Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis Francesca Pisani 1,2 , Thomas Livermore 1 , Giuseppina Rose 2 , Jonathan Robert Chubb 1 , Marco Gaspari 3 , Adolfo Saiardi 1 * 1 Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, United Kingdom, 2 Department of Biology, Ecology and Earth Science, University of Calabria, Rende, Italy, 3 Laboratory of Proteomics and Mass Spectrometry, Department of Experimental and Clinical Medicine, ‘‘Magna Græcia’’ University of Catanzaro, Catanzaro, Italy Abstract The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP 6 or Phytic acid) and its derivative inositol pyrophosphates, IP 7 and IP 8 . Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP 9 in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP 5 ) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP 8 was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP 5 K, doesn’t’ play a major role in the IP 8 developmental increase. Citation: Pisani F, Livermore T, Rose G, Chubb JR, Gaspari M, et al. (2014) Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis. PLoS ONE 9(1): e85533. doi:10.1371/journal.pone.0085533 Editor: Thierry Soldati, Universite ´ de Gene `ve, Switzerland Received October 8, 2013; Accepted November 28, 2013; Published January 9, 2014 Copyright: ß 2014 Pisani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Medical Research Council funding of the Cell Biology Unit. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dmcbado@ucl.ac.uk Introduction The model organism Dictyostelium discoideum, originally developed to study the transition to multicellularity, has subsequently been utilised in several areas of biology from chemotaxis [1] to transcriptional control [2]. Upon exhaustion of nutrients, the Dictyosteliidae slime moulds are able to aggregate into multicel- lular forms, a process regulated by cAMP signaling [3]. The aggregated slugs develop into fruiting bodies (or sporocarp) compromised of two main cell types; stalk cells and thousands of spore cells. Much of the early work with this amoeba focused on this fascinating behaviour. However, in the late 1980s this model organism began to offer insight into the metabolism of inositol phosphates [4]. In fact, it was in D. discoideum that the synthesis of inositol hexakisphosphates (IP 6 ) through direct phosphorylation of inositol was discovered [5]. D. discoideum has also been instrumental in the discovery of inositol pyrophosphates (also known as diphosphoinositol phos- phates) (For reviews see [6,7]) molecules containing highly energetic pyrophosphate moiety(ies) recently implicated into the regulation of cellular homeostasis [8,9,10]. Inositol pyrophos- phates were identified in 1993 in D. discoideum [11] and in mammalian cell [12]. During the 1990s the synthesis of the inositol pyrophosphate IP 7 (diphosphoinositol pentakisphosphate or PP- IP 5 ) and, in particular, IP 8 (bisdiphosphoinositoltetrakisphosphate or (PP) 2 -IP 4 ) was linked to the D. discoideum developmental program [13]. Furthermore, thanks to the high concentration of these molecules in this amoeba, NMR could be used to resolve the isomeric nature of IP 7 and IP 8 extracted from D. discoideum cells. The structure of these isoforms - the 5PP-IP 5 isomer of IP 7 and the 5,6(PP) 2 -IP 4 isomer of IP 8 [14] are, to date, the only resolved structures of inositol pyrophosphates extracted from cells. Despite the influence of this organism, D. discoideum has faded from the attentions of inositol phosphate scientists over time. The last study demonstrating the importance of inositol pyrophosphate in regulating Dictyostelium chemotaxy was published over 10 years ago [15]. This disengagement is in part due to the PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e85533