Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells
(2016) In Journal of Visualized Experiments- Abstract
Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic... (More)
Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.
(Less)
- author
- Tubbs, Emily LU and Rieusset, Jennifer
- organization
- publishing date
- 2016-12-10
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Endoplasmic reticulum, Immunofluorescence, In situ proximity ligation assay, IP3R, Issue 118, Mitochondria, Mitochondria-associated membranes, Molecular biology, VDAC
- in
- Journal of Visualized Experiments
- issue
- 118
- article number
- e54899
- publisher
- JoVE
- external identifiers
-
- scopus:85008700532
- pmid:28060261
- wos:000397846600067
- ISSN
- 1940-087X
- DOI
- 10.3791/54899
- language
- English
- LU publication?
- yes
- id
- 52228361-b45e-41e4-9fea-e4d8ed0db1ce
- date added to LUP
- 2017-01-23 12:33:45
- date last changed
- 2024-04-19 17:29:30
@article{52228361-b45e-41e4-9fea-e4d8ed0db1ce,
abstract = {{<p>Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.</p>}},
author = {{Tubbs, Emily and Rieusset, Jennifer}},
issn = {{1940-087X}},
keywords = {{Endoplasmic reticulum; Immunofluorescence; In situ proximity ligation assay; IP3R; Issue 118; Mitochondria; Mitochondria-associated membranes; Molecular biology; VDAC}},
language = {{eng}},
month = {{12}},
number = {{118}},
publisher = {{JoVE}},
series = {{Journal of Visualized Experiments}},
title = {{Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells}},
url = {{http://dx.doi.org/10.3791/54899}},
doi = {{10.3791/54899}},
year = {{2016}},
}