Using MitER for 3D analysis of mitochondrial morphology and ER contacts

• Published in: Cell reports methods 2024-01, Vol.4 (1), p.100692-100692, Article 100692
• Main Authors: Kichuk, Therese, Dhamankar, Satyen, Malani, Saurabh, Hofstadter, William A., Wegner, Scott A., Cristea, Ileana M., Avalos, José L.
• Format: Article
• Language: English
• Subjects: Animals; Cell Membrane - metabolism; Endoplasmic Reticulum - metabolism; image analysis; imaging; inter-organelle contact; Mammals; Mitochondria; mitochondrial-ER contact; organelle distribution; organelle morphology; Saccharomyces cerevisea; Saccharomyces cerevisiae
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100692

We have developed an open-source workflow that allows for quantitative single-cell analysis of organelle morphology, distribution, and inter-organelle contacts with an emphasis on the analysis of mitochondria and mitochondria-endoplasmic reticulum (mito-ER) contact sites. As the importance of inter-organelle contacts becomes more widely recognized, there is a concomitant increase in demand for tools to analyze subcellular architecture. Here, we describe a workflow we call MitER (pronounced “mightier”), which allows for automated calculation of organelle morphology, distribution, and inter-organelle contacts from 3D renderings by employing the animation software Blender. We then use MitER to quantify the variations in the mito-ER networks of Saccharomyces cerevisiae, revealing significantly more mito-ER contacts within respiring cells compared to fermenting cells. We then demonstrate how this workflow can be applied to mammalian systems and used to monitor mitochondrial dynamics and inter-organelle contact in time-lapse studies. [Display omitted] •MitER is an open-source analysis workflow for three-dimensional organelle renderings•MitER provides automated volume, surface area, and inter-organelle contact measurements•MitER can be used to capture dynamic changes in organelle morphologies Mitochondrial morphology and contact with proximal organelles are important in both healthy and diseased cell states. Quantification of mitochondrial contact with the endoplasmic reticulum (ER) is particularly valuable as these contacts influence numerous cellular processes, such as mitochondrial fission and fusion, mitophagy, and calcium signaling. However, there are limited tools for such quantitative analyses. Current open-source methods solely allow for colocalization measurements, which are sufficient to estimate inter-organelle contact but lack the ability to provide more substantial metrics, such as the surface area and number of discrete contacts. Kichuk et al. introduce MitER, an open-source workflow that allows for quantitative single-cell analysis of organelle morphology, distribution, and inter-organelle contacts. The workflow is used to quantify the variations in the mitochondria-endoplasmic reticulum (mito-ER) networks of Saccharomyces cerevisiae, revealing a significant increase in mito-ER contacts within respiring cells compared to fermenting cells.