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1Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina, 2Curriculum in Neurobiology, Neuroscience Center, University of North Carolina
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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.
Kole, A. J., Knight, E. R., Deshmukh, M. Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c. J. Vis. Exp. (52), e2773, doi:10.3791/2773 (2011).
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.
1. Production of Microinjection Needles
2. Preparation of Protein Mixtures for Injection
3. Cytoplasmic Microinjection of Cytochrome c
4. Representative Results:
The cytoplasmic microinjection of cytochrome c mimics its release from mitochondria during apoptosis. Thus, as expected, fibroblasts rapidly undergo apoptosis upon cytosolic microinjection of bovine cytochrome c (Fig. 1A). To ensure that the injection procedure alone is not responsible for cell death, injection of yeast cytochrome c serves as an important control, since yeast cytochrome c is incapable of activating caspases12.
Interestingly, post-mitotic sympathetic neurons are remarkably resistant to cytosolic cytochrome c (Fig. 1B)8,13. Our lab has identified that the endogenous caspase inhibitor XIAP is a key inhibitor of caspase activation in neurons14. Thus, for neurons to die following cytochrome c injection, XIAP must first become inactivated. For example, microinjection of cytochrome c into xiap-/- sympathetic neurons is sufficient to allow caspase activation and apoptosis in these cells (Fig. 2).
Figure 1. Cytoplasmic microinjection of cytochrome c induces rapid death in fibroblasts, but not neurons. A) Wild-type MEFs or (B) postnatal day 5 wild-type sympathetic neurons were microinjected with bovine cytochrome c (10 mg/mL) together with rhodamine-dextran to mark injected cells. Images show the same field of cells immediately following injection (0 hr), or at the indicated times. Arrows indicate injected cells. Scale bar, 20 μm.
Figure 2. XIAP-deficient neurons are susceptible to cytoplasmic cytochrome c microinjection. Postnatal day 5 sympathetic neurons from XIAP knockout mice were microinjected with bovine cytochrome c (10 mg/mL) together with rhodamine-dextran to mark injected cells. Images show the same field of cells immediately following injection (0 hr), or 5 hours after cytochrome c microinjection (5 hr). Scale bar, 20 μm.
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 microinjection shown here can also be used for other proteins of interest. For example, some investigators have used microinjection of antibodies that target specific proteins, such as cytochrome c or c-Jun in studies of apoptosis13,15.
The most common difficulty during microinjection is the clogging of microinjection needles during the procedure. If the needle becomes clogged, one can use the “clean” function on the microinjector which sends a strong pressure pulse through the needle to expel particles blocking the needle opening. Oftentimes, cleaning the needle is sufficient to unclog the needle. However, if dye is seen exiting the needle during a “clean” but immediately stops again, this indicates that the working pressure on the microinjector may be too low. In this case, increase the working pressure until a continuous flow from the needle is seen again. Cleaning the needle does not always restore flow, in which case the needle most likely needs to be replaced. One technique which can be used as an alternative, especially if the injection material itself is precious, involves gently breaking the tip of the microinjection needle against the bottom of the tissue culture dish. If done carefully, this can enlarge the opening of the needle and will restore flow. However, a large crack in the needle opening will cause a massive release of dye into the culture dish, obscuring the view of cells.
Unfortunately, not all cell types are capable of being microinjected. Some cells (e.g. cerebellar granule neurons) are too small for microinjection. Other cells (e.g. adult cardiomyocytes), while large enough, cannot withstand the injection procedure and die even with control injections. As described above, the microinjection of yeast cytochrome c serves as a useful control since yeast cytochrome c is unable to activate caspases. Cells microinjected with yeast cytochrome c will not activate apoptosis unless it is due to the microinjection procedure itself. Finally, some cells (e.g. fibroblasts) can withstand microinjection but can be difficult to inject because of their flat morphology. As a result, there is a risk that the microinjection needle will break against the bottom of the tissue culture dish with each cell that is injected.
Automated or manual micromanipulators can be used for protein microinjection, with each system having advantages and disadvantages. Automated micromanipulators are convenient because one can set a z-axis level to which the microinjection needle is automatically lowered for injecting cells. This is particularly useful when injecting cells whose height is similar across a cell culture monolayer. However, for cells which are cultured on coated dishes, (e.g. neurons plated on collagen-coated dishes), the three-dimensional nature of these cultures makes setting a specific z-axis level tedious. For these cultures, manual micromanipulators are advantageous since the needle can be rapidly adjusted to the specific height of each cell.
Microinjection can be a difficult technique to master and will require practice. In addition, the technique of microinjection has some limitations. For example, only a small proportion of cells in a tissue culture dish can be injected. Thus, biochemical preparations in which the whole culture is collected (e.g. cell lysates for Western blot) are not an accurate representation of the injected cells alone. Instead, most experiments need to be completed at the single-cell level. However, once the basic techniques presented here are mastered, one can perform a unique set of experiments to test hypotheses that cannot be answered using other methods.
All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina.
This work was supported by NIH grant NS042197 to MD. AJK was supported by grants T32GM008719 and F30NS068006.
|DM IRE2 Inverted Microscope||Leica Microsystems|
|PC-10 Microinjection Needle Puller||Narishige International|
|MWO-202 Micromanipulator||Narishige International|
|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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