CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production

Abstract

Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.This article has an associated First Person interview with the first author of the paper.

Keywords: Agglomeration; Metabolic enzyme; Nucleotide biosynthesis.

Fig. 1.

The CTPS ^H355A mutation prevents CTPS assembly into filaments in vivo. Ovarioles from (A), Flag-CTPS^WT-M1/pCOG-Gal4 or (B) Flag-CTPS^H355A-M2/pCOG-Gal4 flies were stained with anti-FLAG antibody (green) and Propidium Iodide (magenta) to label DNA. Stage 4 and 6 egg chambers are bracketed for comparison. Lower panels show higher magnification of the stage 6 egg chambers from the A and B images: A′ and B′ are the Flag stain and A″ and B″ are Propidium Iodide. (C) Quantification of the percent of stage 4 and 6 egg chambers with CTPS filaments in two independent Flag-CTPS^WT transgenic lines (M1 and M4) and two Flag-CTPS^H355A lines (M1 and M2). *, P<7.5×10⁻³⁸ in comparison with either Flag-CTPS^WT control line by Student's t-test; n, number of egg chambers counted for each genotype. Error bars here and in D represent standard error of the mean (s.e.m.). (D) Relative CTPS RNA expression (endogenous plus transgene) from ovaries of the indicated genotype was quantified by qRT-PCR. *, P-values in comparison to control pCOG-Gal4 flies are 0.029, 0.013, 0.007 (left to right). N denotes the number of independent experiments. Fig. S1 shows similar qRT-PCR data using FLAG-CTPS transgene-specific primers.

Fig. 2.

Transgenic expression of CTPS^H355A dominantly inhibits assembly of endogenous wild-type CTPS. Ovaries from GFP-CTPS-expressing flies also expressing either (A) Flag-CTPS^WT-M1 or (B) Flag-CTPS^H355A-M2 under pCOG-Gal4 control were stained with FLAG-specific antibodies (red) and DAPI (blue). Yellow color indicates overlap between red and green channels. Stage 4, 6 and 8 egg chambers are bracketed. (C) Quantification of the percent of stage 4, 6 and 8 egg chambers with CTPS filaments of the indicated genotypes. *, P<9.98×10⁻¹⁴ in comparison with either Flag-CTPS^WT control line (Student's t-test); n, number of egg chambers counted for each genotype. Error bars here and below represent s.e.m. (D) Expression of the GFP-CTPS reporter was quantified by qRT-PCR from ovaries expressing the indicated transgene. Reporter expression was not significantly different across the four groups (ANOVA P=0.7815).

Fig. 3.

CTPS polymerization in ovarian germline cells supports egg production. Representative fluorescent images of stage 10 egg chambers from (A) Flag-CTPS^WT-M1 or (B) Flag-CTPS^H355A-M1 flies fed standard food stained with Phalloidin-488 (green) to reveal the actin network underlying germline cell plasma membranes. DNA is stained with Propidium Iodide (magenta). (C,D) Similar staining for flies fed food containing the glutamine antimetabolite DON (10 µM). (E) Germline cell nuclear diameter was measured from images of Propidium Iodide-stained nuclei of stage 8 egg chambers of the four genotypes in flies reared on either standard food or DON-containing food. No consistent difference was observed between Flag-CTPS^WT and Flag-CTPS^H355A lines, although DON increased nuclear diameter across all genotypes (Student's t-test for all pairwise comparisons P<1.11×10⁻⁹). Number of nuclei counted for each genotype (n) are shown. Here and below, error bars denote s.e.m. (F,G) GFP-CTPS fluorescence (green) and DNA (magenta) in ovarioles of flies expressing the indicated transgenes and fed 10 µM DON-containing food for 4 days. (H) Eggs laid over 24 h per fly expressing the indicated CTPS transgene driven by pCOG-GAL4 and fed either standard food or DON-containing food were quantified. n indicates the number of times the experiment was replicated. P-values (*) left to right were 0.038 and 0.0014. (I) qRT-PCR quantification of CTPS transgene expression in ovaries in which the indicated transgene was driven by nosGal4:VP16. was quantified by qRT-PCR. Transgene expression was not significantly different (t-test). (J) FLAG western blot of ovaries expressing the indicated FLAG-tagged CTPS transgenes as in I. The lower panel shows a corresponding Coomassie-stained gel of the major yolk proteins as a loading control. (K) Egg-laying assay as in H for nosGal4:VP16-driven Flag-CTPS^WT-M1 and Flag-CTPS^H355A-M1 lines. P-values (*) left to right were 0.0358 and 0.0124. (L) Pupae production. Eggs laid over 7 days were allowed to develop in the presence or absence of DON and pupae were counted after 7 days. P-values (*) left to right were 0.3223 and 0.0158.