Mitochondrial Changes in Apoptosis

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Caspase Cleavage and Activation

Caspase Processing

Caspase Activity

Physiological Caspase Substrates and Inhibitors

Mitochondrial Changes in Apoptosis

Phosphatidylserine Externalization

DNA Fragmentation

The mitochondrial outer membrane permeability (MOMP) is a hallmark of the intrinsic pathway of caspase activation and often considered the point-of-no return. This is because MOMP typically leads to cell death, even in the absence of caspase activation. Activation of BH3-only proteins such as PUMA, BID, and BIM are necessary for the activation and oligomerization of Bax or Bak. These pro-apoptotic proteins form pores in the outer mitochondrial membrane, facilitating the release of multiple proteins into the cytosol.

Browse our reagents to detect Mitochondrial Changes in Apoptosis including:

• Mitochondrial Extraction Kits
• Mitochondria Marker Antibodies
• Cytochrome c Release Kits

Release of Pro-apoptotic Proteins from Mitochondria

• Cytochrome c released from mitochondria, dATP, APAF-1, and pro-Caspase-9 form the apoptosome, which results in the activation of Caspase-9.
• Smac/Diablo and HTRA2/Omi promote caspase activity through interactions with IAP proteins.
• AIF (and possibly endonuclease G) translocate to the nucleus upon mitochondrial release and lead to chromatin condensation and DNA fragmentation.

Cytochrome c Release is an Indicator of MOMP

One of the most prevalent markers to analyze MOMP is the release of Cytochrome c into the cytosol. The extent of Cytochrome c translocation is often determined by Western blot analysis of cytosolic and enriched mitochondrial fractions. Immunofluorescence (IF) staining of fixed cells is an effective approach to visualize these changes at the single-cell level in which the distribution of Cytochrome c shifts from punctate mitochondrial staining to diffuse cytosolic staining during apoptosis. The localization of Cytochrome c that was once challenging to detect in suspension cells or for cells that have rounded up, for example, have been improved with the emergence of high-resolution fluorescence microscopy.

Cytochrome c released from enriched mouse liver mitochondria was quantified with the Rat/Mouse Cytochrome c ELISA (Catalog # MCTC0). Samples (n=3) in this data - Total: mouse liver mitochondria incubated with Triton® X-100, Untreated: untreated mitochondria, BID: mitochondria incubated with casp-8 cleaved human BID (Catalog # 882-B8), BID + Bcl-w: mitochondria incubated with casp-8 cleaved human BID and human Bcl-w (Catalog # 824-BW), BID + Bcl-w + MAB8241: mitochondria incubated with caspase-8 cleaved human BID, human Bcl-w and Bcl-w neutralizing antibody. Note:- Products used in this experiment are available at: rndsystems.com

What is another indicator of MOMP?

MOMP may also be detected by measuring the mitochondrial transmembrane potential (ΔΨm), which is disrupted once the outer membrane is permeabilized. The loss of ΔΨm can be observed using positively charged (cationic) dyes such as TMRE and JC-1 that accumulate in the mitochondrial matrix when ΔΨm is maintained. Accordingly, the mitochondria from healthy cells will fluoresce brighter than cells undergoing apoptotic death or mitochondrial depolarization, e.g. treated with the oxidative phosphorylation uncoupler FCCP.

Cytochrome c WB Protocol