Mitochondria Research

Science Note

[Aug 1, 2023]                                                                                                                                                                                                                            Previous Science Note
Mitochondrial Stress Responses: Iron, ROS, and Mitophagy

In recent years, the discovery of several novel mitochondrial stress responses has attracted much attention. A mitochondrial iron-responsive pathway was discovered that protects cells from iron deficiency. In other breakthroughs, a new anticancer target is identified and the nucleus-to-mitochondria ROS-sensing pathway is implicated in resistance to platinum-based treatments for ovarian cancer. Research also uncovers the role of the protein in promoting mitochondrial fission, essential for effective mitophagy.
Dojindo has a number of unique probes that can be used to evaluate different mitochondrial stress responses: Mitochondrial Fe2+ detection MitoFerro-Green, Mitochondrial ROS Detection mtSOX Deep Red, Mitophagy Detection Kit, and so on. 

A mitochondrial iron-responsive pathway regulated by DELE1
Click here for the original article: Yusuke Sekine, et. al., Molecular Cell, 2023.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway
Click here for the original article: Junbing Zhang, et. al., Cell, 2023.

The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy
Click here for the original article: Tomoyuki Fukuda , et. al., Molecular Cell, 2023.

Point of Interest
- In a steady state, DELE1 is swiftly degraded by the matrix-resident LONP1 post-import.
- When iron is deficient, DELE1 import is halted, leading to its stabilization on mitochondria.
- The stabilized full-length form of DELE1 activates HRI on the surface of mitochondria.
- The DELE1-HRI-ISR pathway serves to protect erythroid cells from cell death induced by iron deficiency.

Point of Interest
- The integration of chemical proteomics and CRISPRi screens identifies ROS-target proteins.
- Nuclear H2O2 oxidizes C408 within the autoinhibitory domain of CHK1, thereby leading to its activation.
- CHK1 regulates mitochondrial translation by suppressing the mtDNA-binding protein, SSBP1.
-  SSBP1 promotes resistance to platinum-based agents and nuclear H2O2 in cases of ovarian cancer.

Point of Interest
- Mitofissin is a mitochondrial fission factor located within the inner mitochondrial space.
- The yeast Mitofissin, known as Atg44, promotes mitochondrial fission, which is essential for mitophagy.
- Atg44 directly induces membrane fragility, facilitating membrane fission.
- The mechanisms and roles of membrane fission performed by Atg44 differ from those of Dnm1.

Related Techniques
           Mitochondria ferrous ion (Fe2+) detection Mito-FerroGreen
           Mitochondrial superoxide detection mtSOX Deep Red NEW
           Mitophagy Detection Mitophagy Detection Kit and Mtphagy Dye
           Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit / MT-1 MitoMP Detection Kit
           Glycolytic/Mitochondrial activity detection Glycolysis/JC-1 MitoMP Assay Kit NEW
           Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
           Mitochondrial staining (Long-Term Visualization) MitoBright LT Green, Red, Deep Red
           Total ROS detection Higher sensitivity or for long-term live cell imaging
 
Related Applications

Lysosomal Function and Mitochondrial ROS

 

 

CCCP and Antimycin are recognized inducers of mitochondrial ROS, linked to the loss of mitochondrial membrane potential. Recent studies have shown that CCCP induces not only mitochondrial ROS but also lysosomal dysfunction. To observe mitochondrial ROS, HeLa cells were labeled with mtSOX Deep Red for Mitochondrial Superoxide Detection, and the lysosomal mass and pH were independently detected with LysoPrime Green and pHLys Red. Co-staining with mtSOX and Lysosomal dyes revealed that CCCP, unlike Antimycin, triggers concurrent lysosomal neutralization and mitochondrial ROS induction.

Reference: Benjamin S Padman, et. al., Autophagy (2013)

Products in Use
   - LysoPrime Green
   - pHLys Red
   - Lysosomal Acidic pH Detection Kit
   - mtSOX Deep Red - Mitochondrial Superoxide Detection


Co-staining of Lysosome and Mitophagy

 

 

We performed fluorescence imaging by stimulating mitophagy induction in SHSY-5Y cells stained with Mitophagy Detection(Code: MD01) and LysoPrime Green or existing products. The fluorescence signal of LysoPrime Green did not decrease and the lysosomal localization over time was confirmed. This means that the co-localization rate of the fluorescent spots of the Mtphagy Dye is higher than that of the existing product, and thus more accurate mitophagy analysis can be performed.

LysoPrime Green: Ex= 488 nm, Em= 500-570 nm
Mtphagy Dye: Ex= 561 nm, Em= 560-650 nm

Products in Use
   - LysoPrime Green
   - Mitophagy Detection Kit and Mtphagy Dye


Inhibition of Mitochondrial Electron Transport Chain

Antimycin stimulation of Jurkat cells was used to evaluate the changes in cellular state upon inhibition of the mitochondrial electron transport chain using a variety of indicators.

The results showed that inhibition of the electron transport chain resulted in (1) a decrease in mitochondrial membrane potential and (2) a decrease in OCR. In addition, (3) the NAD+/NADH ratio of the entire glycolytic pathway decreased due to increased metabolism of pyruvate to lactate to maintain the glycolytic pathway, (4) GSH depletion due to increased reactive oxygen species (ROS), and (6) increase in the NADP+/NADPH ratio due to decreased NADH required for glutathione biosynthesis were observed. 

 


 

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’ (mtSOX, 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 MD01MT02

Lipophilic peroxide / Singlet oxygen / Superoxide

  Lipophilic peroxide Singlet oxygen Superoxide Superoxide
Reagent MitoPeDPP Si-DMA mtSOX 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 MT14

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 mtSOX Deep Red
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

Product Classification

Product Classification