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 death^3-4.

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 cells^6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis^10-11. 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

1. Production of Microinjection Needles

1. Pre-fabricated microinjection needles are available commercially (e.g. Femtotips from Eppendorf) and are useful if one is not performing a large number of microinjections. However, for those who wish to establish long-term capabilities for microinjecting, an alternative is to produce microinjection needles in the lab using thin wall borosilicate glass capillaries and a commercial needle puller. This also allows the shape of needles to be varied, which can be useful for different cell types.
2. With the Narishige PC-10 Microinjection Needle Puller, attach all four weights and use a one-step pulling....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DM IRE2 Inverted Microscope	Leica Microsystems
PC-10 Microinjection Needle Puller	Narishige International
MWO-202 Micromanipulator	Narishige International
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