bims-mikwok 2023-06-04 papers

Methods Mol Biol. 2023 ;2675 97-107

Methods for Monitoring Mitochondrial Biogenesis and Turnover in Cultured Hepatocytes and Mouse Liver Using MitoTimer Reporter Assay.

Xiaowen Ma, Wen-Xing Ding.

Mitochondrial biogenesis and turnover rate are critical to maintain homeostasis of the intracellular mitochondrial pool. Altered mitochondrial biogenesis and mitophagy are closely related to many chronic diseases, highlighting the importance of mitochondrial stasis in various pathological conditions including liver diseases. We describe a detailed protocol for monitoring mitochondrial lifecycle in primary cultured mouse hepatocytes and mouse liver using the dual color fluorescence-based imaging of MitoTimer. Three types of mitochondria were visualized in mouse hepatocytes: green-only mitochondria (newly synthesized mitochondria), red-only mitochondria (old/aging mitochondria), as well as the majority of yellow mitochondria (representing an intermediate stage of mitochondria). The ratio of red/green fluorescence in each cell will be used to track mitochondrial aging. Super-resolution microscopy analysis revealed that majority of mitochondria were spatially heterogeneous with proteins from simultaneous new synthesis, maturation, and turnover in hepatocytes. MitoTimer reporter assay can specifically target to mitochondria and be used to monitor mitochondrial biogenesis and maturation as well as turnover in vitro and in vivo.

Keywords: Hepatocytes; Liver; MitoTimer; Mitophagy; Quality control

DOI: https://doi.org/10.1007/978-1-0716-3247-5_8

J Vis Exp. 2023 05 12.

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels using Live Imaging in HeLa Cells.

Mohammad Fazli, Chantell S Evans.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

DOI: https://doi.org/10.3791/65304

Methods Mol Biol. 2023 ;2675 77-96

Isolation of Mitochondria from Mouse Tissues for Functional Analysis.

Rebeca Acín-Pérez, Katrina P Montales, Kaitlyn B Nguyen, Alexandra J Brownstein, Linsey Stiles, Ajit S Divakaruni.

Methods for isolating mitochondria from different rodent tissues have been established for decades. Although the general principles for crude mitochondrial preparations are largely shared across tissues - tissue disruption followed by differential centrifugation - critical differences exist for isolation from different tissues to optimize mitochondrial yield and function. This protocol offers a unified resource for preparations of isolated mitochondria from mouse liver, kidney, heart, brain, skeletal muscle, and brown and white adipose tissue suitable for functional analysis.

Keywords: Bioenergetics; Brain; Brown adipose tissue; Heart; Kidney; Liver; Mitochondria; Oxidative phosphorylation; Skeletal muscle; White adipose tissue

DOI: https://doi.org/10.1007/978-1-0716-3247-5_7

Stem Cell Rev Rep. 2023 May 27.

MSCs Ameliorate Hepatic IR Injury by Modulating Phenotypic Transformation of Kupffer Cells Through Drp-1 Dependent Mitochondrial Dynamics.

Long-Cheng Shang, Man Wang, Yang Liu, Xinhua Zhu, Shuai Wang.

BACKGROUND: Hepatic ischemia and reperfusion (IR) injury, characterized by reactive oxygen species (ROS) production and immune disorders, leads to exogenous antigen-independent local inflammation and hepatocellular death. Mesenchymal stem cells (MSCs) have been shown to be immunomodulatory, antioxidative and contribute to liver regeneration in fulminant hepatic failure. We aimed to investigate the underlying mechanisms by which MSCs protect against liver IR injury in a mouse model.
METHODS: MSCs suspension was injected 30 min prior to hepatic warm IR. Primary kupffer cells (KCs) were isolated. Hepatic injury, inflammatory responses, innate immunity, KCs phenotypic polarization and mitochondrial dynamics were evaluated with or without KCs Drp-1 overexpression RESULTS: MSCs markedly ameliorated liver injury and attenuated inflammatory responses and innate immunity after liver IR injury. MSCs significantly restrained M1 phenotypic polarization but boosted M2 polarization of KCs extracted from ischemic liver, as demonstrated by lowered transcript levels of iNOS and IL-1β but raised transcript levels of Mrc-1 and Arg-1 combined with p-STAT6 up-regulation and p-STAT1 down-regulation. Moreover, MSCs inhibited KCs mitochondrial fission, as evidenced by decreased Drp1 and Dnm2 levels. We overexpressed Drp-1 in KCs which promote mitochondrial fission during IR injury. the regulation of MSCs towards KCs M1/M2 polarization was abrogated by Drp-1 overexpression after IR injury. Ultimately, in vivo Drp-1 overexpression in KCs hampered the therapeutic effects of MSCs against hepatic IR injury CONCLUSIONS: We revealed that MSCs facilitated M1-M2 phenotypic polarization through inhibiting Drp-1 dependent mitochondrial fission and further attenuated liver IR injury. These results add a new insight into regulating mechanisms of mitochondrial dynamics during hepatic IR injury and may offer novel opportunities for developing therapeutic targets to combat hepatic IR injury.

Keywords: Drp-1; Liver ischemia and reperfusion injury; Macrophage; Mesenchymal stem cells; Mitochondria dynamics

DOI: https://doi.org/10.1007/s12015-023-10566-6

Bio Protoc. 2023 May 20. 13(10): e4680

Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF).

Macy Lozen, Yue Chen, Rebecca A Boisvert, Katharina Schlacher.

Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5'-bromo-2'-deoxyuridine (BrdU) or 5'-ethynyl-2'-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF). Graphical overview Schematic overview of Mitochondrial Replication Assay (MIRA). 5'-ethynyl-2'-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.

Keywords: BRCA2; Fanconi anemia; MIRA; Mitochondria; Mitochondrial DNA; Proximity ligation assay; mtDNA instability; mtDNA replication

DOI: https://doi.org/10.21769/BioProtoc.4680

Sci Adv. 2023 Jun 02. 9(22): eadh4251

Structural basis of purine nucleotide inhibition of human uncoupling protein 1.

Scott A Jones, Prerana Gogoi, Jonathan J Ruprecht, Martin S King, Yang Lee, Thomas Zögg, Els Pardon, Deepak Chand, Stefan Steimle, Danielle M Copeman, Camila A Cotrim, Jan Steyaert, Paul G Crichton, Vera Moiseenkova-Bell, Edmund R S Kunji.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

DOI: https://doi.org/10.1126/sciadv.adh4251