Cargando…

Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological...

Descripción completa

Detalles Bibliográficos
Autores principales: Benhammouda, Sara, Vishwakarma, Anjali, Gatti, Priya, Germain, Marc
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678465/
https://www.ncbi.nlm.nih.gov/pubmed/34926468
http://dx.doi.org/10.3389/fcell.2021.789959
_version_ 1784616311269097472
author Benhammouda, Sara
Vishwakarma, Anjali
Gatti, Priya
Germain, Marc
author_facet Benhammouda, Sara
Vishwakarma, Anjali
Gatti, Priya
Germain, Marc
author_sort Benhammouda, Sara
collection PubMed
description Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.
format Online
Article
Text
id pubmed-8678465
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-86784652021-12-18 Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction Benhammouda, Sara Vishwakarma, Anjali Gatti, Priya Germain, Marc Front Cell Dev Biol Cell and Developmental Biology Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins. Frontiers Media S.A. 2021-12-03 /pmc/articles/PMC8678465/ /pubmed/34926468 http://dx.doi.org/10.3389/fcell.2021.789959 Text en Copyright © 2021 Benhammouda, Vishwakarma, Gatti and Germain. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Benhammouda, Sara
Vishwakarma, Anjali
Gatti, Priya
Germain, Marc
Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title_full Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title_fullStr Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title_full_unstemmed Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title_short Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction
title_sort mitochondria endoplasmic reticulum contact sites (mercs): proximity ligation assay as a tool to study organelle interaction
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678465/
https://www.ncbi.nlm.nih.gov/pubmed/34926468
http://dx.doi.org/10.3389/fcell.2021.789959
work_keys_str_mv AT benhammoudasara mitochondriaendoplasmicreticulumcontactsitesmercsproximityligationassayasatooltostudyorganelleinteraction
AT vishwakarmaanjali mitochondriaendoplasmicreticulumcontactsitesmercsproximityligationassayasatooltostudyorganelleinteraction
AT gattipriya mitochondriaendoplasmicreticulumcontactsitesmercsproximityligationassayasatooltostudyorganelleinteraction
AT germainmarc mitochondriaendoplasmicreticulumcontactsitesmercsproximityligationassayasatooltostudyorganelleinteraction