Probing metazoan polyphosphate biology using Drosophila reveals novel and conserved polyP functions
- Tata Institute of Fundamental Research, India
- Centre for DNA Fingerprinting and Diagnostics, India
- Graduate Studies, Regional Centre for Biotechnology, India
- Institute of Organic Chemistry, Albert-Ludwigs-Universität Freiburg, Germany
- CIBSS – Centre for Integrative Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Germany
Figures
Figure 1 with 1 supplement
Polyphosphate extraction and quantification from flies.
(A) Linear polyphosphate. (B) Polyphosphate (PolyP) quantification from sets of 5, 10, 20 third instar larvae (in Pi terms), N=5 in each set. Statistics: Student’s t-test p>0.001, error bar - s.e.m. Drosophila strain used - CantonS. (C) Quantification of polyphosphate across embryonic stages. n=75 in each time point, N=3. (D) Quantification of polyP across a fly life cycle: embryos (n=75, N=8), first instar larvae (n=25, N=5), second instar larvae (n=25, N=5), feeding third instar larvae (n=10, N=5), non-feeding third instar larvae (n=10, N=5), prepupae (n=20, N=3), one-days-post-pupariation pupae (n=20, N=8), pharate (n=10, N=5), three-days-post-eclosion adult (n=10, N=5). Statistics: Student’s t-test with p>0.001, error bar - s.e.m. Drosophila strain used - CantonS.
Figure 1—figure supplement 1
Schematic of polyP extraction and estimation.
(A) Schematic of citrate-saturated phenol-chloroform-based polyphosphate extraction from Drosophila larvae. (B) Schematic of polyphosphate quantification using Malachite Green.
Figure 2 with 1 supplement
Validation of PolyP Binding Domain (PPBD) as a probe for polyphosphates in fly tissues.
(A) Schematic of Polyphosphate (PolyP) detection using PPBD fused at the N-terminus with a GST tag. (B) PPBD and PPBDMut predicted structural models using ColabFold v1.5.5: AlphaFold2 using MMseqs2 (free online). The amino residues marked in red are changed to Alanine (marked in orange). Overall alignment shows a r.m.s.d. Value of 0.024. (C) Binding characteristic of GST, GST-PPBD, and GST-PPBDMut with polyP100-2X-FITC using MST. Graph showing GST-PPBD fraction bound to polyP, whereas the GST-PPBDMut and GST does not bind to polyP100-2X-FITC. (D) Staining of hemocytes with GST, GST-PPBD, and GST-PPBDMut. Hml-GAL4>UAS GFP reporter was used to identify plasmocytes. Negative control cells incubated with GST and GST-PPBD Mutant proteins separately and stained with anti-GST antibody (GST; orange hot). For polyP staining, cells were incubated with GST-PPBD protein and stained with anti-GST antibody (GST-PPBD-polyphosphate; orange hot). Scale bar - 5 µm. (E) Staining of hemocytes with GST-PPBD in fixed and permeabilised hemocytes with 2 hr pretreatment with buffer (control) and heat inactivated ScPpX1, and active ScPpX1 proteins. GST-PPBD proteins were stained with anti-GST antibody (GST-PPBD-polyphosphate; orange hot), and the cell is marked by Cell Mask Membrane staining dye (green). Scale bar - 5 µm. (F) Graph showing reduced anti-GST staining intensity in hemocytes treated with ScPpX1. (G) Graph showing reduced PPBD punctae intensity in hemocytes treated with ScPpX1. Statistics: One-way Annova with p>0.001, error bar - s.d. Drosophila strain used - CantonS.
Figure 2—figure supplement 1
GST::PolyP Binding Domain (PPBD) staining in fly tissues.
(A) GST::PPBD staining using different fixatives. Salivary glands staining with GST-PPBD and GST control in Bouin’s fixative. Salivary glands stained with GST-PPBD and GST control in methanol fixative. Staining of larval salivary glands with DAPI (nucleus; cyan) and anti-GST antibody (GST; orange hot). The greyscale panel represents the GST channel. Scale bar - 20 µm. Drosophila strain used - CantonS.
Figure 3 with 1 supplement
Polyphosphate staining of Drosophila tissues with PolyP Binding Domain (PPBD).
(A–C) Staining of larval salivary glands with DAPI (nucleus; cyan) and anti-GST antibody (GST; orange hot). (A i, iii, and B) Samples incubated with GST (negative control). (A ii, iv, and C) Samples incubated with GST-PPBD. (B–C) Salivary gland stained with DAPI (cyan) anti-fibrillarin antibody (magenta) and anti-GST antibody (orange hot) to detect polyphosphate colocalisation within the nucleolus. Scale bar for (A i, ii, B-C)- 20 µm, scale bar for (A iii, iv) - 5 µm. Drosophila strain used - CantonS. (D) Staining of fly ovaries. Negative control: Incubated with GST and stained with DAPI (nucleus; cyan) and anti-GST antibody (GST; orange hot). Stages covered - S1-S8. Incubated with GST-PPBD and stained with DAPI (nucleus; cyan) and anti-GST antibody (GST-PPBD-polyphosphate; orange hot). Stages covered - S1-S9. Yellow arrow- follicle cells, Orange arrow- nurse cells. Scale bar - 20 µm. Insets reveal the nucleus and part of the cytoplasm across stages S4-S9, showing reduced polyphosphate signals. Scale bar - 5 µm. Drosophila strain used - CantonS. (E–H) GST-PPBD staining of hemocytes. Hml-GAL4>UAS GFP reporter was used to identify plasmocytes (E–F). Lz-GAL4>UAS mCD8::GFP reporter was used to identify crystal cells (G–H). Negative control (E and G) - cells incubated with GST protein and stained with DAPI (nucleus; blue) and anti-GST antibody (GST; orange hot). For polyP staining (F and H), cells were incubated with GST-PPBD protein and stained with DAPI (nucleus; cyan) and anti-GST antibody (GST-PPBD-polyphosphate; orange hot). Scale bar of (A–D) - 5 µm.
Figure 3—figure supplement 1
Polyphosphate staining of several Drosophila tissues with PolyP Binding Domain (PPBD).
(A, C, E, G, I, K, M, O) Samples were incubated with GST-PPBD and stained with DAPI (nucleus; cyan) and anti-GST antibody (orange hot or greyscale). (B, D, F, H, J, L, N, P) Negative control: tissues were incubated with GST protein and stained with DAPI (nucleus; cyan) and anti-GST antibody (GST; orange hot or greyscale). Scale bar - 20 µm. Drosophila strain used - CantonS.
Figure 4
FLYX- Transgenic fly lines with Polyphosphate (polyP) depletion expressing ScPpX1.
(A) Schematic of creation of FLYX by cloning S. cerevisiae ScPpX1 cDNA into pUAST-attB vector suitable for expression in flies, followed by injection into embryos. (B) Schematic of Drosophila UAS-GAL4-based protein expression system. (C) Schematic of different FLYX lines of the FLYX library- CytoFLYX, Nuc-FLYX, Mito-FLYX, and ER-FLYX. (D) PolyP quantification from third instar larvae of tubulin-GAL4 driven control (AttP40) and Cyto-FLYX, N=10. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. (E) Localisation of HA-ScPpX1 to the target organelles in different FLYX larval muscles- DaGAL4>CytoFLYX, stained for HA in magenta and nucleus (DAPI in green); DaGAL4>Nuc FLYX, stained for HA in magenta and nucleus (DAPI in green); DaGAL4>ER FLYX, stained for HA in magenta and ER (calnexin in green); Mef2GAL4>Mito FLYX, stained for HA in magenta and mitochondria (ATP5A in green). Scale bar - 10 µm.
Figure 5 with 2 supplements
Genetic depletion of Polyphosphate (polyP) shows clotting defects in flies.
(A–D) Clot phenotype analysis of tubulin-GAL4 driven Cyto-FLYX (tubulin-FLYX). The control is tubulin-GAL4>AttP40. (A) Clot structure of control (AttP40) and FLYX. The scale bar is 500 pixels, and the image dimensions in pixels are 2688×2200. (B–D) Quantification of relative clot fibre number density N=3 (B), clot fibre branch point number density N=3 (C) and clot fibre length, N=4 (D) of FLYX line with respect to control. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. (E–P) Analysis of clot fibre number, branching, and length phenotype upon FLYX driven by cg-GAL4 (E–H), hml-GAL4 (I–L), and lz-GAL4 (M–P) with respect to control (GAL4>AttP40). Scale bar- 500 px, Image dimensions in pixels: 2688×2200. For quantifications - N=5. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. (Q–S) Clot phenotype of tubulin-GAL4 driven Cyto-FLYX with exogenous Pi (Q) and polyP65 addition (R) and quantification of fibre number density (S). Scale bar- 500 px, Image dimensions in pixels: 2688×2200.
Figure 5—figure supplement 1
Polyphosphate affects hemolymph clotting.
(A–E) Depiction of hemolymph clotting revealed by fibre formation at the edge of the hemolymph drop. (B–C) Control (AttP40 strain) (B) showing enriched with clot fibres and hml mutant (C) with minor clot fibres at the edges of the drop. (D–E) Quantification of hemolymph clot fibre number (D) and length (E) in Control and hml mutants. (F–V) Hemolymph clotting analysis followed by ex vivo Polyphosphate (polyP) treatment. The relative number of clot fibres formed at the edge of the hemolymph drop incubated with (F) water, N=5, (G) Pi, N=5, (H) PolyP14, N=4, (I) PolyP65, N=5, (J) with PolyP130, N=4. (K–R) Quantifications of fibre numbers and length in F-J. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. (S–V) Hemolymph clot fibre branching analysis. Fold change of clot fibre branches in F-J. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. Scale bar for (J–N)- 500 px, Image dimensions in pixel: 2688×2,200.
Figure 5—figure supplement 2
Hemolymph clotting assay.
(A–E) Clot fibre formation and quantification of fibre phenotype upon 30 min incubation with water (A), Pi (B), P14 (C), P65 (D), P130 (E). Scale bar for (A–E)- 500 pixels. Image dimensions in pixels: 3513×2712. Statistics: Student’s t-test with p>0.001, error bar - s.e.m. (F–I) Clot fibre formation and quantification of fibre phenotype upon 30 min incubation with (F) water, (G) P65 (250 µM Pi terms), (H) P65 (125 µM Pi terms). Statistics: Student’s t-test with p>0.001, error bar - s.e.m.
Figure 6 with 3 supplements
Genetic depletion of Polyphosphate (polyP) shows accelerated eclosion.
(A) Cumulative percentage of eclosion of control and FLYX checked after white prepupa formation (APF), m=213 (control), 268 (FLYX), N=4 (control), N=5(FLYX). (B) Time of 50% eclosion of the control and FLYX flies- ~134 hr APF for control and 120 hr APF for FLYX, m=213 (control), 268 (FLYX), N=4 (control), N=5(FLYX). Statistics: Student’s t-test with p>0.001, error bar - s.e.m. RNA sequencing of tubulin-GAL4 driven Cyto-FLYX (tubulin>FLYX) and tubulin-GAL4>AttP40 control third instar non-feeding wandering age-atched larvae.
Figure 6—figure supplement 1
Phenotypic analysis of Cyto-FLYX w.r.t control.
(A) Cyto-FLYX does not change larval weight, where sets of 10 wandering third instar larvae taken from bottle are ethanol and water washed and weighed in a microcentrifuge tube. m=10, N=5. (B) Cyto-FLYX does not change larval length, where wandering third instar larvae taken from bottles are ethanol and water washed, kept on a slide, and imaged, N=29. (C) Cyto-FLYX does not change adult fly weight, where sets of 10 5-day-old adult flies taken from bottles are weighed in a microcentrifuge tube. m=10, N=6. (D) Cyto-FLYX does not change pupal size (length), where >65 mid pupa (1-day-old pupa) taken from bottles are kept on a slide and imaged, N=30. (E–F) Cyto-FLYX does not change fly fecundity. (E) Expression of pUASp-Cyto-FLYX driven by NosGAL4 in adult ovaries (3-day-old flies) checked by anti-HA staining in magenta showing expression in ovarian stages. (F) Cyto-FLYX does not show any difference in the number of eggs laid per fly every 24 hr w.r.t control flies. 10 sets of five females mated with males for two days were scored for next 24 hr after a 12 hr of acclimatisation. Student’s t-test with p>0.001, error bar - s.e.m.
Figure 6—figure supplement 2
Transcriptomic analysis of TubGAL4 driven Cyto-FLYX w.r.t. control.
(A) qRT-PCR of RNA extracted from third instar non-feeding wandering third instar larvae shows no change in TOR, InR, Ecd related major transcripts between Cyto-FLYX and control. (B) Principal Component Analysis of two biological replicates of samples- Cyto-FLYX and control, both driven by TubGAL4.
Figure 6—figure supplement 3
Transcriptomic analysis of TubGAL4 driven Cyto-FLYX w.r.t. Control, GSEA, and heat map.
(A–C) GO analysis in Cyto-FLYX and control larvae showing Biological processes (A), Cellular components (B), and Molecular functions (C), (D) Heatmap of three gene sets enriched in Cyto-FLYX versus control larval RNA samples from the GO analysis - showing genes from mitochondrial translation, cytoplasmic translation, and ribosome biogenesis processes.
Chemical structure 1
Polydisperse diamine-linked polydisperse PolyP100.
Chemical structure 2
Bis-FITC-labeled PolyP100 (PolyP100-2X-FITC).
Author response image 1
Author response image 2
Author response image 3
Tables
Table 1
Absolute quantification of Polyphosphate (polyP) levels.
| PolyP quantification | |
|---|---|
| Normalised to protein content | 46.80±3.95 picomoles in Pi terms/mg protein |
| Normalised to number of larvae | 419.3±36.83 picomoles in Pi terms/larva |
Table 2
Polyphosphate (PolyP) quantification from embryo (Figure 1C).
| Embryo stages (hours) | polyP (pmoles of Pi/embryo) |
|---|---|
| 1–3 hr | 7.961±3.27 |
| 3–5 hr | 8.825±3.57 |
| 5–7 hr | 7.135±1.39 |
| 7–9 hr | 6.930±1.48 |
| 9–11 hr | 22.47±13.22 |
| 12–14 hr | 14.57±4.49 |
| 14–16 hr | 10.26±3.78 |
| 16–18 hr | 13.67±3.38 |
Table 3
Polyphosphate (PolyP) quantification across fly life cycle (Figure 1D).
| Developmental stages | polyP (pmoles of Pi/mg protein) |
|---|---|
| Embryo | 2.808±1.0 |
| First instar | 1.714±0.94 |
| Second instar | 2.038±1.0 |
| Feeding third instar | 1.002±0.53 |
| Non-feeding third instar (wandering) | 44.06±7.04 |
| Prepupa | 15.63±1.34 |
| One day post-pupariation | 16.88±3.93 |
| Pharate | 1.398±0.24 |
| Three days post-eclosion | 2.445±0.78 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/104841/elife-104841-mdarchecklist1-v1.pdf
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Supplementary file 1
Supporting information.
- https://cdn.elifesciences.org/articles/104841/elife-104841-supp1-v1.docx
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Supplementary file 2
RNA sequencing data.
- https://cdn.elifesciences.org/articles/104841/elife-104841-supp2-v1.xlsx
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Probing metazoan polyphosphate biology using Drosophila reveals novel and conserved polyP functions
eLife 14:RP104841.
https://doi.org/10.7554/eLife.104841.3
