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

Deshmukh, Mohanish

Knight, Elizabeth R.W.

Kole, Adam J.

PubMed

Carolina Digital Repository

Video ArticleActivation of Apoptosis by Cytoplasmic Microinjection of Cytochrome cAdam J. Kole1, Elizabeth R.W. Knight2, Mohanish Deshmukh11Department of Cell and Developmental Biology, Neuroscience Center, University of North Carolina2Curriculum in Neurobiology, Neuroscience Center, University of North CarolinaCorrespondence to: Mohanish Deshmukh at mohanish@med.unc.eduURL: http://www.jove.com/details.php?id=2773DOI: 10.3791/2773Citation: Kole A.J., Knight E.R., Deshmukh M. (2011). Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c. JoVE. 52. http://www.jove.com/details.php?id=2773, doi:10.3791/2773AbstractApoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cellsencounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from themitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapidactivation 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 thepost-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 toenable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of itscDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will besequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool tomimic 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) andprimary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, thetechniques shown here can also be easily adapted for microinjection of other proteins of interest.Protocol1. Production of Microinjection Needles2. Preparation of Protein Mixtures for Injection3. Cytoplasmic Microinjection of Cytochrome cPage 1 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments1. Pre-fabricated microinjection needles are available commercially (e.g. Femtotips from Eppendorf) and are useful if one is not performing alarge number of microinjections. However, for those who wish to establish long-term capabilities for microinjecting, an alternative is toproduce microinjection needles in the lab using thin wall borosilicate glass capillaries and a commercial needle puller. This also allows theshape 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 program (Step 1 setting) with therelative heat setting at 58.0 (No.2 heater). Be sure to place the heating element at the center of each capillary so that the two resultingneedles are a similar length.3. Pull several capillaries (about 2 capillaries for each protein of interest).4. Store needles in a container while being sure not to damage the needle tip. Materials such as foam or Blu-Tack can be used in containers tohold microinjection needles.1. Prepare a 10x microinjection buffer containing 1 M potassium chloride and 0.1 M potassium phosphate (KPi) buffer (equimolar mixture of K2HPO4 and KH2PO4) at a pH of 7.4. This buffer can be stored long-term at room temperature.2. To visualize the injection, a fluorescent dye like rhodamine-dextran needs to be added. Dilute the10x microinjection buffer in water anddissolve rhodamine-dextran powder to make a 5x microinjection buffer solution containing 20-40 mg/mL rhodamine dextran.3. Store this solution in the dark at 4°C. It should be sufficient for up to 100 individual protein solution preparations. Other dyes, like fluoresceinisothiocyanate-dextran can substitute.4. Prepare cytochrome c stocks by dissolving purified cytochrome c in water to a concentration of 20 mg/mL. Store cytochrome c at -80°C forlong-term storage and avoid freeze-thaw cycles by storing cytochrome c in small (~10 μL) aliquots.5. Prepare a 10 μL protein mixture for injection by combining 2 μL 5x microinjection buffer containing rhodamine-dextran with 3 μL water and 5μL cytochrome c for a final concentration of 10 mg/mL cytochrome c in 1x buffer (100 mM KCl, 10 mM KPi, 4-8 mg/mL rhodamine dextran).1. Just prior to microinjection, centrifuge the mixture of cytochrome c and microinjection buffer at 16,000 g for 10 minutes at 4°C to separate anyparticulate matter which may clog microinjection needles.2. During centrifugation, turn on the microinjector to allow air pressure to build.3. Place a dish of cells on the center of the microscope stage and set the focus on the cells. To minimize the amount of time that the cells arekept outside the incubator, cells are typically returned to the incubator within 30 min.4. Representative Results:The cytoplasmic microinjection of cytochrome c mimics its release from mitochondria during apoptosis. Thus, as expected, fibroblasts rapidlyundergo apoptosis upon cytosolic microinjection of bovine cytochrome c (Fig. 1A). To ensure that the injection procedure alone is not responsiblefor 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 theendogenous 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 caspaseactivation 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 markinjected 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 fromXIAP knockout mice were microinjected with bovine cytochrome c (10 mg/mL) together with rhodamine-dextran to mark injected cells. Imagesshow the same field of cells immediately following injection (0 hr), or 5 hours after cytochrome c microinjection (5 hr). Scale bar, 20 μm.Page 2 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments4. Pipette 0.5-1 μL from the top surface of the protein mixture into the blunt end of the capillary. Be careful to not pipette any particles whichhave been centrifuged to the bottom of the tube. Within a minute, the protein mixture will distribute to the needle tip through capillary action.5. Attach the needle firmly to the capillary holder of the micromanipulator and position the needle so that its tip passes through the transmittedlight of the microscope at approximately a 45° angle.6. Adjust the position of the needle tip so that it is located directly in the center of the field of view. To center the needle, use themicromanipulator to move the needle while looking through the microscope eyepiece. The needle shadow should be visible. Adjust the needleso that its shadow is only seen in one half of the field of view, indicating that the needle tip is centered above the cells.7. Set the microinjector to the Continuous Flow mode and set the working pressure to 20 - 100 hPa. Each needle may require a differentworking pressure, and the working pressure will likely need adjustment during the microinjection procedure to maintain a steady flow.8. Lower the needle using the coarse knob to a position just above the cells. To do this, raise the focal plane of the microscope to a position justabove the cells. Then lower the needle towards the cells using the coarse knob until the needle tip is in focus.9. Re-center the needle tip within the field of view and increase magnification by changing the microscope objective.10. Slowly lower the needle using the fine manipulator knob until the needle is just slightly above the focal plane of the cells.11. Check the flow of the protein mixture by looking at the red fluorescence of the rhodamine. The protein mixture should be exiting the needle asa thin, constant stream. Needles should be replaced and re-loaded if the needle is compromised or if the flow is far too strong. Sometimes,the tip of the glass capillary is closed and no flow is seen from the needle. If this occurs, replace the needle or carefully lower it to the bottomof the culture dish to gently rupture the tip of the needle.12. Position the needle tip so that it is pointing towards a cell at approximately a 45° angle. Then with one smooth motion, lower the needle whilemoving it towards the cell. With a second smooth motion, immediately reverse the direction of the needle to remove it from the cell.13. A successfully injected cell will often slightly swell and can be confirmed by visualizing the red fluorescent rhodamine within the cell.Occasionally, a cell will be accidentally injected in the nucleus, which will be visible.14. Continue to inject cells by adjusting the microscope stage until about 50-100 cells are injected. When moving the microscope stage, be sureto raise the microinjection needle so that it clears the top of cells.DiscussionThe microinjection of cytochrome c directly into the cytoplasm of cells is a unique and powerful tool which allows for studies of thepost-mitochondrial regulation of apoptosis. Importantly, this technique allows for the direct activation of apoptosis downstream of mitochondriawithout 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 shownhere can also be used for other proteins of interest. For example, some investigators have used microinjection of antibodies that target specificproteins, 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 theneedle opening. Oftentimes, cleaning the needle is sufficient to unclog the needle. However, if dye is seen exiting the needle during a “clean” butimmediately stops again, this indicates that the working pressure on the microinjector may be too low. In this case, increase the working pressureuntil a continuous flow from the needle is seen again. Cleaning the needle does not always restore flow, in which case the needle most likelyneeds to be replaced. One technique which can be used as an alternative, especially if the injection material itself is precious, involves gentlybreaking the tip of the microinjection needle against the bottom of the tissue culture dish. If done carefully, this can enlarge the opening of theneedle and will restore flow. However, a large crack in the needle opening will cause a massive release of dye into the culture dish, obscuring theview 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. Asdescribed 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 themicroinjection 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 forinjecting cells. This is particularly useful when injecting cells whose height is similar across a cell culture monolayer. However, for cells which arecultured on coated dishes, (e.g. neurons plated on collagen-coated dishes), the three-dimensional nature of these cultures makes setting aspecific z-axis level tedious. For these cultures, manual micromanipulators are advantageous since the needle can be rapidly adjusted to thespecific 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. Forexample, only a small proportion of cells in a tissue culture dish can be injected. Thus, biochemical preparations in which the whole culture iscollected (e.g. cell lysates for Western blot) are not an accurate representation of the injected cells alone. Instead, most experiments need to becompleted at the single-cell level. However, once the basic techniques presented here are mastered, one can perform a unique set ofexperiments to test hypotheses that cannot be answered using other methods.DisclosuresAll experimental procedures on animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina.AcknowledgementsThis work was supported by NIH grant NS042197 to MD. AJK was supported by grants T32GM008719 and F30NS068006.ReferencesPage 3 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments1. Danial, N. N., & Korsmeyer, S. J. Cell death: critical control points. Cell 116, 205-219 (2004).2. Wang, X. The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922-2933 (2001).3. Hengartner, M. O. The biochemistry of apoptosis. Nature 407, 770-776. (2000).4. Fuentes-Prior, P., & Salvesen, G. S. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384,201-232 (2004).5. Tait, S. W., & Green, D. R. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11, 621-632(2010).6. Brustugun, O. T., Fladmark, K. E., Doskeland, S. O., Orrenius, S., & Zhivotovsky, B. Apoptosis induced by microinjection of cytochrome c iscaspase-dependent and is inhibited by Bcl-2. Cell Death Differ. 5, 660-668 (1998).7. Li, F. et al. Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-XL has activity independent of cytochrome c release. J.Biol. Chem. 272, 30299-30305 (1997).8. Deshmukh, M., & Johnson, E. M., Jr. Evidence of a novel event during neuronal death: development of competence-to-die in response tocytoplasmic cytochrome c. Neuron 21, 695-705 (1998).9. Vaughn, A. E., & Deshmukh, M. Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.Nat. Cell Biol. 10, 1477-1483 (2008).10. Yang, J. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132 (1997).11. Gonzales, D. H., & Neupert, W. Biogenesis of mitochondrial c-type cytochromes. J Bioenerg Biomembr 22, 753-768 (1990).12. Ellerby, H. M. et al. Establishment of a cell-free system of neuronal apoptosis - comparison of premitochondrial, mitochondrial, andpostmitochondrial phases. J. Neurosci. 17, 6165-6178 (1997).13. Neame, S. J., Rubin, L. L. & Philpott, K. L. Blocking cytochrome c activity within intact neurons inhibits apoptosis. J. Cell Biol. 142, 1583-1593(1998).14. Potts, P. R., Singh, S., Knezek, M., Thompson, C. B., & Deshmukh, M. Critical function of endogenous XIAP in regulating caspase activationduring sympathetic neuronal apoptosis. J. Cell Biol. 163, 789-799 (2003).Page 4 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments15. Estus, S. et al. Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.J. Cell Biol. 127, 1717-1727 (1994).

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome <em>c</em>

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Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome <em>c</em>

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