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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.

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.

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.

In the original publication [...]

Age‐related skeletal muscle atrophy, known as sarcopenia, is characterized by loss of muscle mass, strength, endurance, and oxidative capacity. Although exercise has been shown to mitigate sarcopenia, the underlying governing mechanisms are poorly understood. Mitochondrial dysfunction is implicated in aging and sarcopenia; however, few studies explore how mitochondrial structure contributes to this dysfunction. In this study, we sought to understand how aging impacts mitochondrial three‐dimensional (3D) structure and its regulators in skeletal muscle. We hypothesized that aging leads to remodeling of mitochondrial 3D architecture permissive to dysfunction and is ameliorated by exercise. Using serial block‐face scanning electron microscopy (SBF‐SEM) and Amira software, mitochondrial 3D reconstructions from patient biopsies were generated and analyzed. Across five human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria are less spherical and more complex, indicating age‐related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age‐related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved, as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age‐related structural changes in mitochondria and further suggest that exercise may mitigate age‐related structural decline through modulation of mitofusin 2. Changes in mitochondrial structure and dynamics during aging provide a mechanism for the development of age‐related sarcopenia, a condition characterized by muscle mass loss. Through the creation of three‐dimensional models of mitochondria from quadriceps muscle tissue taken from old and young humans, a loss in mitochondrial complexity was observed to occur during aging. A decrease in the expression of mitochondrial fusion protein mitofusin 2 (MFN‐2) in older populations may drive these mitochondrial structural changes. A Drosophila model with the MFN‐2 ortholog knocked down demonstrated a loss of mitochondrial complexity and lower quality cristae, which parallel changes in mitochondria observed in older humans. The use of an in vitro cell exercise model showed that the mechanism by which exercise counteracts the effects of sarcopenia, age‐related disease may be due to increased expression of MFN‐2 during exercise.

Molecular dynamics (MD) is a powerful technique that can be used to provide information about a system over time. MD simulations can be used to explore behaviors of many systems such as protein-ligand interactions protein-protein interactions. Additionally, these simulations can be used to generate trajectories of a system to further be used for free energy simulations. The work discussed in Chapter 2 involves the use MD simulations to identify potential drug candidates capable of binding to and stabilizing the nuclear binding domain of the Cystic Fibrosis Transmembrane Conductance Regulator protein. Using ProBiS, we were able to find a potential binding site as well as potential small molecules to bind to said site. The stabilizing effects of these drugs were then tested experimentally using ∆F508- NBD1 expressed in E. coli. DSF experiments were conducted, ultimately revealing that two of the tested small molecules binds to our protein, but showing destabilizing effects rather than the stabilizing effects we’d hoped to see. In Chapter 3, MD simulations were used to assess the stability of a protein-protein complex, 14-3-3 and Raf. Through Molecular Dynamics simulations we found that dephosphorylation of Ser259 affected the stability of the complex, while Raf orientation had minor effects on the complex stability. Lastly, we used MD simulations in Chapter 4, as a starting point to improve free energy simulations by adopting the optimized protocols discovered by our collaborator and employing the use of force matching to improve conformational overlap between the “low level” and “high level” systems. Here, we finally achieved better convergence and configurational overlap betwixt our forcematched parameter sets and those from our higher level of theory (3OB).

Cover legend: The cover image is based on the article 3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights Into MFN‐2‐Mediated Changes by Estevão Scudese et al., https://doi.org/10.1111/acel.70054

Age‐related skeletal muscle atrophy, known as sarcopenia, is characterized by loss of muscle mass, strength, endurance, and oxidative capacity. Although exercise has been shown to mitigate sarcopenia, the underlying governing mechanisms are poorly understood. Mitochondrial dysfunction is implicated in aging and sarcopenia; however, few studies explore how mitochondrial structure contributes to this dysfunction. In this study, we sought to understand how aging impacts mitochondrial three‐dimensional (3D) structure and its regulators in skeletal muscle. We hypothesized that aging leads to remodeling of mitochondrial 3D architecture permissive to dysfunction and is ameliorated by exercise. Using serial block‐face scanning electron microscopy (SBF‐SEM) and Amira software, mitochondrial 3D reconstructions from patient biopsies were generated and analyzed. Across five human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria are less spherical and more complex, indicating age‐related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age‐related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved, as Marf, the MFN2 ortholog in Drosophila , knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age‐related structural changes in mitochondria and further suggest that exercise may mitigate age‐related structural decline through modulation of mitofusin 2.

Many interconnected degradation machineries including autophagosomes, lysosomes, and endosomes work in tandem to conduct autophagy, an intracellular degradation system that is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, identification of specific components of the autophagy machinery has been challenging. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in macro autophagy. We provide methods that help avoid common pitfalls, including a detailed explanation for distinguishing lysosomes and lipid droplets and discuss differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. We also utilize serial block face-scanning electron microscopy (SBF-SEM) to offer a protocol for visualizing 3D morphology of degradation machinery. In addition to TEM and 3D reconstruction, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling that can be utilized to reliably and accurately classify cellular organelles. Our results show how these methods may be used to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1. Competing Interest Statement The authors have declared no competing interest. Footnotes * Changes in figures, data, authors, and main text