title: Activation of apoptosis by cytoplasmic microinjection of cytochrome c.
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.
authors: Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina.
authors: Adam J
authors: AJ
authors: Kole
authors: Elizabeth R W
authors: ER
authors: Knight
authors: Mohanish
authors: M
authors: Deshmukh
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D045304:Cytochromes c / Q000008:administration & dosage / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D051379:Mice; D008845:Microinjections / Q000379:methods*; D011984:Sensory Receptor Cells / Q000166:cytology
chemical_list: D045304:Cytochromes c