Inositol phosphates induce DAPI fluorescence shift

Kolozsvári, Bernadett; Parisi, Federica; Saiardi, Adolfo

doi:10.1042/bj20140237

Cited by 30 publications

(32 citation statements)

References 39 publications

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“…Because phytic acid can also interact and fluoresce with DAPI at the same wavelengths as polyphosphate (24), conditioned medium was treated with the phytic acid-degrading enzymes phytase and DDP1 (25,26). Phytase or DDP1 treatments had no effect on DAPI fluorescence of conditioned medium or on the activity of the stationary phase factor (Fig.…”

Section: Resultsmentioning

confidence: 99%

Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum

Suess

Gomer

2016

Journal of Biological Chemistry

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Polyphosphate is a polymer of phosphate residues linked by high energy phosphoanhydride bonds. Despite being highly conserved throughout nature, its function is poorly understood. Here we show that Dictyostelium cells accumulate extracellular polyphosphate, and this acts to inhibit proliferation at high cell densities. In shaking culture, extracellular polyphosphate concentrations increase as cell density increases, and if the concentration of polyphosphate observed at the stationary phase is added to cells at mid-log, proliferation is halted. Adding an exopolyphosphatase to cell cultures or stationary phase conditioned medium decreases polyphosphate levels and abrogates the anti-proliferative effect. The cells show saturable binding of polyphosphate, suggesting the presence of a cell surface polyphosphate receptor. Extracellular polyphosphate accumulation is potentiated by decreased nutrient levels, potentially as a means to anticipate starvation. Loss of the Dictyostelium polyphosphate kinase DdPpk1 causes intracellular polyphosphate levels to become undetectable and negatively affects fitness, cytokinesis, and germination. However, cells lacking DdPpk1 accumulate ϳ50% normal levels of extracellular polyphosphate, suggesting an additional means of synthesis. We found that cells lacking inositol hexakisphosphate kinase, which is responsible for the synthesis of the inositol pyrophosphates IP7 and IP8, reach abnormally high cell densities and show decreased extracellular polyphosphate levels. Two different enzymes thus appear to mediate the synthesis of Dictyostelium extracellular polyphosphate, which is used as a signal in an autocrine negative feedback loop to regulate cell proliferation.

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Section: Resultsmentioning

confidence: 99%

Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum

Suess

Gomer

2016

Journal of Biological Chemistry

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“…This discrepancy is likely due to the different assays used to quantify polyP: namely the direct visualisation of polyP on gel, or by measuring polyPinduced DAPI (4',6-Diamidino-2-Phenylindole) red fluorescence shift [74]. The latter assay has pitfalls as other phosphate-rich molecules, such as IPs [75] and amorphous calciumphosphate [76], induce the same type of fluorescence. Thus, unlike in yeast, the direct control of polyP synthesis by PP-IPs is not seen in D. discoideum.…”

Section: Inorganic Polyphosphate As a Eukaryote Phosphate Buffermentioning

confidence: 96%

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

Azevedo

Saiardi

2017

Trends in Biochemical Sciences

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129

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“…Several polyP detection methods lack specificity (23). Furthermore quantification of polyP using traditional biochemical assays, such as the PPK assay (8) or measurement of free phosphate (Pi) after polyP hydrolysis, are greatly impaired by the inability to extract polyP without copurifying other phosphate rich molecules (see below).…”

Section: Discoideum Possesses Polypmentioning

confidence: 99%

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Livermore

Chubb

Saiardi

2016

Proc. Natl. Acad. Sci. U.S.A.

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Inorganic polyphosphate (polyP) is composed of linear chains of phosphate groups linked by high-energy phosphoanhydride bonds. However, this simple, ubiquitous molecule remains poorly understood. The use of nonstandardized analytical methods has contributed to this lack of clarity. By using improved polyacrylamide gel electrophoresis we were able to visualize polyP extracted from Dictyostelium discoideum. We established that polyP is undetectable in cells lacking the polyphosphate kinase (DdPpk1). Generation of this ppk1 null strain revealed that polyP is important for the general fitness of the amoebae with the mutant strain displaying a substantial growth defect. We discovered an unprecedented accumulation of polyP during the developmental program, with polyP increasing more than 100-fold. The failure of ppk1 spores to accumulate polyP results in a germination defect. These phenotypes are underpinned by the ability of polyP to regulate basic energetic metabolism, demonstrated by a 2.5-fold decrease in the level of ATP in vegetative ppk1. Finally, the lack of polyP during the development of ppk1 mutant cells is partially offset by an increase of both ATP and inositol pyrophosphates, evidence for a model in which there is a functional interplay between inositol pyrophosphates, ATP, and polyP.inorganic polyphosphate | phosphate | metabolism | inositol pyrophosphate | mitochondria T he simplest biological polymer is inorganic polyphosphate (polyP), which consists of a linear chain of phosphates linked by high-energy phosphoanhydride bonds (1). Regardless of its origin, polyP is ubiquitously present in today's living organisms. The synthesis, enzymology, and role of polyP are best characterized in the bacteria Escherichia coli (2). However, polyP is also present in archaea (3), eukaryotes (4), and human cells (5). Whilst polyP is present in virtually all cellular compartments (6), it accumulates in acidocalcisomes, organelles common to bacteria and mammalian cells (7).PolyP has been implicated in a range of cellular processes over recent years, but its most basic proposed function is to act as a buffer for the level of free phosphate (8). Phosphate buffering is essential for general metabolism, and the synthesis or degradation of polyP is able to safeguard the level of free phosphate in cells. In addition, the polyanionic nature of polyP makes it a strong chelator of bivalent cations; thus polyP plays a key role in cation homeostasis. The chelation of calcium by polyP might even play a role as a calcium sink, regulating calcium signaling (8). Besides these common roles, many specific functions have been attributed to polyP in eukaryotes (see reviews in refs. 9-11). Recently its ability to function as a protein-folding chaperone (12) and to drive a newly discovered posttranslational protein modification, protein polyphosphorylation (13), have provided further insight into the mechanism of action of this simple polymer.Little is known about the synthesis or metabolism of polyP in eukaryotes. The yeast Saccharomyces c...

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Inositol phosphates induce DAPI fluorescence shift

Cited by 30 publications

References 39 publications

Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum

Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

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