Biology subjects

Abel, E. D.

Publications and source records attributed to Abel, E. D..

6 recordsLinked to original sources

bioRxiv2021

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/461807v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c40041org.highwire.dtl.DTLVardef@ecde92org.highwire.dtl.DTLVardef@10870daorg.highwire.dtl.DTLVardef@1290c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

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.

biophysics↗

bioRxiv2021

A comprehensive approach for artifact-free sample preparation and assessment of mitochondrial morphology in tissue and cultured cells

Mitochondrial dynamics (fission, fusion, and the formation of nanotunnels) and morphology are very sensitive to the cellular environment. Mitochondria may be adversely affected by oxidative stress, changes in calcium levels, and hypoxia. Investigating the precise relationship between organelle structure and function requires methods that can adequately preserve mitochondria while providing accurate, quantitative measurements of morphological attributes. Here, we demonstrate a practical approach for preserving and measuring fine structural changes using two-dimensional, high-resolution electron micrographs. This approach is further applicable for three-dimensional volume renderings, obtained using serial block-face and focused ion beam-scanning electron microscopy, highlighting the specific advantages of these techniques. Additionally, this study defines a set of quantifiable metrics that can be applied to measure mitochondrial architecture and other organellar structures. Finally, we validated specimen preparation methods that avoid the introduction of morphological artifacts that may interfere with mitochondrial appearance and do not require whole-animal perfusion.

cell biology↗

bioRxiv2021

A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ

2Transmission electron microscopy (TEM) is a scientific research standard for producing nanometer-resolution ultrastructural images of subcellular components within cells and tissues. Mitochondria, endoplasmic reticulum (ER), lysosomes, and autophagosomes are organelles of particular interest to those investigating metabolic disorders. However, there is no clear consensus amongst regarding the best methods for quantifying the features of organelles in TEM images. In this protocol, we propose a standardized approach to accurately measure the morphology of these important subcellular structures using the free program ImageJ, developed by the National Institutes of Health (NIH). Specifically, we detail procedures for obtaining mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology. We further provide methods for measuring interactions between the mitochondria and ER and measuring the length and width of lysosomes and autophagosomes. This standardized method can be used to quantify key features of organelle morphology, allowing investigators to produce accurate and reproducible measurements of organelle structures in their experimental samples. 1 SUMMARYWe discuss a standardized method for measuring and quantifying organelle features using transmission electron microscopy and accessing for interactions between subcellular structures; organelles of focus include mitochondria, endoplasmic reticulum, lysosomes, and autophagosomes.

biophysics↗

bioRxiv2021

OPA1 Deletion in Brown Adipose Tissue Improves Thermoregulation and Systemic Metabolism via FGF21

Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including proteolytic processing of the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. By deleting OPA1 selectively in BAT (OPA1 BAT KO), we demonstrate that OPA1 is required for cold-induced thermogenesis. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)- dependent manner. BAT-derived FGF21 mediates an adaptive response in OPA1 BAT KO mice, by inducing browning of white adipose tissue (WAT), increasing resting metabolic rates, and improving thermoregulation. However, FGF21 does not mediate the resistance to diet-induced obesity observed in these animals. These findings reveal a requirement for OPA1 in BAT thermogenesis, and uncovers a homeostatic mechanism of BAT-mediated metabolic protection governed by an ATF4-FGF21 axis, that is activated independently of BAT thermogenic function.

physiology↗

bioRxiv2020

SWELL1-LRRC8 complex regulates skeletal muscle cell size, intracellular signalling, adiposity and glucose metabolism

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (LRRC8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. SWELL1 over-expression in SWELL1 KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted SWELL1 KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the SWELL1-LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo.

cell biology↗