Polyphosphate extraction and quantification from flies.

A) Linear polyphosphate. B) 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-day post-pupariation pupae (n=20, N=8), pharate (n=10, N=5), three-day 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.

Polyphosphate staining of Drosophila tissues with 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 colocalization within the nucleolus. Scale bar for (A i,ii,B-C) - 20µm, scale bar for (A iii,iv) - 5µm. 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 - 20µm. Insets reveal the nucleus and part of the cytoplasm across stages S4-S9, showing reduced polyphosphate signals. Scale bar - 5µm. 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) - 5µm. Drosophila strain used - CantonS.

FLYX-Transgenic fly lines with 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 - 10µm

Genetic depletion of 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 x 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 x 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 x 2200.

Genetic depletion of 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 hours APF for control and 120 hours 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 matched larvae. (C-E) GO analysis in Cyto-FLYX and control larvae showing Biological processes (C), Cellular components (D), and Molecular functions (E).