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Nuclear apoptosis induced by isolated mitochondria
Abstract:
We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC-YVAD-CHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.
Insights
Mitochondria can trigger apoptosis in isolated nuclei. Mitochondrial supernatants, even without cytosolic factors, induce nuclear DNA fragmentation and chromatin condensation, mediated by a novel endonuclease.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitochondria play a crucial role in cellular processes, including apoptosis.
- The precise mechanisms by which mitochondria initiate apoptotic events in isolated nuclei are not fully understood.
Purpose of the Study:
- To investigate the ability of isolated mitochondria and their components to induce apoptosis in isolated nuclei.
- To characterize the factors responsible for mitochondrial-induced nuclear apoptosis.
Main Methods:
- Isolation and purification of mitochondria from mouse liver and spinach leaves.
- Incubation of isolated mitochondria with Xenopus laevis egg extracts and isolated mouse liver nuclei.
- Preparation of mitochondrial supernatants through hypotonic treatment and high-speed centrifugation.
- Assessment of apoptotic changes in nuclei, including chromatin condensation, margination, and DNA laddering.
- Inhibition studies using caspase inhibitors (AC-DEVD-CHO, AC-YVAD-CHO).
- Characterization of the nuclear endonuclease activity.
Main Results:
- Isolated mitochondria induced apoptotic changes in mouse liver nuclei within Xenopus egg extracts.
- Mitochondrial supernatants alone, without cytosolic factors, triggered nuclear apoptosis.
- Apoptotic changes included chromatin condensation, margination, and DNA fragmentation.
- Caspase inhibitors partially blocked DNA fragmentation and fully blocked chromatin condensation.
- A novel nuclear endonuclease, distinct from known enzymes like endonuclease G, was identified in the mitochondrial supernatants.
Conclusions:
- Mitochondria contain factors capable of directly inducing apoptosis in isolated nuclei.
- A novel endonuclease, activated or released by mitochondria, plays a key role in DNA fragmentation during this process.
- These findings shed light on the intrinsic pathway of apoptosis initiation by mitochondria.