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In the original publication [...].
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Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.
Assuntos
Microscopia Eletrônica de Transmissão/métodos , Animais , Células Cultivadas , Retículo Endoplasmático/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , SoftwareRESUMO
Transmembrane proteins (TMEMs) are integral proteins that span biological membranes. TMEMs function as cellular membrane gates by modifying their conformation to control the influx and efflux of signals and molecules. TMEMs also reside in and interact with the membranes of various intracellular organelles. Despite much knowledge about the biological importance of TMEMs, their role in metabolic regulation is poorly understood. This review highlights the role of a single TMEM, transmembrane protein 135 (TMEM135). TMEM135 is thought to regulate the balance between mitochondrial fusion and fission and plays a role in regulating lipid droplet formation/tethering, fatty acid metabolism, and peroxisomal function. This review highlights our current understanding of the various roles of TMEM135 in cellular processes, organelle function, calcium dynamics, and metabolism.
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Saúde , Proteínas de Membrana/metabolismo , Dinâmica Mitocondrial , Sequência de Aminoácidos , Transporte Biológico , Cálcio/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peroxissomos/metabolismoRESUMO
High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.
