Featured Cover Vue, Zer; Garza‐Lopez, Edgar; Neikirk, Kit ...
Aging cell,
December 2023, Letnik:
22, Številka:
12
Journal Article
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Cover legend: The cover image is based on the Research Article 3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex by Zer Vue et al., ...https://doi.org/10.1111/acel.14009
Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct ...mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.
Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy ...contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1.
Macroautophagy is a dynamic process that regulates cellular mechanisms It can be difficult to identify machinery and quantify them. Here, the use of transmission electron microscopy, immunogold labeling, immunofluorescence, and 3D reconstruction serial block face‐scanning electron microscopy to identify and quantify lysosomes, autophagosomes, and lipid droplets under drug response conditions and experimental knockouts is described.
3D Reconstruction
Macroautophagy is a dynamic process that regulates cellular mechanisms. It can be difficult to identify machinery and quantify them. In article 2200221, Antentor O. Hinton Jr. and ...co‐workers demonstrate the use of transmission electron microscopy, immunogold labeling, immunofluorescence, and 3D reconstruction serial block face‐scanning electron microscopy to identify and quantify lysosomes, autophagosomes, and lipid droplets under drug response conditions and experimental knockouts.
Neisseria gonorrhoeae has been shown to form biofilms during cervical infection. Thus, biofilm formation may play an important role in the infection of women. The ability of N. gonorrhoeae to form ...membrane blebs is crucial to biofilm formation. Blebs contain DNA and outer membrane structures, which have been shown to be major constituents of the biofilm matrix. The organism expresses a DNA thermonuclease that is involved in remodeling of the biofilm matrix. Comparison of the transcriptional profiles of gonococcal biofilms and planktonic runoff indicate that genes involved in anaerobic metabolism and oxidative stress tolerance are more highly expressed in biofilm. The expression of aniA, ccp, and norB, which encode nitrite reductase, cytochrome c peroxidase, and nitric oxide reductase respectively, is required for mature biofilm formation over glass and human cervical cells. In addition, anaerobic respiration occurs in the substratum of gonococcal biofilms and disruption of the norB gene required for anaerobic respiration, results in a severe biofilm attenuation phenotype. It has been demonstrated that accumulation of nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard biofilm formation. However, NO can also enhance biofilm formation, and this is largely dependent on the concentration and donation rate or steady-state kinetics of NO. The majority of the genes involved in gonococcal oxidative stress tolerance are also required for normal biofilm formation, as mutations in the following genes result in attenuated biofilm formation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation appears to be an adaptation for coping with the environmental stresses present in the female genitourinary tract. Therefore, this review will discuss the studies, which describe the composition and metabolic phenotype of gonococcal biofilms.
The Shenguang-II Upgrade (SG-II Up) facility is an under-construction high-power laser driver with eight beams, 24 kJ energy, 3 ns pulse duration and ultraviolet laser output, in the Shanghai ...Institute of Optics and Fine Mechanics, China. The prototype design and experimental research of the prototype final optics assembly (FOA), which is one of the most important parts of the SG-II Up facility, have been completed on the ninth beam of the SG-II facility. Thirty-three shots were fired using 1-${\it\omega}$ energy from 1000 to 4500 J and 3-${\it\omega}$ energy from 500 to 2403 J with a 3 ns square pulse. During the experiments, emphasis was given to the process of optical damage and to the effects of clean-gas control. A numerical model of the FOA generated by the Integrated Computer Engineering and Manufacturing code for Computational Fluid Dynamics (ICEMCFD) demonstrated that a flux within $1{-}5~\text{l s}^{-1}$ and a 180 s period is effectual to avoid contaminant sputtering to the optics. The presence of surface ‘mooning’ damage and surface spots located outside the clear aperture are induced by contaminants such as wire, silica gel and millimeter order fiber and metal.
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