Glutamine analogs promote cytoophidium assembly in human and Drosophila cells

CTP synthase is compartmentalized within a subcellular structure,termed the cytoophidium,in a range of organisms including bacteria, yeast,fruit fly and rat.Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells.Surprisingly,the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-L-norleucine（DON）,an inhibitor of glutamine-dependent enzymes including CTP synthase.Experiments in flies confirmed that DON globally promotes cytoophidium assembly.Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia.Moreover,DON facilitates cytoophidium assembly even when CTP synthase level is low.A second glutamine analog azaserine also promotes cytoophidum formation.Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.

Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe

The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.

mTOR-S6K1 pathway mediates cytoophidium assembly

CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin(mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1(mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6 K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6 K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing m TOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells.Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.

川楝素对人胃癌细胞MKN-45中CTPS细胞蛇形成的影响及其机制

目的:本研究旨在对川楝素(TSN)与胃癌细胞MKN-45中CTPS细胞蛇形成的联系进行初步探究。方法:以人胃癌细胞MKN-45为实验材料,设置7个处理组分别为:0、20、40、60、80、100、120 nmol/L TSN。每组3次重复,分别作用24 h、48 h、72 h,利用CCK-8法检测川楝素对MKN-45细胞增殖抑制作用,使用免疫荧光检测之后再通过激光共聚焦显微镜观察细胞内CTPS细胞蛇形态,qRT-PCR检测川楝素对MYC基因表达的影响。另外设置2个处理组为1 mmol/L DON和1 mmol/L MPA,每组3次重复,作用6 h然后采用免疫荧光检测细胞蛇形态。结果:免疫荧光结果显示,分别利用1 mmol/L DON和1 mmol/L MPA处理MKN-45细胞后CTPS形成丝状的细胞蛇结构,意味着该细胞具有形成CTPS细胞蛇的能力;川楝素处理组的细胞增殖率明显低于0 nmol/L TSN组(P<0.01);免疫荧光结果显示80 nmol/L的川楝素作用72 h时MKN-45细胞中CTPS细胞蛇形成数量最多;qRT-PCR检测结果显示,80 nmol/L川楝素作用24 h细胞内MYC表达明显降低(P<0.05),48 h后细胞内MYC的表达量明显增多(P<0.01)随后表达降低。结论:川楝素可能通过调节MYC的表达影响细胞内细胞蛇的组装。

细胞蛇的研究进展

细胞蛇是近年来细胞生物学研究的热门方向之一,由于其在细胞的增殖、代谢和发育上具有一定的生物学功能,因此,对一些疾病如癌症等的临床诊断或治疗具有一定的指导意义。细胞蛇是由三磷酸胞苷合成酶(cytidine triphosphate synthetase, CTPS)聚合而成的无膜细胞器,其形成过程及功能在不同类型的细胞中不尽相同。例如:细胞蛇能促进癌细胞增殖,并使患者病情恶化;过表达的细胞蛇可抑制神经干细胞增殖,影响大脑皮层发育;在卵泡细胞中,细胞蛇相当于CTPS的存储库,在卵子发生过程起到促进细胞增殖和代谢的作用。本文就细胞蛇的形成过程及其在不同类型细胞中的功能进行阐述,以期人们进一步认识细胞蛇,并探索其潜在作用机制和应用价值。

Filamentation and inhibition of prokaryotic CTP synthase with ligands

Cytidine triphosphate synthase(CTPS)plays a pivotal role in the de novo synthesis of cytidine triphosphate(CTP),a fundamental building block for RNA and DNA that is essential for life.CTPS is capable of directly binding to all four nucleotide triphosphates:adenine triphosphate,uridine triphosphate,CTP,and guanidine triphosphate.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 viral or prokaryotic pathogens.Utilizing cryo-electron microscopy,we determined the structure of Escherichia coli CTPS(ecCTPS)filament in complex with CTP,nicotinamide adenine dinucleotide(NADH),and the covalent inhibitor 6-diazo-5-oxo-L-norleucine(DON),achieving a resolution of 2.9A.We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis,providing an evolutionary perspective on CTPS filament formation.Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding.Through comparative structural analysis,we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts.Combining biochemical assays and structural analysis,we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS.Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

细胞蛇

随着成像技术以及分析技术的发展,人们对细胞的认识也越来越深入。在经典的有膜细胞器发现几十年甚至一百年以后,越来越多的无膜细胞器被发现。这篇文章着重阐述一个新型的无膜细胞器细胞蛇的发现、分布和进化保守性,并探讨细胞蛇和代谢纤维的结构、功能和应用。

Intracellular compartmentation of CTP synthase in Drosophila

Compartmentation is essential for the localization of biological processes within a eukaryotic cell. ATP synthase localizes to organelles such as mitochondria and chloroplasts. By contrast, little is known about the subcellular distribution of CTP synthase, the critical enzyme in the production of CTP, a high-energy molecule similar to ATP. Here I describe the identification of a novel intracellular structure con- taining CTP synthase, termed the cytoophidium, in Drosophila cells. I find that cytoophidia are present in all major cell types in the ovary and exist in a wide range of tissues such as brain, gut, trachea, testis, accessory gland, salivary gland and lymph gland. In addition, I find CTP synthase-containing cytoophidia in other fruit fly species. The observation of compartmentation of CTP synthase now permits a broad range of questions to be addressed concerning not only the structure and function of cytoophidia but also the organization and regulation of CTP synthesis.

Drosophila CTP synthase can form distinct substrate-and product-bound filaments

Intracellular compartmentation is a key strategy for the functioning of a cell.In 2010,several studies revealed that the metabolic enzyme CTP synthase(CTPS)can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells.However,recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells.In this study,using negative staining and cryo-electron microscopy,we demonstrate that Drosophila CTPS,whether in substrate-bound or product-bound form,can form filaments.Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species.We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.

CTP Synthase Is Required for Optic Lobe Homeostasis in Drosophila

CTP synthase （CTPsyn） is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTE Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in bacteria, yeast, fruit flies and humans. However, it remains elusive whether and how CTPsyn and cytoophidia play a role during development. Here, we show that cytoophidia are abundant in the neuroepithelial stem cells in Drosophila optic lobes. Optic lobes are underdeveloped in CTPsyn mutants as well as in CTPsyn RNAi. Moreover, overexpressing CTPsyn impairs the development of optic lobes, specifically by blocking the transition from neuro- epithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.

The atlas of cytoophidia in Drosophila larvae

In 2010,cytidine 50-triphosphate synthase(CTPS)was reported to form the filamentous or serpentine structure in Drosophila,which we termed the cytoophidium.In the last decade,CTPS filaments/cytoophidia have been found in bacteria,budding yeast,human cells,mice,fission yeast,plants,and archaea,indicating that this mechanism is highly conserved in evolution.In addition to CTPS,other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures,demonstrating that this is a basic strategy of cells.Nevertheless,our understanding of the physiological function of the cytoophidium remains incomplete and elusive.Here,we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development.We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues.Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.

CTP synthase forms cytoophidia in archaea

CTP synthase(CTPS)is an important metabolic enzyme that catalyzes the rate-Iimiting reaction of nucleotide CrP de novo synthesis.Since 2010,a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes,which are termed cytoophidia.However,it is unknown whether cytoophidia exist in the third domain of life,archaea.Using Haloarcula hispanica as a model system,here we demonstrate that CTPS forms distinct intracellular compartments in archaea.Under stimulated emission depletion microscopy,we find that the structures of H.hispanica CTPS are elongated,similar to cytoophidia in bacteria and eukaryotes.When Haloarcula cells are cultured in lowsalt medium,the occurrence of cytoophidia increases dramatically.In addition,treatment of H.hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly.Our study reveals that CTPS can fo rm cytoophidia in all three domains of life,suggesting that forming cytoophidia is an ancient property of CTPS.

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase(P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.

Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae

Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filamentforming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frameGFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases（Ura7p and Ura8p） and two asparagine synthetases（Asn1p and Asn2p） form filaments both in the cytoplasm and in the nucleus.Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.