# An Update on Polyphosphate In Vivo Activities
## 8 PolyP Signalling  
Established functions of polyP in prokaryotic cells include storage for energy, P\({}_{i}\) and Ca\({}^{2+}\), biofilm formation, biomineralisation, and bone formation [2; 32; 129; 130] (Table 2). Importantly, intracellularly, polyP activates mTOR and, thus, promotes cell proliferation and growth [131]. The exopolynophostatase h-prune provides another link of polyP and mitogenic activity. The binding of h-prune to mm23-h1, a metastasis suppressor, inhibits h-prone phosphatase activity [52]. Defective phosphatase activity, at least in yeast, increases polyP content and might provide a rationale for h-prune expression in cancer associated with poor prognosis via augmented metastasis and tumour growth [52; 80]. The precise function of polyP in cancer is likely complex and might be cell-type specific. In myeloma cancer cells, the intracellular polyP level is significantly higher than that of \"healthy\" primary plasma cells. The addition of synthetic polyP initiates apoptosis of myeloma cells [132]. However, the underlying mechanisms are unclear and might include a change in pH, complexation of surface-bound ions, activation of specific signalling pathways, or local increase in P\({}_{i}\). In addition to regulating basal cellular functions, it has been suggested that polyP is covalently attached to target proteins [133], e.g., nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1; polyphosphorylation), on lysine residues within conserved N-terminal polyacidic serine and lysin- (PASK) or histidine-rich clusters [134; 135]. However, recent studies have challenged the hypothesis of polyP being a novel noncovalently attached post-translational modification [136; 137]. Lysine and histidine residues are positively charged at physiological pH, and these amino acids bind tightly electrostatically (but not covalently) to the negatively charged polyP backbone. The conserved histidine a-helical domain (CHAD) is an example of a polyP-binding protein found in several procaryotic cells [138]. Extracellular polyP has emerged as a procoagulant and proinflammatory mediator. Due to its high biological activity and link to disease states, the polymer has potential diagnostic implications as a (predictive) biomarker with multiple methods for polymer analysis (Table 3). Intercalating dyes can visualise polyP in histology upon separation in agarose and urea gels and individual cells by flow cytometry (FACS) [1]. Specificity for polyP upon UREA-PAGE separation is increased by photobleaching (negative DAPI staining), where polyP appears dark and DNA or proteoglycans turn whitish [36; 77; 132]. DAPI binds both to polyP and DNA but differs in its emission wavelength. DAPI bound to DNA emits a greenish colour around 461 nm, while DAPI/polyP is bluish at 525 nm [139]. As the dyes bind to helical polyP portions, the polymer backbone needs to exceed >15 P\({}_{i}\) to allow for detection. Dyes JC-D7 and JC-D8 differ in emission spectrum intensity depending on the polyP chain length with >15 P\({}_{i}\)[140]. Elegant work using FITC-labelled polyP has shown that cells endocytose polyP microparticles. Using FITC-polyP allows for intracellular polyP staining and the analyses of transport and enrichment of (labelled) polyP to distinct cellular compartments depending on cell activation, metabolic state, and environment [141]. For flow cytometry and histology, neutral red was initially used to visualise polyP [142]. The polyP-binding domain of PPX (PPBD, synonymous to PPX_\(\Delta\)12) selectively binds to polyP with chain length > 38 P\({}_{i}\) units [142]. PPBD [143] has been used in histology and FACS to visualise and quantify the polymer in an experimental setting and might provide a lead structure for a diagnostic polyP probe with clinical implications. Furthermore, CHADs bind to polyP-rich granules in vivo and may provide an alternative investigation for polyP [144].