Mitochondria Research

Science Note

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

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

Previous Science Note
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

Why is Mitochondrial Research Important?

Mitochondrial analysis is essential for understanding cellular energy metabolism because mitochondria regulate ATP production, redox balance, and signaling pathways. Thus, assessment of mitochondrial functions such as membrane potential, mitophagy, and respiration provides insight into cellular abnormalities, and these studies are critical in neurodegenerative diseases, cancer biology, and metabolic disorders, helping to uncover therapeutic targets and disease mechanisms.

Selection Guide for Mitochondria-related Reagents

Mitochondria research is very multi-faceted, because the multi-functional organelle is not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.

Selection Guide of Reagents

The following table lists reagents for mitochondrial research designed to stain and detect mitochondria (MitoBright LT, MitoTracker, etc.), mitochondrial membrane potential (JC-1, TMRM, TMRE, etc.), reactive oxygen species AKA ‘ROS’ (MitoBright ROS, MitoSOX, etc.), mitophagy, and lipid peroxides.

Mitophagy

Mitophagy
Reagent Mtphagy Dye Keima-Red
Principle Mtphagy Dye (included in Mitophagy Detection Kit) is a pH-sensitive fluorescent probe that accumulates in mitochondria and emits red fluorescence due to acidic conditions in a lysosome. A pH-sentitive ratiometric fluorescent protein. The excitation spectrum changes accoring to pH. This protein shows high fluorescence ratio (Ex. 550 nm/440 nm) values in a lysozome.
Fixed cell staining – –
Live-cell staining Yes Yes
Fixation after live-cell staining – –
Staining time > 30 min –
Ex / Em 530 / 700 440, 550 / 620
Product code MD01, MT02 –

Lipophilic peroxide / Singlet oxygen / Superoxide

  Lipophilic peroxide Singlet oxygen Superoxide Superoxide
Reagent MitoPeDPP Si-DMA MitoBright ROS Deep Red MitoSOX
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with lipophilic peroxides in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with singlet oxigen generated in mitochondria to emit red fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit red fluorescence.
Fixed cell staining – – – –
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining – – – –
Staining time > 15 min > 45 min > 10 min > 10 min
Ex / Em 452 / 470 644 / 670 540 / 670 510 / 590
Product code M466 MT05 MT16 –

Membrane potential

Membrane potential
Reagent JC-1 MT-1 TMRM, TMRE
Principle A cell-permeant ratiometric fluorescent dye that accumulates in intact mitochondria due to the membrane potential. The excitation spectrum changes according to the mitochondria membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. MT-1 is extremely photostable and more sensitive than JC-1 and can provide equivalent detection sensitivity to TMRE. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Diffusion of the probes occurs in a damaged mitochondria that has the decreased membrane potential.
Fixed cell staining – – –
Live-cell staining Yes Yes Yes
Fixation after live-cell staining – Yes –
Staining time 30-60 min 30 min 30-60 min
Ex / Em Monomer: 514 / 529
J-aggregation: 585/590		 530-560 / 570-640 550 / 575
Product code MT09 MT13 –

Mitochondria staining

Mitochondria staining
Reagent MitoBright LT series MitoBright IM Red MitoTracker series Rhodamine 123
Principle Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential and covalently binds to proteins and other biomolecules. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dye that accumulates in intact mitochondria due to the membrane potential.
Fixed cell staining – – – –
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining Yes Yes – –
Staining time 30 min 30 min 15-45 min > 15 min
Ex / Em 493 / 508, 547 / 563, 643 / 663 548 / 566 490 / 516 ~
644 / 665		 507 / 529
Product code MT10, MT11,MT12 MT15 – R233

Metal Ion Detection

   Iron ion (Fe2+) Calcium ion (Ca2+)
Reagent Mito-FerroGreen Rhod2-AM
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with ferrous ion in mitochondria to emit green fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with calcium ion in mitochondria to emit red fluorescence.
Fixed cell staining – –
Live-cell staining Yes Yes
Fixation after live-cell staining – –
Staining time 30 min 30-60 min
Ex / Em 505 / 535 553 / 576
Product code M489 R002 
Application Products
Mitophagy Detection Mitophagy Detection Kit
Mitochondrial Phospholipid Peroxidase Detection MitoPeDPP
Mitochondrial Ferrous Ion Detection Mito-FerroGreen
Mitochondrial Superoxide MitoBright ROS - Mitochondrial Superoxide Detection
Mitochondrial Singlet Oxygen Detection Si-DMA for Mitochondrial Singlet Oxygen Imaging
Mitochondrial Membrane Potential JC-1 MitoMP Detection Kit
MT-1 MitoMP Detection Kit
Mitochondria Staining MitoBright LT Green
MitoBright LT Red
MitoBright LT Deep Red
MitoBright IM Red for Immunostaining

Cancer, Cell Death, and Mitochondria

Apoptosis is a protective defense mechanism that effectively suppresses tumor growth and eliminates tumor cells.
One of the main mechanisms that trigger apoptosis is the increase in mitochondrial metabolic activity, which leads to elevated ROS levels in cancer cells. Excessive ROS damage mitochondrial function, causing mitochondrial membrane depolarization, which subsequently activates the intrinsic apoptosis pathway. Tumor cell immune evasion is a key feature of tumor pathophysiology, and mitochondria play a central role in both inhibiting and promoting immune evasion within the  complex mechanism    s involved in immune responses.1)

The Potential of Ferroptosis in Cancer Therapy: Many studies have found that ferroptosis sensitivity can be used to target tumors resistant to conventional therapies (such as triple-negative breast cancer and glioblastoma).2)

Ferroptosis and the Immune Microenvironment: Neutrophils in the tumour microenvironment die spontaneously by ferotosis and the lipid peroxide released suppresses T-cell activity, thereby suppressing tumour immunity.3)

Reference
1) Gao, J., Cancer Gene Therapy, 2024, 31, 970-983
2) Yang, F., Cell Metabolism, 2023, 35(1), 84-100
3) Kim, R., Nature, 2022, 612, 338-346


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Mitochondrial Function and Cellular Senescence

In senescent cells, due to mitochondrial dysfunction, ATP is primarily generated through the anaerobic glycolysis pathway, leading to an increase in lactate production2). DNA damage is one of the causes of mitochondrial dysfunction in cellular aging. The accumulation of DNA damage activates DNA repair mechanisms and increases NAD+ consumption. The decrease in NAD+ levels reduces SIRT1 activity, an important factor in maintaining mitochondrial function, leading to impaired mitochondrial function (inhibition of electron transfer → ATP production / reduction of NAD+ levels)1),3).

Reference:

1. J. Wu, Z. Jin, H. Zheng and L. Yan, “Sources and implications of NADH/NAD+redox imbalance in diabetes and its complications”, Diabetes Metab. Syndr. Obes., 2016, 9, 145

2. Z. Feng, R. W. Hanson, N. A. Berger and A. Trubitsyn, “Reprogramming of energy metabolism as a driver of aging”, Oncotarget., 2016, 7(13), 15410.

3. S. Imai and L. Guarente, “NAD+ and sirtuins in aging and disease”, Trends in Cell Biology, 2014, 24(8), 464.

Neurodegeneration-Autophagy and Mitochondrial Damage

Indicator Mitophagy Mitochondrial fission abnormalities ROS MPTP (Mitochondrial Permeability Transition Pore) MMP （Mitochondrial Membrane Potential） ATP
Detection Kit MD01 Mitophagy Detection Kit MT10/MT11/MT12 MitoBright LT Green/Red/Deep Red R252/R253
ROS Assay Kit		 - MT09:JC-1 MT13:MT-1 A550 ATP Assay Kit-Luminescence
Alzheimer's disease (AD) ↓ ✓ ↑ ↑ ↓ ↓
Parkinson's disease (PD) - -
Huntington's disease (HD) - - -
Amyotrophic lateral sclerosis (ALS) - ↓

Refernce :

Role of Mitochondria in NeurodegenerativeDiseases: From an Epigenetic Perspective
 

Mitophagy / Autophagy Analysis Products

Product Name Probe 1
Dyes and Fluorescence Properties		 Probe 2
Dyes and Fluorescence Properties
Mitophagy Detection Kit Mtphagy Dye
Ex: 500-560 nm, Em: 670-730 nm		 Lyso Dye
Ex: 350-450 nm, Em: 500-560 nm
Mtphagy Dye Mtphagy Dye
Ex: 500-560 nm / Em: 690-750 nm		  
Autophagic Flux Assay Kit* DAPRed
Ex: 500-560 nm / Em: 690-750 nm		 DALGreen
Ex: 350-450 nm / Em: 500-560 nm

   *Autophagic Flux Assay Kit includes lysosome acidification Inhibitor as a negative control.

 

 

 

 

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