at the diffraction-limited resolution or anisotropic super-resolution with structured illumination. Here we present meta-learning-empowered reflective lattice light-sheet virtual structured illumination microscopy (Meta-rLLS-VSIM), which upgrades lattice light-sheet microscopy to a near-isotropic super resolution of ~120 nm laterally and ~160 nm axially without modifications of the core optical system or loss of other live-cell imaging metrics. Moreover, we devised an adaptive online training approach by synergizing the front-end imaging system and back-end meta-learning framework, which alleviated the demand for training data by tenfold and reduced the total time for data acquisition and model training down to tens of seconds. We demonstrate the versatile functionalities of Meta-rLLS-VSIM by imaging a variety of bioprocesses with ultrahigh spatiotemporal resolution for hundreds of multicolor volumes, delineating the nanoscale distributions, dynamics and interaction patterns of multiple organelles in embryos and eukaryotic cells.

Cyclic-oligonucleotide-based antiphage signaling systems (CBASS), a widespread antiviral bacterial immune system homologous to the mammalian cGAS-STING pathway, synthesizes cyclic nucleotide signals and triggers effector proteins to induce cell death and prevent viral propagation. Among various CBASS effectors, phospholipase effectors are the first to be discovered and are one of the most widespread families that sense cyclic dinucleotides to degrade cell membrane phospholipids. Here, we report that CBASS phospholipases assemble from a dimeric inactive state into active higher-order filamentous oligomers upon sensing cyclic dinucleotides. Using a combined approach of cryo-electron microscopy and X-ray crystallography, we have determined the structures of CBASS phospholipase in the inactive dimeric state, the cyclic-dinucleotide-bound active higher-order state, and the substrate-analog-bound catalytic mimicry state, thereby visualizing the complete conformational reorganization process. Complemented by functional assays of intermolecular binding, phospholipase enzymatic activity, in vitro membrane disruption, and in vivo antiphage efficiency, our work elucidates the mechanisms of assembly and activation of CBASS phospholipases.

Deep-learning-based structured illumination microscopy (SIM) has demonstrated substantial potential in long-term super-resolution imaging of biostructures, enabling the study of subcellular dynamics and interactions in live cells. However, the acquisition of ground-truth (GT) data for training poses inherent challenges, limiting its universal applicability. Current approaches without using GT training data compromise reconstruction fidelity and resolution, and the lack of physical priors in end-to-end networks further limits these qualities. Here we developed self-supervised reconstruction (SSR)-SIM by combining statistical analysis of reconstruction artifacts with structured light modulation priors to eliminate the need for GT and improve reconstruction precision. We validated SSR-SIM on common biological datasets and demonstrated that SSR-SIM enabled long-term recording of dynamic events, including cytoskeletal remodeling in cell adhesion, mitochondrial cristae remodeling, interactions between viral glycoprotein and endoplasmic reticulum, endocytic recycling of transferrin receptors, vaccinia-virus-induced actin comet remodeling, and mitochondrial intercellular transfer through tunneling nanotubes.

The Endoplasmic/sarcoplasmic reticulum (ER/SR) is central to calcium (Ca2+) signaling, yet current genetically encoded Ca2+ indicators (GECIs) cannot detect elementary Ca2+ release events from ER/SR, particularly in muscle cells. Here, we report NEMOer, a set of organellar GECIs, to efficiently capture ER Ca2+ dynamics with increased sensitivity and responsiveness. NEMOer indicators exhibit dynamic ranges an order of magnitude larger than G-CEPIA1er, enabling 2.7-fold more sensitive detection of Ca2+ transients in both non-excitable and excitable cells. The ratiometric version further allows super-resolution monitoring of local ER Ca2+ homeostasis and dynamics. Notably, NEMOer-f enabled the inaugural detection of Ca2+ blinks, elementary Ca2+ releasing signals from the SR of cardiomyocytes, as well as in vivo spontaneous SR Ca2+ releases in zebrafish. In summary, the highly dynamic NEMOer sensors expand the repertoire of organellar Ca2+ sensors that allow real-time monitoring of intricate Ca2+ dynamics and homeostasis in live cells with high spatiotemporal resolution.

Obesity is a global epidemic that threatens public health. Castration promotes the deposition of subcutaneous fat. However, the underlying mechanism remains unclear. The gut microbiota and their associated metabolites may regulate castration-induced subcutaneous fat deposition. In this study, we found surgical castration significantly increased subcutaneous fat deposition, adipocyte size, and fatty acid abundance in mice. Castration affected the β diversity of cecal bacteria and changed the interaction between bacteria and fungi. Castration enhanced cecal glycerolipid metabolism, which was significantly positively correlated with clavispora, Galactomyces, Ligilactobacillus, Adlercreutzia, Anaerovorax, Christensenella, and the Prevotellaceae NK3B3l group. Castration enhanced the abundance of glycerol, sn-glycerol 3-phosphate, dihydroxyacetone phosphate, and lipoteichoic acid in the serum, which influenced the expression of Gpat4, Lpin1, Gpat3, Gpam, Akr1b10, Agpat1, and Akr1a1 in the subcutaneous fat. These genes were involved in glycerolipid metabolism and the regulation of lipid droplet formation. Furthermore, they showed a significant positive correlation with subcutaneous fat weight. Gene set enrichment analysis confirmed that castration enhanced lipid droplet storage and fatty acid synthesis in the subcutaneous fat. These results confirm that glycerolipid metabolism regulates subcutaneous fat deposition in mice after castration from the gut-serum-subcutaneous fat axis.