Abstract
Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.
Highlights
Inorganic phosphate (Pi) is a component of nucleic acids and phospholipids, plays key roles in signal transduction cascades, and is a substrate for the generation of ATP via glycolysis and oxidative phosphorylation
The use of a membrane-targeted sensor may lead to intermolecular fluorescence resonance energy transfer (FRET), but this would contribute to baseline FRET and have minimal effect on measurements of changes in Pi dependent FRET
Previous studies showed that intestinal Pi metabolism is critical for growth and development of C. elegans [24,25,26]
Summary
Inorganic phosphate (Pi) is a component of nucleic acids and phospholipids, plays key roles in signal transduction cascades, and is a substrate for the generation of ATP via glycolysis and oxidative phosphorylation. The concentrations of Pi in different cells and both intra- and extra-cellular compartments must, be maintained within certain limits, despite fluctuations in dietary supply and metabolic demand. Multiple Pi transporters, as well as metabolic recycling and excretory activities have been identified in animals [1, 2]. A comprehensive understanding of their mechanisms and how these are integrated to achieve Pi. PLOS ONE | DOI:10.1371/journal.pone.0141128. In Vivo Phosphate Imaging in C. elegans
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