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Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging

The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organi...

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Autores principales: Mahajan, Divyanshu, Tie, Hieng Chiong, Lu, Lei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Bio-Protocol 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10127052/
https://www.ncbi.nlm.nih.gov/pubmed/37113336
http://dx.doi.org/10.21769/BioProtoc.4658
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author Mahajan, Divyanshu
Tie, Hieng Chiong
Lu, Lei
author_facet Mahajan, Divyanshu
Tie, Hieng Chiong
Lu, Lei
author_sort Mahajan, Divyanshu
collection PubMed
description The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging. Graphical abstract [Image: see text]
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spelling pubmed-101270522023-04-26 Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging Mahajan, Divyanshu Tie, Hieng Chiong Lu, Lei Bio Protoc Methods Article The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging. Graphical abstract [Image: see text] Bio-Protocol 2023-04-20 /pmc/articles/PMC10127052/ /pubmed/37113336 http://dx.doi.org/10.21769/BioProtoc.4658 Text en Copyright © 2023 The Authors; https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
spellingShingle Methods Article
Mahajan, Divyanshu
Tie, Hieng Chiong
Lu, Lei
Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title_full Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title_fullStr Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title_full_unstemmed Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title_short Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
title_sort visualizing the cisternal organization of golgi ministacks in hela cells by side-averaging
topic Methods Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10127052/
https://www.ncbi.nlm.nih.gov/pubmed/37113336
http://dx.doi.org/10.21769/BioProtoc.4658
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