Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery

Kit Neikirk; Zer Vue; Prasanna Katti; Ben I Rodriguez; Salem Omer; Jianqiang Shao; Trace Christensen; Edgar Garza Lopez; Andrea Marshall; Caroline B Palavicino-Maggio; Jessica Ponce; Ahmad F Alghanem; Larry Vang; Taylor Barongan; Heather K Beasley; Taylor Rodman; Dominique Stephens; Margaret Mungai; Marcelo Correia; Vernat Exil; Steven Damo; Sandra A. Murray; Amber Crabtree; Brian Glancy; Renata O Pereira; E. Dale Abel; Antentor O Hinton Jr

doi:10.1002/adbi.202200221

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Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery

Journal article

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Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery

Kit Neikirk, Zer Vue, Prasanna Katti, Ben I Rodriguez, Salem Omer, Jianqiang Shao, Trace Christensen, Edgar Garza Lopez, Andrea Marshall, Caroline B Palavicino-Maggio, …

Advanced biology, Vol.7(6), e2200221

06/2023

DOI: 10.1002/adbi.202200221

PMID: 36869426

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Abstract

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.

Materials Science

Technology

Materials Science, Biomaterials

Science & Technology

Details

Title: Subtitle: Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery
Creators: Kit Neikirk - University of Hawaii at Hilo
Zer Vue - Vanderbilt University
Prasanna Katti - National Heart Lung and Blood Institute
Ben I Rodriguez - Vanderbilt University
Salem Omer - Vanderbilt University
Jianqiang Shao - University of Iowa
Trace Christensen - Mayo Clinic
Edgar Garza Lopez - Vanderbilt University
Andrea Marshall - Vanderbilt University
Caroline B Palavicino-Maggio - Harvard University
Jessica Ponce - University of Utah
Ahmad F Alghanem - King Abdullah International Medical Research Center
Larry Vang - Vanderbilt University
Taylor Barongan - Vanderbilt University
Heather K Beasley - Vanderbilt University
Taylor Rodman - Vanderbilt University
Dominique Stephens - Vanderbilt University
Margaret Mungai - University of California, Berkeley
Marcelo Correia - University of Iowa
Vernat Exil - University of Iowa
Steven Damo - Fisk University
Sandra A. Murray - University of Pittsburgh
Amber Crabtree - Vanderbilt University
Brian Glancy - National Heart Lung and Blood Institute
Renata O Pereira - University of Iowa
E. Dale Abel - University of Iowa
Antentor O Hinton Jr - Vanderbilt University
Resource Type: Journal article
Publication Details: Advanced biology, Vol.7(6), e2200221
DOI: 10.1002/adbi.202200221
PMID: 36869426
NLM abbreviation: Adv Biol (Weinh)
ISSN: 2701-0198
eISSN: 2701-0198
Publisher: Wiley
Number of pages: 18
Grant note: DK007563 / National Institute of Health (NIH) NIDDK; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) 1021868.01 / Burroughs Wellcome Fund Career Awards at the Scientific Interface Award; Burroughs Wellcome Fund Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer's Disease Pilot & Feasibility Program United Negro College Fund/Bristol-Myers Squibb EE Just Faculty Fund 1022376 / UNCF/BMS EE United Negro College Fund/Bristol-Myers Squibb EE Just Postgraduate Fellowship in the Life Sciences Fellowship PDEP 2022-253529; 2022-253614 / CZI Science Diversity Leadership grant from the Chan-Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation T32 DK101003 / Integrated Training in Engneering and Diabetes 1022355 / Burroughs Wellcome Fund Postdoctoral Enchricment Program; Burroughs Wellcome Fund MCB 2011577I; EES2112556; EES1817282; MCB1955975 / NSF; National Science Foundation (NSF) R01HL108379; R01DK092065; T32 5T32GM133353 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA Burroughs Wellcome Fund Ad-hoc Award; Burroughs Wellcome Fund 5R25HL106365-12; DK020593 / NIH Small Research Pilot Subaward from the NIH PRIDE Program; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
Language: English
Electronic publication date: 03/03/2023
Date published: 06/2023
Academic Unit: Stead Family Department of Pediatrics; Fraternal Order of Eagles Diabetes Research Center; Endocrinology and Metabolism; Internal Medicine
Record Identifier: 9984420842302771

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