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Prudente de Mello, N. ; Fecher, C.* ; Pastor, A.M.* ; Perocchi, F. ; Misgeld, T.*

Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.

Nat. Protoc. 18, 2181-2220 (2023)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6–8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1–2 h.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Messenger-rna; Bioenergetics; Calcium; Muscle; Recombination; Proteomics; Transport; Dynamics; Driver; Lines
ISSN (print) / ISBN 1754-2189
e-ISSN 1750-2799
Journal Nature Protocols
Quellenangaben Volume: 18, Issue: 7, Pages: 2181-2220 Article Number: , Supplement: ,
Publisher Nature Publishing Group
Publishing Place Heidelberger Platz 3, Berlin, 14197, Germany
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Diabetes and Obesity (IDO)
Grants European Research Council (ERC)
ExNet-0041-Phase2-3 (SyNergy-HMGU') through the Initiative and Network Fund of the Helmholtz Association
Munich Center for Systems Neurology (SyNergy EXC 2145)
German Center for Neurodegenerative Diseases(DZNE)
ERC
DFG