Cytidine 5′-triphosphate

CTPS and IMPDH form cytoophidia in developmental thymocytes

Min Peng a, 1, Chia-Chun Chang b, 1, Ji-Long Liu b,*, Li-Ying Sung a, c, d,**
a Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan
b School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
c Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan
d Animal Resource Center, National Taiwan University, Taipei, 106, Taiwan


The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphos- phate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c- Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium struc- tures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physio- logical roles of the cytoophidium in thymocyte development.
Keywords: CTPS IMPDH Cytoophidium Thymocyte Glycolysis

1. Introduction

Intracellular compartmentalization of enzymes has emerged as ma- chineries responsible for delicate regulation of diverse biological func- tions. Most of membraneless organelles, such as P-bodies, Cajal bodies and stress granules, comprise RNA and protein components and display spherical appearance. Given structures construct barriers differentiating local environments and so to create specialized niches to facilitate particular biochemical reactions [1]. In contrast, a group of metabolic enzymes have been found to polymerize and further assemble into a larger filamentous structure, termed the cytoophidium (“cellular snake” in Greek, plural: cytoophidia), in the nucleus and cytoplasm of cells in various organisms [2–4,11,41,42,47,48]. In mammalian models, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehy- drogenase (IMPDH) are the most characterized cytoophidium-forming enzymes that catalyze the rate-liming steps in de novo CTP and GTP biosynthesis, respectively. Previous structural and biochemical studies have revealed the molecular regulation and conformational changes of IMPDH and CTPS in their polymeric states. Promoted by the binding of ATP and IMP, polymerization of IMPDH has been shown to desensitize the enzymatic inhibitory effect by GTP binding, thereby accelerating GTP biosynthesis in physiological conditions [5,6]. Similarly, CTPS polymers display higher catalytic activity than non-polymerized CTPS tetramers in the presence of CTP in physiological concentrations [7–9].
Moreover, a recent study demonstrates that the cytoophidium can pro- tect CTPS proteins from degradation, shedding the light on novel func- tions of cytoophidia in cell metabolism [10].
Formation of the cytoophidium is correlated with active mTORC pathway and the filaments are more frequently observed in highly proliferative cell types [11,12,44]. For instance, in mouse and human T-lymphocytes, IMPDH cytoophidium formation could be induced by T cell activation in vitro and in vivo, implying that the cytoophidium par- ticipates in metabolism of proliferative cells [13,14].
Previous research on the CTPS cytoophidium was mostly based on Drosophila models, in which the filaments are present in several tissues without additional manipulation [2,15,45,46]. A recent study demon- strates the atlas of CTPS cytoophidium in Drosophila larvae, providing valuable information for future research [15]. In mammals, although we previously reported that CTPS cytoophidia are present in some human hepatocellular carcinoma tissues, the physiological roles of the cytoo- phidium are still unknown [16].
Aiming to understand the physiological relevance of CTPS cytoo- phidia in mammalian tissues, here we survey the presence of CTPS cytoophidia in major organs of mice and find a subset of thymocytes displaying both CTPS and IMPDH cytoophidia. According to the CD markers and distribution of given cells in the thymus, we identify cytoophidium-presenting cells (cytoophidium+ cells) are thymocytes between stages DN3 (double-negative) to early DP (double-positive). Additionally, our data suggest that the assembly of the cytoophidium may associate with active glycolytic metabolism, which is a well-known feature of highly proliferative thymocytes under the regulation of pre- TCR signaling. Collectively, we show that CTPS and IMPDH cytoophi- dium assembly is present in a particular population of thymocytes, presumably coupling with their active metabolism. Considering the putative functions of the cytoophidium, we propose that cytoophidium formation may facilitate nucleotide biosynthesis, hence benefiting the rapid proliferation of developmental thymocytes.

2. Results

2.1. CTPS and IMPDH form the cytoophidium in mouse thymus

Human CTPS1 and CTPS2 tetramers can polymerize in vitro and so to reduce the competitive inhibition effect from CTP binding [7,8]. CTPS polymers are known as subunits of a subcellular filament, the cytoo- phidium. CTPS cytoophidia are observed in various tissues in the fruit fly and in multiple human cancer tissues [2,16]. However, a systemic sur- vey for the presence of CTPS cytoophidia under normal physiological states in mammalian models has not yet been reported. In order to un- derstand the physiological roles of the cytoophidium in vivo, we first performed immunofluorescence on sections of some major organs of mouse, including brain (cerebrum), retina, heart, skeletal muscle (leg), lung, adipose tissues, liver, gallbladder, pancreas, skin, stomach, intes- tine, colon, kidney, prostate, ovary, testis, prostate, skin, spleen, lymph node and thymus, to select a target for further investigation. CTPS cytoophidia were rarely detected in most tissues except for the thymus (Fig. 1A). The thymus is composed of cortical and medullary areas. CD8 expression is markedly more abundant in the cortex than in the medulla. Therefore, the contrast and boundary between cortex and medulla could be clearly detectable by immunofluorescence. We observed that most cytoophidia are concentrated in the outer cortex region (Fig. 1B). Pre- vious studies have shown that IMPDH cytoophidium assembles in pro- liferative lymphocytes in lymph node, spleen, thymus and circulation [13]. We also performed immunostaining for IMPDH in these tissues and observed IMPDH cytoophidia in the white pulp of spleen, in some cells in lymph node and cells at the cortex of thymus (Fig. 2A, C). However, CTPS cytoophidia were only observed in the thymus (Fig. 2B). We subsequently collected thymi from mice at ages of E17.5 and 8 months, representing the developmental and regressing states of the tissue. Despite the difference in sizes of the organ, the distribution and abun- dance of CTPS cytoophidia were consistent in the thymus of all ages (Fig. 3).
IMPDH and CTPS cytoophidia are substantially different structures, but frequently exhibit a spatial association in the cell [17,18]. We compared the distribution of CTPS and IMPDH cytoophidia in the thymus and found around 75% of cytoophidia are labeled by both antibodies, indicating that most of cytoophidium+ cells have both CTPS and IMPDH in the structure (Fig. 2D).

2.2. CTPS cytoophidium+ cells in the thymus are DN3, DP4 and early DP thymocytes

Maturation of T cells goes through a few developmental stages (Fig. 1C). Derived from the bone marrow, T cell progenitors arrive the thymus and acquire the stimulation of Notch signaling before proceeding to CD25+ DN2 thymocytes. To subsequently progress to the CD44-/ CD25+ DN3 stage, thymocytes require correct rearrangement of the T cell receptor (TCR) α and β loci. Synthesis of rearranged TCR chains results in the assembly of functional pre-TCR, comprising pTα, TCRβ and CD3, and triggers pre-TCR signaling. Signals emanating from the pre-TCR induces transient self-renewal (β-selection), and promotes sur- vival and differentiation to the DN4 and CD4/CD8 DP thymocytes [19].
In order to determine at which developmental stages the thymocytes would display the cytoophidia, we performed double-staining for cytoophidia and CD markers for each developmental stage. Cytoophidia in DN1 and DN2 thymocytes were unlikely since cytoophidium was rarely detected in CD44+ thymocytes (Fig. 4A, E). CD25, the marker for DN2 and DN3 thymocytes, was positive in about 20% of cells with cytoophidia (Fig. 4B, E). Meanwhile, CD4 and CD8, markers for DP cells, labeled most of cells at thymus cortex but only 20–25% of cytoophidium+ cells were also positive for CD4 or CD8. (Fig. 4C and D). This implies that roughly 50% of cytoophidium+ cells are DN3 and DP cells, while the other 50% of cytoophidium+ cells are probably DN4 thymocytes, which is the transient stage between DN3 and DP express- ing none of CD markers described above (Fig. 4E). Notably, the majority of cytoophidium+ DP cells showed relatively weaker CD8 and CD4 and located along the blur line between DN and DP thymocytes, supporting the notion that cytoophidia are enriched in cells during the transient states between DN3 and ‘early’ DP thymocytes (Fig. 4C and D).

2.3. Cytoophidium+ thymocytes are highly proliferative

Assembly of the cytoophidium is proposed to enhance nucleotide biosynthesis as CTPS and IMPDH are more resistant to the end-product inhibition effect in their polymeric forms [6,7,9]. It is also proposed that the functions of cytoophidia might be particularly important for cells undergoing a metabolic transition. For instance, massive IMPDH cytoophidium assembly could be stimulated by T cell activation [13]; CTPS cytoophidia are observed in neuroblasts of Drosophila larvae [20].
With the cytoophidium, the cell may produce CTP/GTP efficiently to meet the increasing demands for cell proliferation, and save energy from generating more CTPS and IMPDH proteins. Conversely, cells could downregulate CTP and GTP production by disassembling cytoophidia rather than diminishing the enzymes when cellular demands drop. This prompted us to investigate the relationship between cytoophidium formation and DNA replication in thymocytes. We gave mice 200 μg of EdU through intraperitoneal injection and collected their thymi for cryosection in 2 h of treatment. EdU labeled cells were mainly located at the cortex region, generally colocalizing with cytoophidium+ cells.
ApproXimately 55.4% of cytoophidium+ cells were positive for EdU, indicating the active DNA replication and cell proliferation in these cells (Fig. 5A). To further clarify the distribution of proliferating cells, the percentages of EdU+ cells marked with specific CD marker were calculated. As a consequence, 5% of EdU+ cells were CD25 positive and around 15% of EdU+ cells presented CD8 or CD4 (Fig. 5B and C), which
CD markers. (D) A model for the T cell development with different cell surface markers expressed at the different stages as well as those proliferating cells being labeled with EdU. The percentage of CTPS cytoophidium-containing cells calculated from Fig. 3E shows half of the cytoophidia corresponds to the DN4 stage when the proliferative expansion occurs.

2.4. Regulation of the cytoophidium is correlated with active glycolysis

Based on the notion that cytoophidium+ thymocyte ranges between DN3 to early DP stages, we suspected cytoophidium assembly is asso- ciated with pre-TCR signaling metabolic signatures. It has been demonstrated that pre-TCR signaling is related to the upregulation of mTORC1 signaling, glycolysis and the expression of c-Myc (Myc) [22, 23].
The transcription factor, Myc, whose expression is dysregulated in many cancers, controls the expression of more than a thousand genes involved in cell cycle progression and various cell metabolic pathways, including purine and pyrimidine metabolism [24]. In fact, human CTPS1 and IMPDH1 genes are among the long list of Myc direct targets [25,26]. Additionally, our previous work has revealed that CTPS cytoophidium assembly in Drosophila egg chambers is highly correlated with changes of Myc expression levels during egg maturation [27]. Thus, we sought to investigate whether cytoophidium+ thymocytes display higher expression levels of Myc, CTPS and IMPDH. Immunofluorescence for Myc and CTPS was performed on thymus sections. Thymocytes at cortex region showed heterogenous expression patterns of Myc. We observed that higher Myc intensity levels are present in cytoophidium+ cells than the other area (Fig. 7). We also performed immunohisto- chemistry to determine expression levels of CTPS and IMPDH in the thymus. All three antibodies against CTPS1, CTPS2 and IMPDH2 gave higher intensity at the cortex than those at the medulla. With image-based quantification, we compared the signal intensity between high (cortex) and low (medulla) expression cells and revealed 2.2-fold, 1.6-fold and 1.8-fold increases of CTPS1, CTPS2 and IMPDH2 at cor- tex region, respectively (Fig. 8). Although 74% of identical amino acid sequence shared by CTPS isoforms and 84% of conserved amino acid sequence existed in IMPDH isoforms might lead to a cross-reactivity between isoforms in each protein, our results strongly indicate that the cell population with cytoophidia has higher levels of CTPS, IMPDH and Myc.
It has been shown that nearly all genes encoding glycolytic enzymes are under the control of Myc [24]. Glycolytic pathway begins with the utilization of glucose and generates various intermediates, which sub- sequently serve as substrates of TCA cycle, amino acid synthesis and nucleotide synthesis. Many proliferative cell types, such as lymphocytes, pluripotent stem cells and cancers, feature active glycolysis and form many IMPDH cytoophidia [13,14,28,29]. Moreover, thymocytes have been shown to acquire active glycolysis upon β selection in a PI3K/AKT pathway dependent manner [23,30–32]. These prompted us to assess whether cytoophidium regulation is relevant to active glycolysis. Phosphorylation of AKT at Ser473 is well-known to activate AKT activity and it is also known that increase in AKT activity is sufficient to stim- ulate glycolysis [33]. Therefore, we examined the relationship between active glycolysis and IMPDH cytoophidia in mouse thymus by double-labeling phospho-Akt (p-Akt, Ser473) and IMPDH. A generally higher intensity of p-Akt at cortex than medulla of the thymus was observed. However, heterogenous intensity of p-Akt was seen at cortex as well (Fig. 9A–C). With the fire lookup table being applied to p-Akt channel in ImageJ software, the brighter signals are, the whiter will be exhibited; the dimmer signals are, the bluer will be displayed (Fig. 9B). As the mean intensity of a randomly picked up medulla region being assigned a value of 1-fold, various expression levels of p-Akt could be detected in the different outer cortex regions. Accordingly, we defined 5-fold change as a threshold to determine the areas are relatively high or low p-Akt levels (Fig. 10). Similar to the colocalization with higher Myc signal, we found a positive correlation between p-Akt level and the cytoophidium abundance and length (Fig. 7D–F). Moreover, as a key regulatory enzyme in glycolysis, the expression level of phosphofruc- tokinase (PFK) was universally higher in the outer cortex region, in which IMPDH cytoophidia were observed (Fig. 7G and H).
Next, we wondered if cytoophidia would be attenuated by distur- bance of glycolytic pathway. 2DG is a glucose analog that inhibits hexokinase, a key enzyme of glycolysis, thereby impeding glycolytic pathway. We treated mice with 1500 mg/kg of 2DG by intraperitoneal injection for 2 h and subsequently gave EdU for another 2 h before sample collection. After a total 4-h treatment of 2DG, both EdU+ and cytoophidium+ cells were significantly reduced, showing a strong cor- relation between cell proliferation, cytoophidium formation and glycolysis (Fig. 11). In the lymph node and spleen, however, there was no obvious change in the formation of cytoophidium and patterns of EdU labeling, suggesting distinct dependence on glycolysis of various cell types (Fig. 12). Taken together, our findings support the direct linkage between active glycolysis and the cytoophidium assembly, which is under the control of pre-TCR signaling, and also expand our understanding of the coordination of cell metabolism in developmental thymocytes.

3. Discussion

The filament-forming ability of IMPDH and CTPS was demonstrated more than a decade ago [2,3,47]. To date, the formation of IMPDH cytoophidia has been found naturally in pancreatic β cells, spleen and thymus of mice [11,13]. However, to our knowledge, the observation of CTPS cytoophidia in healthy mammals has not yet been reported. We were motivated to search for the presence of CTPS cytoophidia in mouse model and to further investigate the physiological process that CTPS cytoophidia might participate in. After screening of multiple mouse tissues by immunofluorescence, abundant CTPS cytoophidia were found in the subset of cells in all thymi collected from young and old mice. These results reveal that the formation of CTPS cytoophidia is an inherent phenomenon in the mouse thymus. Moreover, the detection of cytoophidia in a particular region denotes that the filamentation of CTPS may correlate with specific physiological events.
The cytoophidium is a dynamic structure in response to specific metabolic cues of the cell. Previous studies have demonstrated that the polymerization of both CTPS and IMPDH can reduce the catalytic inhibitory effects from their end products CTP and GTP [5–7].
Lengths of IMPDH cytoophidia in the mouse thymus with different expression levels of p-Akt. (***p value < 0.001; Student’s t-test) (E) and (F) highlight the localization of IMPDH cytoophidia and p-Akt shown in (C′) and (C′′), representing regions with relatively high and low p-Akt levels, respectively. (G) Immuno- histochemistry for PFK, the key regulatory enzyme in glycolysis, in the mouse thymus. Scale bars: 50 μm. (H) Immunofluorescence for IMPDH and PFK in the mouse thymus and scale bar: 50 μm. (H′) and (H′′) are magnified images of selected areas shown in (H), representing regions with relatively high and low PFK levels, respectively. Scale bars: 10 μm. Here we report that both CTPS and IMPDH form cytoophidia in developmental mouse thymocytes at stages between DN3 to early DP, matching the time point of pre-TCR signaling activation. It has been shown that DN3, DN4 and ISP (immature single-positive) thymocytes display significant higher oXygen consumption rate (OCR) than quies- cent DP thymocytes, indicating higher levels of oXidative phosphoryla- tion (OXPHOS) in the cells. Furthermore, extracellular acidification rate (ECAR), which denotes glycolytic activity of the cell, is progressively upregulated during the transition from DN3 to ISP cells, and drops dramatically in DP thymocytes [23]. Moreover, the presence of cytoophidia is accompanied by the elevated expression levels of Myc, p-Akt and PFK, which are markers for active cell metabolism and glycolysis. This is consistent with previous studies arguing that IMPDH cytoophidia are frequently found in cell types with active glycolytic activity, such as activated T cells, embryonic stem cells and some cancer cells [13,14,28,40]. When mice were treated with a glycolysis inhibitor, 2DG, cytoophidia disappeared along with a dramatic reduction of proliferating cells at the cortex region of the thymus within few hours. These results suggest a positive correlation between glycolysis and cytoophidium formation. Both CTPS and IMPDH protein levels are higher in the cortex region, where cytoophidia are enriched, than those in the medulla region of the thymus. In murine T cells, the IMPDH expression level as well as cytoophidium structures significantly increase upon activation [14]. These might be attributed to the upregulation of upstream metabolic regulators since both CTPS and IMPDH are demonstratedto be direct targets of Myc [25,26]. On the other hand, cytoophidium formation has been shown to prolong the half-life of the CTPS protein and lead to CTPS accumulation [10]. It is possible that the cytoophidium could also pro- tect the IMPDH protein from degradation. We propose that the assembly of cytoophidia coordinate nucleotide production in rapidly proliferating thymocytes not only by enhancing activities of CTPS and IMPDH but also by reducing the degradation of functional CTPS and IMPDH Our results shed the light on the putative roles of the CTPS cytoo- phidium in normal mouse tissues, expanding our understanding of pre- TCR signaling and β selection. Unfortunately, there is no drug that has been identified to block the formation of CTPS and IMPDH cytoophidia specifically. We are currently unable to determine the significance of cytoophidium structures in mouse thymocyte development. Specific point mutations, such as H355A on human CTPS1 and Y12A on human IMPDH2, are proved to abolish polymerization of the enzymes and so to prevent cytoophidium assembly without directly disturbing the catalytic activity, making them ideal tools for cytoophidium research [5–7]. Future studies on animal models carrying point mutations that disrupt cytoophidium assembly are required for exploring the physiological roles of CTPS and IMPDH cytoophidium and for assessing their clinical potential in the immune system and particular diseases. Taken together, we have observed the formation of CTPS and IMPDH cytoophidia in developmental thymocytes between stages DN3 to early DP in the mouse thymus. These cytoophidium-presenting cells are exclusively proliferative and associated with higher levels of CTPS, IMPDH, c-Myc, p-Akt and PFK, indicating an active glycolytic meta- bolism of thymocytes (Fig. 13). These findings provide insight into the roles of cytoophidium in thymocyte development. 4. Materials and methods 4.1. Animals All animal maintenance, care and use procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Na- tional Taiwan University, Taiwan according to the protocol number NTU-107-EL-216. Mice were 7 to 19-week-old ICR males or females. 4.2. Frozen sections Brain (cerebrum), retina, heart, skeletal muscle (leg), lung, adipose tissues, liver, gallbladder, pancreas, skin, stomach, intestine, colon, kidney, prostate, ovary, testis, prostate, skin, spleen, lymph node, and thymus removed after euthanasia were fiXed in 4% paraformaldehyde overnight at 4 ◦C. After incubating stepwise from 10% to 30% sucrose (S1888, Sigma) at 4 ◦C until tissue sinks, embed the tissues in OCT mounting medium (4583, Tissue-Tek) and freeze at - 80 ◦C until use. Then cut 7 μm thick tissue sections using Leica CM1950 cryostat. 4.3. EdU labeling For EdU incorporation, animals were injected intraperitoneally with 200 μg of EdU 2 h before sacrifice. Tissue sections were prepared as described above. After washing in PBS, a Click-iT® azide-based reaction was performed to bind Alexa Flour 647 molecule to the EdU incorpo- rated to newly synthesized DNA. All procedures followed according to instructions provided by manufactures (Thermo). 4.4. Immunofluorescence Tissue sections were incubated with primary antibody in PBS with 2.5% bovine serum albumin (A9647, Sigma-Aldrich) and 0.25% Triton- X100 (X100, Sigma-Aldrich) for at least 2 h at room temperature. After washing with PBS, samples were incubated with secondary antibody, which is diluted in the same solution as used in primary antibody dilution. At least 2 h after the secondary antibody reaction, samples were washed and mounted with PBS. All tissue staining was performed in parallel with control sections stained with only secondary antibodies (Fig. 14). Antibodies used in this study include: rabbit anti-CTPS1 IgG (15914-1-AP, ProteinTech), mouse anti-IMPDH1 IgG (ab55297, ABcam), rabbit anti-IMPDH2 IgG (12948-1-AP ProteinTech), rat anti- CD4 IgG (1:250, MCA4635T, BIO-RAD), rat anti-CD8 IgG (MCA1768T, BIO-RAD), rat anti- CD25 IgG (1:250, MA5-17812, Thermo), rat anti- CD44 IgG (14–0441, eBioscience), goat anti-Myc IgG (AF3696, R&D Systems), rabbit anti-p-AKT1-S473 IgG (AP0637, ABclonal), and rabbit anti-phosphofructokinase (PFK, 55028-1-AP, Proteintech), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, invitrogen), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (A21207, invitrogen), Alexa Fluor 647-conjugated goat anti-rabbit IgG (A21244, invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (A11029, invitrogen), Alexa Fluor 488-conjugated donkey anti-rat IgG (112-545-167, Jackson ImmunoResearch), and Alexa Fluor 488-conjugated donkey anti-goat IgG (A-11055, invitrogen). Antibodies without mention were applied at 1:500 dilution. Images were acquired on a laser-scanning confocal microscope (Leica TCS SP5 II confocal microscope) with Alexa Fluor 488 being excited by argon-ion laser (488 nm), Alexa Fluor 594 being excited by orange/yellow helium-neon laser (594 nm), and Alexa Fluor 647 being excited by red helium-neon laser (647 nm). Images were then processed by the software of the Leica Application Suite. 4.5. Immunohistochemistry Frozen tissue sections were covered with 0.3% H O for 5 min and Declaration of competing interest subsequently incubated in normal blocking serum. After washing with PBS, samples were incubated with primary antibody in PBS with 2.5% bovine serum albumin (A9647, Sigma-Aldrich) and 0.25% Triton X-100 (X100, Sigma-Aldrich) for at least 2 h at room temperature. After washing with PBS, samples were incubated in biotinylated secondary antibody for at least 2 h followed by another PBS wash. 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