Figure 1.
The Drosophila melanogaster CG6854/CTPsyn gene locus encodes three isoforms.
(A) Genome Browser view of the CG6854/CTPsyn gene locus (www.flybase.org). In two protein trap lines (CA06746 and CA07332), GFP was trapped between the first and second exons of the CTPsyn isoform. (B) The protein map of three isoforms of CTPsyn. UTP binding sites are indicated by red triangles.
Figure 2.
Expression profiles of Drosophila CTPsyn isoforms as revealed by qPCR.
(A) embryos and larvae. (B) S2 cells and pupae. (C) Adult flies and tissues. WPP, white pre-pupae. P1–P4, pupal stages 1–4.
Figure 3.
Overexpression of different CTPsyn isoforms in germline cells using nos-GAL4::VP16 driver.
(A–C) Cytoophidia revealed by CTPsyn-GFP in an egg chamber from a protein trap line CA06746. (D–F) CTPsyn isoform A is localized in punctate structures in the nucleus and does not affect endogenous cytoophidia. (G–I) CTPsyn isoform B is dispersed in the cytoplasm of the egg chamber and does not affect endogenous cytoophidia. (J–L) C-terminal-tagged CTPsyn isoform C induced longer and more curved cytoophidia. (M–O) N-terminal-tagged CTPsyn isoform C affects cytoophidia morphology. Iso, Isoform. Scale bars, 20 µm.
Figure 4.
(A) Genetic map of CTPsyn and the insertion site of p-element in three mutants CTPsynd6099, CTPsyne01207 and CTPsynd07411. (B) Comparison of the expressions of CTPsyn isoforms in larvae from y w and three CTPsyn mutants (CTPsynd6099, CTPsyne01207 and CTPsynd07411). While dramatic decrease of isofrom C expression occurs in CTPsynd6099 and CTPsyne01207, isoform A expression is hugely diminished in CTPsynd07411.
Figure 5.
CTPsyn mutants survive until larval stages.
CTPsynd6099 homozygous mutants (−/− in A–E) and CTPsyne01207 homozygous mutants (−/− in F–J) survive until 7 days after egg deposition (DAE) with slower growth compared to heterozygous mutants (+/−) or y w (+/+). (K–M) CTPsynd07411 homozygous mutants (−/−) show severely delayed development, in comparison with heterozygous mutants (+/−and y w (+/+). CTPsynd07411 (−/−) mutant larvae only survive until 5 days after egg deposition (DAE) with very little growth. Heterozygous mutants were balanced with TM6B Tb (+/− in all panels).
Figure 6.
Clonal analysis of three CTPsyn mutations in adult ovaries.
(A–C) CTPsynd6099. (D–F) CTPsyne01207. (G–I) CTPsynd07411 In all three mutations, no cytoophidia are detected in mutant mitotic clone (dotted lines) as indicated by lack of GFP, while cytoophidia (labelled by an antibody against CTPsyn in red) are clearly seen in adjacent wild-type germline cells (GFP positive). Scale bar, 20 µm.
Figure 7.
Overexpression of CTPsyn isoform C induces the assembly of cytoophidia in Drosophila follicle cells.
(A, B) Cytoophidia revealed by CTPsyn-GFP in protein trap line CA06746. (C, D) Long cytoophidia are induced in stage-10 follicle cells overexpressing CTPsyn isoform C. (E, F) Each follicle cell contains only one long cytoophidium in a stage-7 egg chamber. The cell boundary is outlined by membrane protein Hu-li tai shao (Hts). (G) Within the same stage 10 egg chamber, cytoophidia in follicle cells overexpressing CTPsyn isoform C (GFP+ cells) are much longer and thicker than those in neighbouring wild-type follicle cells (GFP− cells). While the average length of cytoophidia in GFP− cells is only 3.97±0.61 µm (n = 41), cytoophidia in GFP+ cells are 20.76±5.60 µm long on average (n = 32). Note that cytoophidia in GFP+ cells in E are much longer than those in C and D, which could be due to different expression of GAL4 drivers in follicle cells (actin-GAL4 in C, D and tubulin-GAL4 in E). Scale bars, 20 µm.
Figure 8.
Overexpression of CTPsyn isoform C induces macro-cytoophidia in Drosophila embryos.
(A, C, E) No macro-cytoophidium formation can be detected in y w embryos at stage 1 (A), 12 (C) and 15 (E). (B, D, F) Overexpressing CTPsyn isoform C induces macro-cytoophidia in embryos at stage 1 (B), 12 (D), and 15 (F). Segments in embryos are labeled by a transcription factor Engrailed (red). IsoC, isoform C. Scale bars, 20 µm.
Figure 9.
Ectopic expression of transgenes in egg chambers using MTD-GAL4 driver.
(A–C) UAS-Venus-N-term which consists of the 56 amino acids shows dispersed cytoplasmic expression. (D–I) UAS-Trun isoC-Venus forms punctate structures in the cytoplasm. The punctate structures are more visible in egg chambers at later stages (G–I) than in those at early stages (D–F). (J–L) UAS-Venus-Trun isoC shows dispersed distribution in the cytoplasm. (M–O) Ectopic expression of UAS-SD-Venus disrupts endogenous cytoophidium formation in the germline cells. Note that there is no expression of UAS-SD-Venus in follicle cells in which cytoophida are not affected. (P–R) UAS-GAT-Venus shows disperse distribution in the cytoplasm. See Figure S6 for the structures of the constructs. N-term, a N-terminal segment (56-aa) of CTPsyn isoform C; Trun IsoC, truncated isoform C. SD, synthetase domain; GAT, type 1 glutamine amidotransferase domain. Scale bars, 20 µm.
Figure 10.
Clonal expression of CTPsyn domains in follicle cells using inducible driver.
(A–F) Ectopic expression of UAS-SD-Venus disrupts endogenous cytoophidium formation in follicle cells (GFP+ cells), while cytoophidia are detectable in wild-type follicle cells (GFP− cells, within dotlined-area). (A–C) Surface view. (D–F) Lateral view. (G–I) Ectopic expression of UAS-Venus-GAT has no obvious effect on cytoophidium formation in follicle cells. Note that cytoophidia are detectable in both GFP+ (where the transgene is expressed) and GFP− cells (dotlined, wild-type). SD, synthetase domain; GAT, type 1 glutamine amidotransferase domain. Venus (C), cytoplasmic signal from Venus. GFP (N), nuclear signal from GFP. Scale bars, 20 µm.