Assessment of Apoptosis in Fractionated Extract

2.5.1. Reconstituted Extract:

Cytosolic and Mitochondria/Heavy Membrane Fractions

1. Cytosolic extract.

2. Mitochondria/heavy membrane fraction.

3. 0.2 M Phosphocreatine.

4. 5 mg/mL Creatine kinase.

2.5.2. Assessment of Cytochrome c-Induced Apoptosis

1. Cytosolic extract.

2. 0.2 M Phosphocreatine.

3. 5 mg/mL Creatine kinase.

5. Purified cytochrome c (from equine heart; Sigma); make stock solution in water at 1 mg/mL and store at -80°C for up to 1 mo.

2.6. Immunodepletion, Addition of Recombinant Proteins, and Antibody Neutralization

1. Antibody/antiserum.

2. Preimmune serum or immunoglobulin G (IgG).

3. Protein A/G-Sepharose.

4. Phosphate-buffered saline PBS: 8 g NaCl, 0.2 g KCl, 0.1 g CaCl2, 0.1 g MgCl2-6 H2O, 1.15 g NaH2PO4-H2O, 0.2 g KH2PO4; add water to 1 L; store at room temperature.

6. Bio-Spin Disposable 1-mL chromatography columns (Bio-Rad).

7. Extract buffer (XB): 50 mM sucrose, 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM potassium HEPES, pH 7.7; prepare fresh.

8. Recombinant protein of interest.

3. Methods

The methods described in this chapter summarize: (1) how to obtain eggs for interphase extract preparation and how to prepare sperm chromatin for nuclear formation to monitor nuclear morphology during apoptosis; (2) the preparation of crude interphase egg extract and assessment of apoptosis in the interphase egg extract; (3) the separation of the crude interphase extract into cytosolic and membrane fractions and the assessment of apoptosis using fractionated extract; (4) the modification of the extract through immunodepletion or supplementation with recombinant proteins of interest or neutralizing antibodies.

3.1. Collection of Xenopus Eggs and Preparation of Sperm Chromatin for Nuclei Formation

3.1.1. Collection of Eggs

Xenopus laevis mature females are housed in clean, dechlorinated tap water at a maximal density of one frog per liter (lower densities are desirable). Dechlorination can be achieved by passing tap water through a carbon filter. If chloramines are absent in the tap water, it can be stored in an open tank for 24 h with aeration. The frogs should be housed at 18°C to achieve optimal egg production. To promote egg laying, frogs are first primed with PMSG, and 2 d later are injected with hCG (see Note 1).

1. Prepare a tank containing 100 mM NaCl to house frogs after injection. Keep frog density at one frog per liter.

2. Using a 25-gage needle attached to a 1-mL syringe, inject frogs subcutaneously into the dorsal lymph sac (the upper part of the frog leg) with 0.5 mL of 200 U/mL PMSG (total 100 U) (see Fig. 1). Place the frogs in the tank containing 100 mM NaCl. The frogs will be ready to induce egg laying in 2 to 3 d. Alternatively, the PMSG-injected frogs can be housed for up to 10 d before induction of egg laying with hCG.

3. To induce egg laying, inject the frogs as described in step 2 with 0.5 mL of 1000 U/mL hCG (a total of 500 U).

4. Place the injected frog into a tank containing 5 L of 100 mM NaCl. Each injected frog should be housed in a separate tank as the quality of eggs varies between frogs.

5. At 16 to 22 h after injection of hCG, the eggs will be ready to collect. Remove the frog to a clean tank containing 100 mM NaCl and pour the eggs into a 500-mL beaker.

Subcutaneous Lymph Sac Frog
Fig. 1. Injection of frogs to obtain eggs. The frog is first primed with 100 U PMSG by subcutaneous injection into the dorsal lymph sac, marked by the asterisk in the picture. A second subcutaneous injection into the dorsal lymph sac (this time with 500 U hCG) 2 to 3 d later induces egg laying.

3.1.2. Preparation of Demembranated Sperm Chromatin

Fully functional nuclei are formed within 30 min of adding sperm chromatin to interphase egg extract. The formation of nuclei is driven by the binding and fusion of membrane vesicles to the chromatin. The resultant synthetic nuclei incorporate nuclear pore components and are competent to undergo bidirectional nuclear transport, DNA replication, mitosis, and apoptosis (1,4,5).

1. Anesthetize four or five male frogs by immersion in 0.1% Tricaine and sacrifice either by cervical dislocation or by pithing, followed by cutting through the spinal cord. Using dissecting scissors, cut through the skin into the peritoneum along the midline of the frog. The testes are beige 0.5-cm organs located along the midline in the center of the abdominal cavity.

2. After pushing the liver aside, grasp the fatty material at midabdomen and pull gently. The testes will emerge on either side of the midline. Gently cut them free of adherent tissue with either forceps or scissors and place in a 60-mm glass Petri dish containing extraction buffer.

3. Using two pairs of sharp forceps, mince the testes into small pieces and transfer them to a 15-mL conical polypropylene tube.

4. Vortex the minced testes vigorously and pellet the larger pieces by gentle centrifugation (~10 s at 200g in a clinical centrifuge).

5. Transfer the supernatant to a new tube and add 3 mL extraction buffer supplemented with 200 mM sucrose to pellet. Vortex for 1 min and recentrifuge for 10 s at 200g. Combine the supernatants and repeat the extraction of the pellets two or three times until the supernatant is no longer cloudy.

6. Centrifuge the combined supernatants 50 s at 450g at room temperature to pellet any remaining large pieces of tissue. Transfer the supernatants to a 15-mL tube and centrifuge 10 min at 2600g (4000 rpm in a Sorvall HB4 rotor).

7. Prepare gradients by adding 0.2 mL extraction buffer containing 2.5 M sucrose to each of four 2.5-mL Beckman Ultra-Clear tubes and overlaying with 1.7 mL extraction buffer containing 2.3 M sucrose.

8. Resuspend sperm in 0.8 mL extraction buffer containing 2 M sucrose and overlay gently on the top of the sucrose gradients (0.2 mL per tube). After stirring the interface between the sperm and the 2.3 M sucrose with a pipet tip, centrifuge the sucrose gradients in a TL-100 tabletop ultracentrifuge, 25 min at 93,000g (33,000 rpm in a TLS-55 rotor) at 4°C.

9. Aspirate the top half of the gradient, which contains contaminating red blood cells. Transfer the lower half of the gradient to a new tube (see Note 2).

10. Dilute sperm to 12 mL with extraction buffer containing 200 mM sucrose and centrifuge for 10 min in a swinging bucket rotor at 4100g (5000 rpm in a Sorvall HB4 or HB6 rotor or equivalent).

11. To demembranate the sperm, resuspend the pellet in 1 mL extraction buffer supplemented with 200 mM sucrose, 5 |lg/mL aprotinin, 5 |lg/mL leupeptin, 1 mM DTT, 0.4% (v/v) Triton X-100. Incubate on ice for 30 min.

12. Prepare sucrose cushions by adding 0.5 M sucrose, 3% (w/v) BSA fraction V, 5 |g/mL aprotinin, 5 |g/mL leupeptin, and 1 mM DTT to the extraction buffer. Prepare the sucrose cushion in two 1.5-mL microcentrifuge tubes and then overlay them with one-half of the sperm preparation from step 11. Pellet the sperm by centrifugation for 10 min in a clinical tabletop centrifuge at 870g at room temperature.

13. Prepare extraction buffer containing: 200 mM sucrose, 3% (w/v) BSA fraction V, 5 |g/mL aprotinin, 5 | g/mL leupeptin, and 1 mM DTT.

14. After centrifugation, remove the supernatant from the pellets and resuspend the sperm in 0.1 mL freshly prepared extraction buffer (step 13). Transfer the resuspended sperm to a clean microcentrifuge tube. Be very careful not to contaminate the sperm with residual Triton X-100 from the sides of the original tube.

15. Add extraction buffer prepared in step 13 so that the final volume of the sperm preparation is 0.25 mL. Count the sperm using a hemacytometer and dilute to 1 x 105/|L (this is 100X). Snap freeze the sperm in small aliquots in liquid nitrogen and store at -70°C. Usually, a sperm preparation yields 1 to 2 x 107 sperm per frog.

3.2. Preparation of Crude Interphase Egg Extract and Assessment of Apoptosis

The preparation and assessment of apoptosis in crude interphase egg extracts is described in Subheadings 3.2.1. to 3.2.4. The mature Xenopus eggs are arrested in metaphase of meiosis II and therefore have high mitotic kinase (cyclin B/cdc2) activity. On crushing of the eggs by centrifugation, there is a transient release of Ca2+ from intracellular stores. This release of Ca2+ promotes the degradation of cyclin B, subsequent loss of cdc2 kinase activity, and entry into interphase of the cell cycle. The extract is retained in interphase by addition of cycloheximide, which prevents new protein synthesis. When the egg extract is prevented from entering interphase by the addition of EGTA so Ca2+ release is not allowed, the resultant cytostatic factor (CSF) is extremely refractory to apoptosis (6). This is attributed to the high levels of mitogen-activated protein kinase, which phosphorylates and inhibits the activity of caspase 9 (6,7). Prolonged incubation of the crude interphase extract (containing mitochondria) at room temperature induces a spontaneous apoptosis program that is characterized by the release of cytochrome c from the mitochondria, activation of apoptotic proteases (caspases), and condensation and fragmentation of added DNA.

3.2.1. Preparation of Crude Interphase Egg Extract and Induction of Spontaneous Apoptosis Program

1. Collect eggs in a 200-mL glass beaker and pour off any excess solution.

2. Rinse eggs once in 2% L-cysteine, pH 8.0, then continue to incubate in 100 mL 2% L-cysteine solution per frog for approx 5 min (or until the eggs pack very closely) at room temperature, gently swirling the eggs periodically (see Note 3).

3. Pour the cysteine solution off the dejellied eggs and wash the eggs in 0.25X MMR (1/40 dilution of 10X stock) by adding buffer, swirling the eggs, then pouring off the buffer. Repeat twice. Pour eggs into a 100-mm glass Petri dish.

4. Remove the "bad eggs" with a Pasteur pipet. Bad eggs appear as white puffy balls, with an obscure boundary between the dark and light hemispheres. This is in contrast with a "good egg," which has an even unmottled appearance and a tiny white spot (indicative of oocyte maturation) in the center of the darkly pigmented hemisphere of the mature egg (see Note 4).

5. Wash the eggs three times with ELB by swirling the eggs in buffer, then pouring off the buffer.

6. Transfer the eggs with an inverted Pasteur pipet (the bulb on the pointed end so the eggs are not prematurely lysed) into a 15-mL polypropylene tube.

7. To pack the eggs, centrifuge for 15 s in a clinical centrifuge at 400g. Remove any extra buffer from the top of the packed eggs with a Pasteur pipet. It is critical to remove as much buffer as possible to ensure a concentrated egg extract. Sacrifice eggs to achieve this.

8. Determine the volume of the packed eggs, then add 5 |lg/mL aprotinin (1/1000), 5 |lg/mL leupeptin (1/1000), 5 |g/mL cytochalasin B (1/1000), 5 |L/mL of 10 mg/mL cyclo-hexamide (50 | g/| L final) directly to the top of the packed eggs. For example, if you have 8 mL packed eggs, add 8 | L of each of these items.

9. Crush the eggs by centrifuging for 15 min at 12,000g, 4°C, in a Sorvall HB-4 swinging bucket rotor. The lysed eggs will be separated into three layers: a yellow lipid layer on top, the crude interphase extract in the middle, and a dark pellet containing pigment granules (see Fig. 2A).

10. Remove the crude extract by piercing the side of the polypropylene tube with an 18-gage needle attached to a 5-mL syringe (see Note 5).

11. Add 1/10 dilution of 0.2 M phosphocreatine (20 mM final) and 1/100 of 0.5 mg/mL creatine kinase (50 |g/mL final) and 0.2 M ATP (20 |M final) to create an ATP-regenerating system and sperm nuclei to a concentration of 1000/|L (1/100 dilution). Incubate this extract at room temperature for 4 to 8 h. The onset of apoptosis can be assayed in this system by observing the morphology of sperm nuclei stained with Hoechst 33528 (Subheading 3.2.4.), measuring cytochrome c release (Subheading 3.2.2.) or assessing caspase activity (Subheading 3.2.3.).

Fig. 2. (A) Crude interphase extract. After the eggs are crushed by centrifugation at 12,000 rpm, the eggs are separated into three layers. The top yellow layer consists of lipids, and the middle layer contains the cytosol and membrane fractions; the dark bottom layer contains pigment granules and yolk proteins and glycogen. (B) After ultracentrifugation of the crude extract, it is separated into cytosolic and membrane fractions. The light membrane fraction, which is pale yellow, lies just beneath the cytosolic fraction. The heavy membrane fraction enriched in mitochondria lies just beneath the light membrane layer; glycogen, ribosomes, and the like pellet at the bottom of the tube.

Fig. 2. (A) Crude interphase extract. After the eggs are crushed by centrifugation at 12,000 rpm, the eggs are separated into three layers. The top yellow layer consists of lipids, and the middle layer contains the cytosol and membrane fractions; the dark bottom layer contains pigment granules and yolk proteins and glycogen. (B) After ultracentrifugation of the crude extract, it is separated into cytosolic and membrane fractions. The light membrane fraction, which is pale yellow, lies just beneath the cytosolic fraction. The heavy membrane fraction enriched in mitochondria lies just beneath the light membrane layer; glycogen, ribosomes, and the like pellet at the bottom of the tube.

3.2.2. Cytochrome c Release Assays

Execution of the intrinsic apoptotic program depends on the release of cytochrome c from the mitochondria. Once released, cytochrome c is involved in the formation of a large proapoptotic proteolytic complex, known as the apoptosome. The apoptosome promotes apoptosis by activating caspases. Because cytochrome c serves to activate caspases, the transit of cytochrome c from the mitochondria to cytosol is indicative of apoptosis. This release can be monitored directly by immunoblotting of cytochrome c in the cytosolic fraction after removal of mitochondria by filtration.

1. Remove 25 ||L egg extract containing ATP-regenerating mix (Subheading 3.2.1.) at timed intervals (30- to 60-min intervals) and pass through a 0.1 mM Ultrafree MC filter. This process removes the mitochondria. The supernatant that flows through the filter will contain any cytochrome c that has been released into the cytosol.

2. Subject 7 ||L of sample to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis using a 17.5% acrylamide gel.

3. Immunoblot with anti-cytochrome c antibody (see Note 6).

3.2.3. Biochemical Assessment of Apoptosis: Measuring Caspase Activation

The apoptotic program culminates in the activation of proteases, known as caspases, that cleave intracellular substrates, resulting in orderly dismantling of the cell. Caspase activity can be measured easily in vitro using a colorimetric assay and thus provides a quantitative readout of apoptosis in the egg extract. The in vitro caspase assay described here measures the cleavage of a synthetic peptide substrate (Ac-DEVD-pNA) of caspase 3 that is linked to a chromophore (see Note 7). When the egg extract, containing active caspase 3, is incubated with the substrate, the substrate is cleaved. Caspase-mediated cleavage results in the release of the chromophore from the substrate, causing a color change which is measured spectrophotometrically.

1. Add 85 |L assay buffer to a well of a 96-well microtiter plate for each sample/time-point to be assayed. Keep this plate at room temperature.

2. Add 5 |L egg extract containing ATP-regenerating mix (Subheading 3.2.1.) to the well.

3. Add 10 |L caspase substrate (Ac-DEVD-pNA). Tap the sides of the plate to mix and incubate at 37°C for 1 h (see Note 8).

4. Read the plate at 405 nm using a microplate reader.

3.2.4. Nuclear Morphology

A hallmark of apoptosis is nuclear condensation and fragmentation. This apoptotic phenotype can be readily observed in synthetic nuclei formed in the egg extract because apoptotic nuclei appear as condensed and fragmented (see Fig. 3).

1. Prepare crude interphase egg extract with ATP-regenerating mix (Subheading 3.2.1.).

2. Add sperm chromatin to the egg extract at a 1/100 dilution (1000 sperm/|L) and incubate at room temperature. Nuclei will start to form after 20 min and should be well formed after 30 min. The sperm chromatin will change shape from coiled and stringy to kidney bean shape and round. Well-formed nuclei appear round and are homogeneous (see Fig. 3).

3. Nuclei should be observed at 30-min intervals after 2.5 h of incubation in egg extract at room temperature because extracts undergo spontaneous apoptosis at varying rates. Remove 2 |L extract and mix with 2 |L Hoecsht dye on a microscope slide and then add a cover slip. Observe nuclei using the ultraviolet filter on a fluorescent microscope. Apoptotic nuclei are condensed and fragmented or blebbed (see Fig. 3).

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