The Interplay between Myc and CTP Synthase in Drosophila

Abstract

CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.

Fig 1. Cytoophidium formation correlates with Myc expression in Drosophila follicle cells.

(A) Schematic representation of the stages of Drosophila oogenesis within an ovariole. (B-H) Immunostaining of Drosophila follicle cells at different stages with antibodies against Myc and CTPsyn. Myc expression is observed at Region 2 of the germarium (B) and is continuously expressed at early- (C) and mid-oogenesis (D, E) until Stage 10a (F). Myc expression in follicle cells drops from Stage 10b (G) to late-stage egg chambers (H). The appearance of cytoophidia correlates with Myc expression. Cytoophidia are detectable from Region 2 to stage 10a (B-F). (B) Cytoophidia are first observed at region 2 of the germarium concomitant with high Myc expression. (C) Two egg chambers at stages 2–5. Note large filamentous structures are macro-cytoophidia in germline cells. (D) An egg chamber at Stage 7. Note large filamentous structures are macro-cytoophidia in germline cells. (E) Follicle cells of a Stage-9 egg chamber. (F) Follicle cells of a Stage-10a egg chamber. (G) Follicle cells of a Stage-10b egg chamber. In stage 10b follicle cells, Myc expression is low and cytoophidia are hardly detectable. (H) Follicle cells of a Stage-12 egg chamber. In stage-12 follicle cells, Myc expression is low and no cytoophidia are detectable. (I) Quantification of follicle cell cytoophidia lengths at various stages of oogenesis. Quantification represents the mean cytoophidium length from > 50 cells in > 3 egg chambers at each stage in w¹¹¹⁸ flies. See S1–S7 Figs for individual channels of images shown.

Fig 2. Myc knockdown reduces cytoophidium formation in follicle cells.

UAS-Myc-RNAi^JF01761 clones (i.e. Myc RNAi) marked with GFP (B, outlined in yellow in A-H) have decreased levels of Myc (C) and have no detectable cytoophidia as indicated by an antibody against CTP synthase (CTPsyn). DNA staining shows that nuclei of clonal cells (green cells in E-F) are smaller than those of neighbouring cells (non-green cells in E-F). (I) Quantification of nuclear area shows that Myc RNAi decreases nuclear size significantly. (J) Myc RNAi decreases cytoophidium length significantly. ***P<0.001. Error bars show SEM.

Fig 3. Myc overexpression promotes cytoophidium formation in follicle cells.

(A-D) In stage-8 egg chambers, UAS-Myc overexpression clones marked with GFP (B, outlined in yellow in A-D) have longer cytoophidia, as indicated by CTPsyn staining (C), than non-GFP cells. DNA staining shows that the nuclei in clones (green cells in D) are larger than those of neighbouring cells (non-green cells in D). (E) Cytoophidia in Myc overexpression (UAS-Myc) cells increase significantly in length, compared with those in neighbouring cells. ***P<0.001. Error bars show SEM.

Fig 4. Myc overexpression is sufficient to induce cytoophidia formation in Stage 10b follicle cells.

(A-E) In this stage-10b egg chamber, non-clonal cells (cells that are lack of GFP in B) have low levels of Myc (D) and only have very short cytoophidia, as indicated by an antibody against CTPsyn (C). UAS-Myc overexpression clones (i.e. Myc overexpression) marked with GFP (B, outlined in yellow in A-E) have increased levels of Myc (D) and much longer cytoophidia than non-clonal (nc, i.e. non-green) cells (C). DNA staining show that the nuclei in clones (green cells in E) are larger than those of nc cells (non-green cells in D). (F) Quantification shows that cytoophidia in Myc overexpression (UAS-Myc) cells are significantly increased in length, compared to those in nc cells. ***P<0.001. Error bars show SEM.

Fig 5. CTPsyn knockdown supresses Myc-induced overgrowth phenotype in Drosophila follicle cells.

(A-E) The nuclei of cells both overexpressing Myc and knocking down CTPsyn (UAS-Myc, CTPsyn^RNAi) are marked by GFP (B, outlined by yellow lines in A-E). Myc overexpression is verified by immunostaining with an antibody against Myc (D). CTPsyn knockdown is verified by immunostaining with an antibody against CTPsyn (C). Note that no cytoophidia are detectable in the clonal cells even when Myc is overexpressed. (F) Quantification of mid-stage follicle cells shows that Myc overexpression (UAS-Myc) alone increases nuclear size significantly. Follicle cells in UAS-Myc, CTPsyn^RNAi cells have similar nuclear size compared to non-clonal (nc) cells. CTPsyn^RNAi or CTPsyn overexpression show no significant difference in nuclear area (see S10 and S11 Figs for representative images). Quantification represents the mean nuclear areas from > 50 cells in > 3 egg chambers per genotype. ***P<0.001. n.s. = not significant. Error bars show SEM.

Fig 6. Myc overexpression increases cytoophidia length in Drosophila follicle cells.

(A-E) The nuclei of cells overexpressing Myc (UAS-Myc) are marked by GFP (B, outlined by yellow lines in A-E). Myc overexpression is verified by immunostaining with an antibody against Myc (C). Note that cytoophidia in the clone cells are not only detectable but also longer than those in wild-type cells (comparing those in Fig 5).