Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c
Summary
In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.
Abstract
Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.
Protocol
Discussion
The microinjection of cytochrome c directly into the cytoplasm of cells is a unique and powerful tool which allows for studies of the post-mitochondrial regulation of apoptosis. Importantly, this technique allows for the direct activation of apoptosis downstream of mitochondria without the use of agents which cause cellular or mitochondrial damage.
While this protocol has focused on microinjection of cytochrome c for studies on apoptosis, the general principles of protein …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant NS042197 to MD. AJK was supported by grants T32GM008719 and F30NS068006.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| DM IRE2 Inverted Microscope | Leica | ||
| PC-10 Microinjection Needle Puller | Narishige | ||
| MWO-202 Micromanipulator | Narishige | ||
| FemtoJet Microinjector | Eppendorf | ||
| Thin-wall Boroscilicate Capillary Glass with Microfilament | A-M Systems | 615000 | 4 inch length, 1.00 mm outer diameter, 0.75 mm inner diameter |
| Rhodamine B isothiocyanate-Dextran | Sigma-Aldrich | R9379 | Average molecular weight ~70,000 Da |
| Bovine Cytochrome c Protein | Sigma-Aldrich | C3131 |
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