Rebuilding Mitochondria: Lipids, Contacts, and Actin [May 27, 2025] DOJINDO LABORATORIES

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Mitochondria under stress activate distinct recovery pathways involving lipid mobilization, organelle contact sites, and cytoskeletal dynamics. This Science Note highlights recent studies revealing how cells coordinate lipid metabolism, mitochondria–ER connections, and actin polymerization to restore mitochondrial structure and function.

Triacylglycerol mobilization underpins mitochondrial stress recovery (Nature Cell Biology, 2025)
Summary: Mitochondria are essential for cellular energy and function. This study found that, when under stress, cells use the stored triacylglycerol in lipid droplets to aid recovery. TAG lipase breaks down the triacylglycerol to release fatty acids, which are used to make cardiolipin, a key lipid for building mitochondrial membranes and supporting mitochondrial function.

Highlighted technique: Researchers labeled TAG stores in yeast with carbon-labeled oleic acid to track fat mobilization after mitochondrial stress. This helped show that fatty acids from TAG, released by Tgl3-5p lipases, are used to make cardiolipin during mitochondrial recovery.

Related technique Lipid Droplet Detection, OCR Measurement

ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress (Nature Communications, 2025)
Summary: When mitochondria are damaged, cells increase MERCs (mitochondria-ER contacts) to export harmful lipid radicals and prevent cell death. This study reveals that MERCs formation depends on phosphorylated RMDN3 binding to VAPB, and disrupting this tethering leads to lipid radical buildup and cell death.

Highlighted technique: NanoBiT is a split-luciferase system that emits light when two protein fragments come close, allowing real-time tracking of molecular interactions in living cells. In this study, the authors created MERBiT by tagging mitochondrial TOMM20 with SmBiT and ER-localized Sec61β with LgBiT in HeLa cells, enabling quantitative monitoring of reversible MERCs during mitochondrial stress.

Related technique Mitochondrial Staining, Mitochondrial Lipid Peroxide Detection (used in this article)

Mitochondria- and ER-associated actin are required for mitochondrial fusion (Nature Communications, 2025)

Summary: This study reveals that actin polymerization is essential for mitochondrial fusion, uncovering a new role in organelle dynamics. It shows that fusion requires Arp2/3 (a complex that builds branched actin), INF2 (a protein that drives actin filament growth), and that actin appears at fusion sites before MFN2 (a key fusion protein), suggesting actin actively guides the fusion process.

Highlighted technique: In this study, PA-GFP (photoactivatable GFP), a protein that remains non-fluorescent until exposed to 405 nm light, was used to label specific regions of the mitochondrial network. This allowed the authors to track fluorescence spread through fused mitochondria and quantitatively demonstrate that mitochondrial fusion depends on actin polymerization.

Related technique Mitochondrial Membrane Potential Detection

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Related Techniques (click to open/close)

Target	Kit & Probes
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial Staining	MitoBright LT Green / Red / Deep Red
Lipid peroxidation detection	Liperfluo(Intracellular), MitoPeDPP(Mitochondrial)
Mitochondrial superoxide detection	MitoBright ROS Deep Red
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Lipid Droplet Staining	Lipi-Blue/ Green/ Red/ Deep Red
Glycolysis/Oxidative phosphorylation Assay	Glycolysis/OXPHOS Assay Kit
Mitophagy detection	Mitophagy Detection Kit
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST

Application Note I (click to open/close)
> Induction of Mitophagy in Parkin Expressed HeLa cells

After HeLa cells were washed with HBSS, co-stained with MitoBright ROS Deep Red and mitochondrial membrane potential staining dye (JC-1: code MT09), and the generated mitochondrial ROS and membrane potential were observed simultaneously. As a result, the decrease in mitochondrial membrane potential and the generation of mitochondrial ROS are simultaneously observed.
＜Imaging Conditions＞(Confocal microscopy) JC-1: Green Ex = 488, Em = 490-520 nm, Red: Ex = 561, Em = 560-600 nm MitoBright ROS ：Ex = 633 nm, Em = 640-700 nm Scale bar: 10 μm	＜Examination Conditions＞(Plate Reader)Tecan, Infinite M200 Pro JC-1: Green Ex=480-490 nm, Em=525-545 nm; Red: Ex= 530-540 nm, Em=585-605 nm MitoBright ROS: Ex=545-555 nm, Em = 665-685 nm