Morphometric analysis of autophagy-related structures in Saccharomyces cerevisiae |
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Authors: | Tatsuya Kawaoka Shinsuke Ohnuki Yoshikazu Ohya |
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Institution: | Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan |
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Abstract: | When macroautophagy (autophagy) is induced by nutrient starvation or rapamycin treatment, Atg (autophagy-related) proteins are assembled at a restricted region close to the vacuole. Subsequently, the phagophore expands to form a closed autophagosome. In Saccharomyces cerevisiae cells overexpressing precursor Ape1 (prApe1), a specific autophagosome cargo protein, the phagophore can be visualized as a cup-shaped structure labeled with green fluorescent protein (GFP)-tagged Atg8. Previously, our group has shown that the maximum length of GFP-Atg8-labeled structures reflects the magnitude of bulk autophagy. In that study, the morphological parameters of the autophagy-related structures were extracted manually, requiring a great deal of time. Moreover, only well-expanded phagophores were subjected to further analysis. Here we report Qautas (Quantitative autophagy-related structure analysis system), a high-throughput and comprehensive system for morphological analysis of autophagy-related structures using a combination of image processing and machine learning. We describe both the manual method and Qautas in detail. |
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Keywords: | autophagy yeast machine learning isolation membrane image processing morphological analysis ImageJ R fluorescence microscopy |
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