Biology subjects

Liu, J.-L.

Publications and source records attributed to Liu, J.-L..

At least 19 recordsLinked to original sources

bioRxiv2025

Cytoskeleton Regulates Cytoophidium Dynamics in Drosophila Ovaries

Cytoophidia are filamentous structures composed of CTP synthase (CTPS) and were initially discovered in ovarian cells of Drosophila. As a highly conserved membraneless organelle present across all three domains of life, cytoophidia display dynamic behaviors that are crucial for cellular homeostasis and function. Previous research has shown that cytoophidia are actively transported from nurse cells to oocytes, implicating their potential role in Drosophila oogenesis. Nevertheless, the molecular and cellular mechanisms governing cytoophidium dynamics remain largely unclear. In this study, we employ live-cell imaging to systematically characterize the spatiotemporal dynamics of cytoophidia and to explore the underlying regulatory mechanisms. Our findings demonstrate that cytoophidium dynamics are dependent on the cytoskeletal components-- microtubules, microfilaments (actin filaments), and myosin II. Notably, disruption of either microtubules or microfilaments resulted in the disassembly or depolymerization of large cytoophidia (macro-cytoophidia), highlighting an essential role of the cytoskeleton in maintaining cytoophidium integrity and assembly. Together, these results indicate that microtubules, microfilaments, and myosin II play pivotal roles in regulating cytoophidium dynamics. This study provides new insights into the mechanisms underlying cytoophidium transport and assembly, and lays a foundation for further investigation of their functional significance in Drosophila oogenesis.

cell biology↗

bioRxiv2025

Superficial stromal cell population in the mouse uterus require METTL14 for development and functional competence to support embryo implantation

Uterine stromal cells are critical for pregnancy, coordinating embryo implantation, placental development, and maintenance of gestation. Single-cell RNA-seq analyses have identified two distinct stromal cell populations in the mouse uterus: superficial and deep stromal cells. However, their functional specialization remains unresolved. Here, we show that mice lacking methyltransferase-like 14 (METTL14), a core enzyme of the m6A methyltransferase complex, exhibit a selective depletion of superficial stromal cells while preserving deep stromal cells. This genetic model allowed us to investigate the physiological role of superficial stromal cells in uterine function. Paralleled by the aberrant development and loss of superficial stromal cells, Mettl14 deletion impaired embryo implantation and decidualization. Transcriptomic profiling revealed that Mettl14 deficiency disrupts hormonal signaling by upregulating estrogen (E2)-responsive genes and downregulating progesterone (P4)-responsive genes, resulting in aberrant development and loss of superficial stromal cells. Mechanistically, Mettl14 ablation reduces m6A methylation in the 3'-UTR of Esr1 mRNA, thereby impairing m6A-dependent mRNA degradation and elevating ESR1 expression, which amplifies E2 signaling. This study underscores the essential role of METTL14 in maintaining the superficial stromal cell population required for embryo implantation by regulating Esr1 mRNA stability through m6A modification.

physiology↗

bioRxiv2025

Programmed downregulation of METTL3 is essential for decidualization in both humans and mice

N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in regulating gene expression. Our prior research, alongside that of others, demonstrated that conditional uterine knockout of methyltransferase-like 3 (METTL3), the enzyme responsible for m6A modification, led to complete failure of embryo implantation and decidualization. Intriguingly, METTL3 expression is downregulated rather than upregulated in human endometrial stromal cells (HESCs) during in vitro decidualization and in mouse decidual tissues during pregnancy. We hypothesized that this decline in METTL3 expression is indispensable for successful decidualization. To test this hypothesis, we overexpressed METTL3 in HESCs and observed impaired decidualization in vitro. Additionally, we generated genetically engineered mice with uterine-specific METTL3 overexpression using Pgr-Cre, which exhibited subfertility mainly due to impaired decidualization. Further investigation revealed a marked decrease in HAND2, a well-established regulator of decidualization, following METTL3 overexpression. Mechanistically, we uncovered that METTL3 destabilizes HAND2 mRNA via m6A modification at the 5'-UTR. In summary, our study underscores the critical role of programmed METTL3 downregulation in decidualization by sustaining HAND2 expression.

physiology↗

bioRxiv2025

CTCF is crucial for decidualization of uterine stromal cells through facilitating HOXA11 expression

CCCTC-binding factor (CTCF), a highly versatile transcriptional regulator and chromatin architectural protein, plays a pivotal role in mammalian development. In this study, we investigated the function of CTCF in uterine biology during pregnancy. We observed that CTCF expression in the human endometrium decreases from the proliferative to the secretory phase of the menstrual cycle. Similarly, in cultured human endometrial stromal cells (hESCs), CTCF expression declines during in vitro decidualization. Using siRNA-mediated knockdown, we demonstrated that CTCF is essential for the early phase of decidualization but dispensable in later stages. Notably, CTCF depletion reduces the expression of HOXA11, a well-established regulator of decidualization, and overexpression of HOXA11 rescues the decidualization defects caused by CTCF loss, identifying HOXA11 as a key downstream effector of CTCF. Mechanistically, CTCF binds to the HOXA11 promoter to facilitate its transcriptional activation. Furthermore, uterine-specific knockout of CTCF in Pgr-Cre mice leads to female infertility, characterized by diminished HOXA11 expression, impaired uterine receptivity, and defective decidualization. Collectively, our findings highlight the critical role of the CTCF-HOXA11 axis in decidualization in both humans and mice, providing novel insights into the molecular regulation of this process and potential therapeutic targets for reproductive disorders.

physiology↗

bioRxiv2025

NAT10 governs uterine function and fertility by stabilizing progesterone receptor mRNA via ac4C modification

The N4-acetylcytidine (ac4C) modification is one of the most abundant chemical modifications in mammalian transcriptome, which plays a crucial role in regulating gene expression across various biological processes. In this study, we investigate the role of N4-acetyltransferase 10 (NAT10), the sole enzyme responsible for catalyzing the ac4C modification, in uterine function. Our findings revealed that knockdown of NAT10 in cultured human endometrial stromal cells leads to decreased progesterone receptor (PGR) expression and compromised decidualization. Mechanistically, NAT10 is found to enhance the stability of PGR mRNA through its ac4C modification in the coding sequence (CDS). Furthermore, conditional deletion of Nat10 in the mouse uterus using Pgr-Cre resulted in defective pubertal uterine development, characterized by a thinner stroma and reduced endometrial gland number. These mice were infertile in adulthood, experiencing failures in both implantation and decidualization. However, heterozygous Nat10 conditional knockout mice showed reduced NAT10 expression in the uterus without affecting uterine development or fertility. To bridge the gap between heterozygous and homozygous knockout conditions, we employed Remodelin, an inhibitor of NAT10, in adult female mice, to mimic an intermediate gene dosage. Our results demonstrated that inhibiting NAT10 with Remodelin preserved uterine structures but disrupted the PGR signaling pathway, leading to impaired uterine function during pregnancy. In conclusion, our study provides compelling evidence that NAT10 safeguards uterine function and fertility by regulating the ac4C modification of PGR mRNA. SignificanceHere we identified NAT10-catalyzed ac4C modification of progesterone receptor (PGR) mRNA as a crucial mechanism regulating PGR expression by enhancing its mRNA stability. In vitro studies using human endometrial stromal cells show that the NAT10-PGR axis is required for decidualization. Additionally, genetic and pharmacologic manipulations in mice further demonstrate the necessity of NAT10-dependent P4/PGR signaling for uterine function during implantation and decidualization. Our findings highlight the importance of NAT10-mediated ac4C modification in maintaining PGR expression and uterine function.

physiology↗

bioRxiv2025

FMR1 KH0-KH1 Domains Coordinate m6A Binding and Phase Separation in Fragile X Syndrome

The Fragile X Mental Retardation Protein (FMR1) regulates neurodevelopment via m6A RNA interactions, but domain-specific roles of KH0 and KH1 in RNA binding and pathology remain unclear. Through mutagenesis and AlphaFold3 modeling, we demonstrate that KH1 serves as the primary m6A-binding interface, whereas the KH0 domain (e.g., Arg138) modulates liquid-liquid phase separation (LLPS). Pathogenic mutations in KH0 disrupt RNA binding and enhance LLPS aggregation, while m6A RNA suppresses LLPS at KH0. Structural simulations reveal synergistic cooperation between KH0 and KH1 through hydrophobic and electrostatic networks. These dual-domain interactions establish a mechanistic link between m6A dysregulation, aberrant phase separation, and Fragile X syndrome pathogenesis. Our findings propose KH0 as a therapeutic target for RNA-driven neurodevelopmental disorders.

cell biology↗

bioRxiv2025

CTP Drives Human CTPS1 Filamentation

CTP synthase (CTPS) is a key enzyme in de novo CTP synthesis, playing a critical role in nucleotide metabolism and cellular proliferation. Human CTPS1 (hCTPS1), one of the two CTPS isoforms, is essential for immune responses and is highly expressed in proliferating cells, making it a promising therapeutic target for immune-related diseases and cancer. Despite its importance, the regulatory mechanisms governing hCTPS1 activity remain poorly understood. Here, we reveal that CTP, the product of CTPS, acts as a key regulator for hCTPS1 filamentation. Using cryo-electron microscopy (cryo-EM), we resolve the high-resolution structure of CTP-bound hCTPS1 filaments, uncovering the molecular details of CTP binding and its role in filament assembly. Importantly, we demonstrate that CTP generated from the enzymatic reaction does not trigger filament disassembly, suggesting a conserved regulatory pattern. Furthermore, by analyzing the binding modes of two distinct CTP-binding pockets, we provide evidence that this filamentation mechanism is evolutionarily conserved across species, particularly in eukaryotic CTPS. Our findings not only elucidate a novel regulatory mechanism of hCTPS1 activity but also deepen the understanding of how metabolic enzymes utilize filamentation as a conserved strategy for functional regulation. This study opens new avenues for targeting hCTPS1 in therapeutic interventions.

biochemistry↗

bioRxiv2025

IMPDH Cytoophidia in the Pancreas: Morphological Dynamics and Metabolic Implications

Cytoophidia are filamentous structures formed by metabolic enzymes, playing critical roles in cellular metabolism and regulation. Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo nucleotide synthesis, is known to form cytoophidia and is implicated in cell proliferation, nutrient sensing, and metabolic stress responses. Despite their significance, the behavior of IMPDH cytoophidia under different metabolic conditions, particularly in diabetes, remains poorly understood. This study investigates the distribution, morphology, and assembly patterns of IMPDH cytoophidia in the pancreas of normal and diabetic models. Using immunofluorescence staining, pharmacological interventions (e.g., mycophenolic acid), and glucose tolerance tests, we demonstrate that IMPDH cytoophidia exhibit distinct morphological changes in pancreatic islets under diabetic conditions. Notably, cytoophidia elongation is associated with impaired glucose tolerance, suggesting a potential link between cytoophidia dynamics and metabolic dysfunction. Our findings provide novel insights into the role of IMPDH cytoophidia in metabolic regulation and highlight their potential as therapeutic targets for diabetes and related metabolic disorders.

cell biology↗

bioRxiv2025

VitisC: Visualizing interested target via integrated scaffold of cytoophidium

Cryo-electron microscopy (cryo-EM) single-particle analysis has become a widely used technique for high-resolution structural determination of biological macromolecules and complexes. However, the determination of the structure of small molecule proteins remains a limitation of this technology. To address this issue, here we develop a novel approach termed Visualizing interested target via integrated scaffold of cytoophidium (VitisC). We use a filamentous structure formed by Escherichia coli CTP synthase (CTPS) as the scaffold, termed the scaffold of cytoophidium. Through artificial design and modification, small proteins can be attached to this symmetrical scaffold, which is very suitable for cryo-EM imaging. The formation conditions for stable filament structures of the fusion protein are optimized in vitro, and the three-dimensional structure of the fusion protein is reconstructed using cryo-EM single-particle analysis, achieving an overall resolution of 3.39 [A]. Therefore, cryo-EM is successfully applied to visualize small proteins. VitisC not only demonstrates the feasibility of employing cytoophidia as scaffolds, but also provides an approach for high-resolution structural analysis of small proteins using Cryo-EM.

biochemistry↗

bioRxiv2024

Filamentation and proline inhibition of glutamate kinase

Glutamate kinase (GK) is the first committed enzyme in the proline biosynthesis pathway. Belonging to amino acid kinase (AAK) superfamily, most prokaryotic GKs have an additional PseudoUridine synthase and Archaeosine transglycosylase (PUA) domain at the C-terminus, while the function of the PUA domain in GK is poorly understood. Here, we find that Escherichia coli GK (EcGK) assembles into filaments and bundles in the state of apo and proline binding. Using cryogenic electron microscopy, we determine the high-resolution structures of EcGK filaments and bundles. The PUA domain is necessary for EcGK filaments and bundles, and the main interfaces have been clearly defined. The feedback inhibitor proline binds at the same pocket as substrate glutamate, inducing conformational changes on nearby regulatory loop which facilitate proline binding. The PUA domain stabilizes the regulatory loop and contributes to proline feedback inhibition. This study reports the special filament-based assembly of EcGK at apo and proline binding state. The first proline binding structure in the GK family illustrates the feedback inhibition mechanism. Intriguingly, the PUA domain is involved in both filamentation and feedback inhibition of EcGK, revealing the versatility of this ancient domain.

biochemistry↗

bioRxiv2024

Cytoophidium complexes resonate with cell fates

Metabolism is a fundamental characteristic of life. In 2010, we discovered that the metabolic enzyme CTP synthase (CTPS) can assemble a snake like structure inside cells, which we call the cytoophidium. Including CTPS, an increasing number of metabolic enzymes have been found to form cytoophidia in cells. However, the distribution and relationship among cytoophidia formed by different metabolic enzymes remain elusive. Here we investigate five metabolic enzymes that can form cytoophidia, namely Asn1, Bna5, CTPS (ie. Ura7), Glt1, and Prs5 in Saccharomyces cerevisiae. We find that multiple cytoophidia can be assembled into cytoophidium complexes by docking one after another. Glt1 cytoophidia tend to assemble in non-quiescent cells, while CTPS cytoophidia are more abundant in quiescent cells and form complexes with Prs5 and Asn1 cytoophidia. Blocking CTPS cytoophidium assembly can lead to a non-quiescent phenotype and increase the assembly of Glt1 cytoophidia, Bna5 cytoophidia, and cytoophidium complexes. Blocking CTPS cytoophidium assembly also inhibits the NAD biosynthesis pathway, which includes Bna5 and Sir2. Consistent with this result, the non-quiescent phenotype caused by blocking CTPS cytoophidium assembly can be rescued by blocking Glt1 cytoophidium assembly, supplementing nicotinic acid, or overexpressing Sir2. Our results indicate that the assembly of cytoophidium complexes with different compositions resonates with distinct cell fates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/614056v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a297bcorg.highwire.dtl.DTLVardef@197c0aforg.highwire.dtl.DTLVardef@1eb46e8org.highwire.dtl.DTLVardef@17c2e6e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIMultiple cytoophidia can be assembled into cytoophidium complexes by docking one after another. C_LIO_LICTPS cytoophidia blockage impedes quiescence and boosts Glt1-Bna5 cytoophidium complex. C_LIO_LIGlt1 cytoophidium blockage restores quiescence and Bna5 inhibition in NAD pathway. C_LIO_LIActivating NAD utilizing SIR2 rescues CTPS cytoophidium blockage induced non-quiescence. C_LI

cell biology↗

bioRxiv2024

Differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe

The de novo synthesis of cytidine 5-triphosphate (CTP) is catalyzed by the enzyme CTP synthase (CTPS), which is known to form cytoophidia across all three domains of life. In this study, we use the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as model organisms to compare cytoophidium assembly under external environmental and intracellular CTPS alterations. We observe that under low and high temperature conditions, cytoophidia in fission yeast gradually disassembled, while cytoophidia in budding yeast remain unaffected. The effect of pH changes on cytoophidia maintenance in the two yeast species is different. When cultured in the yeast saturated cultured medium, cytoophidia in fission yeast disassemble, while cytoophidia in budding yeast gradually form. Overexpression of CTPS leads to the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demostrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.

cell biology↗

bioRxiv2024

Dynamic cytoophidia during Drosophila late oogenesis

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live cell imaging to capture the continuous dynamics of cytoophidia in Stages 10-12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Our observation provides a systematic understanding of the dynamics of cytoophidia during Drosophila late oogenesis.

developmental biology↗

bioRxiv2023

Connecting Hippo pathway and cytoophidia in Drosophila posterior follicle cells

CTP synthase (CTPS), the rate-limiting enzyme in de novo synthesis of CTP, assembles into filamentous structures termed cytoophidia. Here we study the impact of Hippo pathway on the posterior follicle cells (PFCs) in Drosophila egg chambers. We find that the inactivation of Hippo pathway correlates with a reduction in cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigates hpo (Hippo)-associated over-proliferation. In summary, our results suggest a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.

genetics↗

bioRxiv2023

Filamentation and inhibition of prokaryotic CTP synthase

CTP synthase (CTPS) plays a pivotal role in the de novo synthesis of CTP, a fundamental building block for RNA and DNA, which is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: ATP, UTP, CTP, and GTP. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by virus or prokaryotic pathogens. Utilizing cryo-electron microscopy, we have determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, NADH, and the covalent inhibitor DON, achieving a resolution of 2.8[A]. We construct a phylogenetic tree based on differences in filament-forming interfaces and design a variant to validate our hypothesis, providing an evolutionary perspective on the CTPS filament formation. Our computational analysis reveals a solvent-accessible ammonia tunnel upon DON binding. By comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS, differing from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determine and validate the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

biochemistry↗

bioRxiv2023

Cytoophidia influence cell cycle and size in Schizosaccharomyces pombe

CTP synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific point mutations, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutant. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

cell biology↗

bioRxiv2023

Dynamic atP5CS2 Filament Facilitates Substrate Channeling

In plants, the rapid accumulation of proline is a common response to combat abiotic stress. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalyzing the initial two-step conversion from glutamate to proline. Here, we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS2 (atP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of atP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveals that filament-based substrate channeling is essential for maintaining the high catalytic efficiency of atP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of P5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response. Therefore these findings provide potential avenues for crop genetically modified breeding.

biochemistry↗

bioRxiv2023

Structural basis for activation of glutaminase

Glutaminase is a rate-limiting enzyme in glutaminolysis, which produces glutamate from glutamine and enters the TCA cycle[1]. In addition, it plays a key role in redox homeostasis[2], autophagy[3], immune system regulation[4], central nervous system maintenance[5], and senolysis[6]. Therefore, the allosteric regulation of glutaminase is a fascinating topic that has broad implications for our understanding of glutamine metabolism and related diseases[7-9]. Phosphate was discovered as a natural agonist for glutaminase in 1947[10], but the structural basis and mechanism for this regulation remains unclear. Using cryo-electron microscopy, here we determine the structure of human glutaminase with phosphate. This structure allows us to capture phosphate binding at the dimer-dimer interface at near atomic resolution, revealing an allosteric activation mechanism by remodelling the catalytic pocket. Surprisingly, we find that phosphate antagonizes BPTES (a classical antagonist) and CB-839 (the current subject of several phase II clinical trials). Accurate identification of phosphate binding sites lays the foundation for the design of glutaminase agonists and antagonists with broad pharmaceutical significance.

biochemistry↗