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.     

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.     

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.     

Ack kinase regulates CTP synthase filaments during Drosophila oogenesis

The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.     

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 S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR 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.     

Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5′-triphosphate

While enzyme activity is often regulated by a combination of substrate/effector availability and quaternary structure, many cytosolic enzymes may be further regulated through oligomerization into filaments. Cytidine-5′-triphosphate (CTP) synthase (CTPS) forms such filaments—a process that is promoted by the product CTP. The CTP analog and active chemotherapeutic metabolite gemcitabine-5′-triphosphate (dF-dCTP) is a potent inhibitor of CTPS; however, its effect on the enzyme's ability to form filaments is unknown. Alongside electron microscopy studies, dynamic light scattering showed that dF-dCTP induces Escherichia coli CTPS (EcCTPS) to form filaments in solution with lengths ≥ 30 nm in the presence of CTP or dF-dCTP. The substrate UTP blocks formation of filaments and effects their disassembly. EcCTPS variants were constructed to investigate the role of CTP-binding determinants in CTP- and dF-dCTP-dependent filament formation. Substitution of Glu 149 (i.e., E149D), which interacts with the ribose of CTP, caused reduced affinity for both CTP and dF-dCTP, and obviated filament formation. Phe 227 appears to interact with CTP through an edge-on interaction with the cytosine ring, yet the F227A and F227L variants bound CTP and dF-dCTP. F227A EcCTPS did not form filaments, while F227L EcCTPS formed shorter filaments in the presence of CTP or dF-dCTP. Hence, Phe 227 plays a role in filament formation, although replacement by a bulky hydrophobic amino acid is sufficient for limited filament formation. That dF-dCTP can induce filament formation highlights the fact that nucleotide analogs employed as chemotherapeutic agents may affect the filamentous states of enzymes and potentially alter their regulation in vivo.     

Assembly of IMPDH2-Based,CTPS-Based,and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

Inhibition of guanosine triphosphate(GTP)and cytidine triphosphate(CTP)biosynthetic pathways induces cells to assemble rod/ring(RR)structures,also named cytoophidia,which consist of the enzymes cytidine triphosphate synthase(CTPS)and inosine-50-monophosphate dehydrogenase 2(IMPDH2).We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures.He La and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine(DON),ribavirin,or mycophenolic acid(MPA).Over 90%of DON-treated cells presented RR structures.In He La cells,35%of the RR structures were positive for IMPDH2alone,26%were CTPS alone,and 31%were IMPDH2/CTPS mixed,while in COS-7 cells,42%of RR were IMPDH2 alone,41%were CTPS alone,and 10%were IMPDH2/CTPS mixed.Ribavirin and MPA treatments induced only IMPDH2-based RR.Cells were also transfected with an N-terminal hemagglutinin(NHA)-tagged CTPS1 construct.Over 95%of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100%presented CTPS1-based RR;when treated with ribavirin,over 94%of transfected cells presented IMPDH2-based RR and 37%presented CTPS1-based RR,whereas 2%of untreated transfected cells presented IMPDH2-based RR and 28%presented CTPS1-based RR.These results may help in understanding the relationship between CTP and GTP biosynthetic pathways,especially concerning the formation of filamentous RR structures.     

Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.     

Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments

Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.     

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 CTP. 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 neuroepithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.     

Structural Differences Explain Diverse Functions of Plasmodium Actins

Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.     

Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a K_D value of 210 nM in SPR analysis and CTPS1-selective inhibition with an IC₅₀ value of 110 nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor.     

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.     

Growth-saturation in vitro of Salmonella flagella

At physiological ionic strength and pH, short fragments of Salmonella flagella (seeds) grow longer in the presence of monomeric flagellin and there exists a one-to-one correspondence between the seeds and fully grown filaments (Asakura et al., 1964). In this study it was shown that when monomer and seed derived from a preparation of flagella (strain SJ25) were mixed in a protein ratio r larger than 20, the filaments stopped growing or became inactive for a long period of time, and the average length of inactive filaments was independent of the value of r. The phenomenon was called growth-saturation. The antibody-labelling technique (Asakura et al., 1968) made it possible to show that, though active filaments having equal lengths grew at various rates ranging between 0 and 0.16 μm/min, the average value of growth rate depended little on length. On the other hand, it was found that the proportion of inactive filament in the total filament increased rapidly as the value of r was increased continuously from 0 to 10. The dependence of the proportion of inactive filament on r suggested that filaments became inactive with a probability independent of their length. The rate of inactivation (or the probability with which a filament becomes inactive during growth by a unit length) had various values when different preparations of flagella were used as starting materials. The distribution of length for an assembly of inactive filaments was determined by low-magnification electron microscopy. The result could be approximated by an exponential distribution: the number-average length was 4.54 μm and the rate of inactivation was 0.224 μm^?1.     

Fine (2-5-nm) filaments: new types of cytoskeletal structures

Over the past 30 years filaments 2-5 nm in diameter have been found in a number of different types of eukaryotic cells. As a group, these fine filaments lack the similarity of composition and function that characterize the three major classes of cytoskeletal elements--microfilaments, microtubules, and intermediate filaments. Six different proteins that form fine filaments have been identified; proposed functions for these fibers range from cell motility to cytoarchitecture. Recent studies, however, have revealed filaments with similar compositions and/or functions in otherwise different cells, suggesting that the fine filaments may eventually fit into a limited number of subgroups.     

Bactofilins,a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.     

Human cytidine triphosphate synthetase 1 interacting proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

A Nucleotide-Dependent Conformational Switch Controls the Polymerization of Human IMP Dehydrogenases to Modulate their Catalytic Activity

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades, but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity. In vitro reconstituted micron-length cytoophidia-like structures show catalytic activity comparable to unassembled IMPDH but, in turn, are more resistant to GTP/GDP allosteric inhibition. Therefore, IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires it. Finally, we demonstrate that most of the IMPDH retinopathy-associated mutations abrogate GTP/GDP-induced allosteric inhibition and alter cytoophidia dynamics.     

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.