Quantitative imaging of inositol distribution in yeast using multi-isotope imaging mass spectrometry (MIMS).

Despite the widely recognized importance of the several species of inositol polyphosphates in cell biology, inositol has not been successfully imaged and quantified inside cells using traditional spectrophotometry. Multi-isotope imaging mass spectrometry (MIMS) technology, however, has facilitated direct imaging and measurement of cellular inositol. After pulsing cells with inositol labeled with the stable isotope Carbon-13 ((13)C), the label was detected in subcellular volumes by MIMS. The tridimensional localization of (13)C within the cell illustrated cellular distribution and local accumulation of inositol. In parallel, we performed control experiments with (13)C-Glucose to compare a different (13)C distribution pattern. Because many functions recently attributed to inositol polyphosphates are localized in the nucleus, we analyzed its relative nuclear concentration. We engineered yeast with human thymidine permease and viral thymidine kinase, then fed them with (15)N-thymidine. This permitted direct analysis of the nuclear DNA through the detection of the (15)N isotopic signal. We found practically no co-localization between inositol signal ((13)C-isotope) and nuclear signal ((15)N-isotope). The (13)C-tag (inositol) accumulation was highest at the plasma membrane and in cytoplasmic domains. In time-course labeling experiments performed with wild type yeast (WT) or modified yeast unable to synthesize inositol from glucose (ino1Δ), the half-time of labeled inositol accumulation was ~1 hour in WT and longer in ino1Δ. These studies should serve as a template to study metabolism and physiological role of inositol using genetically modified yeasts.