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

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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 (13C), the label was detected in subcellular volumes by MIMS. The tridimensional localization of 13C within the cell illustrated cellular distribution and local accumulation of inositol. In parallel, we performed control experiments with 13C-glucose to compare a different 13C 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 15N-thymidine. This permitted direct analysis of the nuclear DNA through the detection of the 15N isotopic signal. We found practically no co-localization between inositol signal (13C-isotope) and nuclear signal (15N-isotope). The 13C-tag (inositol) accumulation was highest at the plasma membrane and in cytoplasmic domains. In time-course labeling experiments performed with wild-type (WT) yeast or modified yeast unable to synthesize inositol from glucose (ino1$Δ$), the halftime of labeled inositol accumulation was \~{}1 h in WT and longer in ino1$Δ$. These studies should serve as a template to study metabolism and physiological role of inositol using genetically modified yeasts.