The Cytoophidium and Its Kind: Filamentation and Compartmentation of Metabolic Enzymes.

Compartmentation is essential for the localization of biological processes within a cell. In 2010, three groups independently reported that cytidine triphosphate synthase (CTPS), a metabolic enzyme for de novo synthesis of the nucleotide CTP, is compartmentalized in cytoophidia (Greek for "cellular snakes") in bacteria, yeast, and fruit flies. Subsequent studies demonstrate that CTPS can also form filaments in human cells. Thus, the cytoophidium represents a new type of intracellular compartment that is strikingly conserved across prokaryotes and eukaryotes. Multiple lines of evidence have recently suggested that polymerization of metabolic enzymes such as CTPS and inosine monophosphate dehydrogenase into filamentous cytoophidia modulates enzymatic activity. With many more metabolic enzymes found to form the cytoophidium and its kind, compartmentation via filamentation may serve as a general mechanism for the regulation of metabolism.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

eCRUIS captures RNA-protein interaction in vitro and in vivo.

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From its synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interaction between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to rapidly label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications for studying RNA-protein interactions.

Architecture of CTPS filament networks revealed by cryo-electron tomography.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments.

The IMPDH cytoophidium couples metabolism and fetal development in mice.

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.

Motor defects in a Drosophila model for spinal muscular atrophy result from SMN depletion during early neurogenesis.

Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disease, and is characterised by spinal motor neuron loss, impaired motor function and, often, premature death. Mutations and deletions in the widely expressed survival motor neuron 1 (SMN1) gene cause SMA; however, the mechanisms underlying the selectivity of motor neuron degeneration are not well understood. Although SMA is degenerative in nature, SMN function during embryonic and early postnatal development appears to be essential for motor neuron survival in animal models and humans. Notwithstanding, how developmental defects contribute to the subversion of postnatal and adult motor function remains elusive. Here, in a Drosophila SMA model, we show that neurodevelopmental defects precede gross locomotor dysfunction in larvae. Furthermore, to specifically address the relevance of SMN during neurogenesis and in neurogenic cell types, we show that SMN knockdown using neuroblast-specific and pan-neuronal drivers, but not differentiated neuron or glial cell drivers, impairs adult motor function. Using targeted knockdown, we further restricted SMN manipulation in neuroblasts to a defined time window. Our aim was to express specifically in the neuronal progenitor cell types that have not formed synapses, and thus a time that precedes neuromuscular junction formation and maturation. By restoring SMN levels in these distinct neuronal population, we partially rescue the larval locomotor defects of Smn mutants. Finally, combinatorial SMN knockdown in immature and mature neurons synergistically enhances the locomotor and survival phenotypes. Our in-vivo study is the first to directly rescue the motor defects of an SMA model by expressing Smn in an identifiable population of Drosophila neuroblasts and developing neurons, highlighting that neuronal sensitivity to SMN loss may arise before synapse establishment and nerve cell maturation.

P300 increases CSNK2A1 expression which accelerates colorectal cancer progression through activation of the PI3K-AKT-mTOR axis.

Casein kinase 2 alpha 1 (CSNK2A1) is a known oncogene, but its role in the progression of colorectal cancer (CRC) remain undefined. Here, we investigated the effects of CSNK2A1 during CRC development. In the current study, CSNK2A1 expression in the colorectal cancer cell lines (HCT116, SW480, HT29, SW620 and Lovo) vs. normal colorectal cell line (CCD841 CoN) were compared via RT-qPCR and western blotting. The role of CSNK2A1 on CRC growth and metastases were investigated through Transwell assay. Immunofluorescence analysis was used to investigate the expression of EMT-related proteins. The association between P300/H3K27ac and CSNK2A1 were analyzed using UCSC bioinformatics and Chromatin-immunoprecipitation (Ch-IP) assays. Results revealed that both the mRNA and protein levels of CSNK2A1 in HCT116, SW480, HT29, SW620 and Lovo cells were upregulated. Additionally, P300-mediated H3K27ac activation at the CSNK2A1 promoter was found to drive the increase in CSNK2A1 expression. Transwell assay showed that CSNK2A1 overexpression increased the migration and invasion of HCT116 and SW480 cells, which decreased following CSNK2A1 silencing. CSNK2A1 was also found to facilitate EMT in HCT116 cells, evidenced by the increases of N-cadherin, Snail and Vimentin expression, and loss of E-cadherin. Importantly, the levels of p-AKT-S473/AKT, p-AKT-T308/AKT, and p-mTOR/mTOR in cells overexpressing CSNK2A1 were high, but significantly decreased following CSNK2A silencing. The PI3K inhibitor BAY-806946 could reverse the increase in p-AKT-S473/AKT, p-AKT-T308/AKT, p-mTOR/mTOR induced by CSNK2A1 overexpression and suppress CRC cell migration and invasion. In conclusion, we report a positive feedback mechanism through which P300 enhances CSNK2A1 expression and accelerates CRC progression through the activation of the PI3K-AKT-mTOR axis.

Cytoophidia safeguard binucleation of Drosophila male accessory gland cells.

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. Here we observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, we find that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, our data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.

Structural basis for ligand binding modes of CTP synthase.

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

The Impact of Developmental and Metabolic Cues on Cytoophidium Formation.

The cytoophidium, composed mainly of CTP synthase (CTPS), is a newly discovered dynamic filamentous structure in various organisms such as archaea, bacteria, and humans. These filamentous structures represent a fascinating example of intracellular compartmentation and dynamic regulation of metabolic enzymes. Currently, cytoophidia have been proven to be tightly regulated and highly dynamic, responding rapidly to developmental and metabolic cues and playing a critical role in maintaining cellular homeostasis. In this review, we would like to discuss in detail the characteristics, mechanisms, functions, and potential applications of this conservative but promising organelle.

Dynamic Cytoophidia during Late-Stage Drosophila 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. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis.

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 disassemble, 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 results in the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demonstrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.

Cytoophidia Influence Cell Cycle and Size in Schizosaccharomyces pombe.

Cytidine triphosphate 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 mutation, 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 histone chaperone slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutants. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

Connecting Hippo Pathway and Cytoophidia in Drosophila Posterior Follicle Cells.

CTP synthase (CTPS), the rate-limiting enzyme in the de novo synthesis of CTP, assembles into a filamentous structure termed the cytoophidium. The Hippo pathway regulates cell proliferation and apoptosis. The relationship of the nucleotide metabolism with the Hippo pathway is little known. Here, we study the impact of the Hippo pathway on the cytoophidium in Drosophila melanogaster posterior follicle cells (PFCs). We find that the inactivation of the Hippo pathway correlates with reduced 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 that there is a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.

Cytoophidia and filaments: you must unlearn what you have learned.

The nucleotide CTP can be synthesized de novo from UTP via the metabolic enzyme CTP synthase (CTPS). As a textbook enzyme, CTPS has been extensively studied for seven decades. However, it came as a surprise when CTPS was found to form snake-shaped mesoscale cytoophidia in fruit fly cells. Since 2010, more and more studies have demonstrated that CTPS can form cytoophidia within the cells across all three domains of life. Oligomers of CTPS form filaments that are undetectable under light microscopy. This review summarizes our current understanding of cytoophidia and filaments, highlighting some basic features such as conservation, morphology and functions of the two levels of CTPS structures.

CTP synthase does not form cytoophidia in Drosophila interfollicular stalks.

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of the nucleotide CTP. In 2010, CTPS has been found to form filamentous structures termed cytoophidia in Drosophila follicle cells and germline cells. Subsequently, cytoophidia have been reported in many species across three domains of life: bacteria, eukaryotes and archaea. Forming cytoophidia appears to be a highly conserved and ancient property of CTPS. To our surprise, here we find that polar cells and stalk cells, two specialized types of cells composing Drosophila interfollicular stalks, do not possess obvious cytoophidia. We show that Myc level is low in these two types of cells. Treatment with a glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), increases cytoophidium assembly in main follicle cells, but not in polar cells or stalk cells. Moreover, overexpressing Myc induces cytoophidium formation in stalk cells. When CTPS is overexpressed, cytoophidia can be observed both in stalk cells and polar cells. Our findings provide an interesting paradigm for the in vivo study of cytoophidium assembly and disassembly among different populations of follicle cells.

Connecting Ras and CTP synthase in Drosophila.

CTP synthase (CTPS), the enzyme responsible for the last step of de novo synthesis of CTP, forms filamentous structures termed cytoophidia in all three domains of life. Here we report that oncogenic Ras regulates cytoophidium formation in Drosophila intestines. Overexpressing active Ras induces elongate and abundant cytoophidia in intestinal stem cells (ISCs) and enteroblasts (EBs). Knocking-down CTPS in ISCs/EBs suppresses the over- proliferation phenotype induced by ectopic expression of active Ras. Moreover, disrupting cytoophidium formation increases the number of proliferating cells in the background of overexpressing active Ras. Therefore, our results demonstrate a link between Ras and CTPS.

Ubiquitination regulates cytoophidium assembly in Schizosaccharomyces pombe.

CTP synthase (CTPS), a metabolic enzyme responsible for the de novo synthesis of CTP, can form filamentous structures termed cytoophidia, which are evolutionarily conserved from bacteria to humans. Here we used Schizosaccharomyces pombe to study the cytoophidium assembly regulation by ubiquitination. We tested the CTP synthase's capacity to be post-translationally modified by ubiquitin or be affected by the ubiquitination state of the cell and showed that ubiquitination is important for the maintenance of the CTPS filamentous structure in fission yeast. We have identified proteins which are in complex with CTPS, including specific ubiquitination regulators which significantly affect CTPS filamentation, and mapped probable ubiquitination targets on CTPS. Furthermore, we discovered that a cohort of deubiquitinating enzymes is important for the regulation of cytoophidium's filamentous morphology. Our study provides a framework for the analysis of the effects that ubiquitination and deubiquitination have on the formation of cytoophidia.

Molecular crowding facilitates bundling of IMPDH polymers and cytoophidium formation.

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.

Cytoophidia coupling adipose architecture and metabolism.

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IV collagen (Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. Our results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.