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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

¹Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, University of Ljubljana, Ljubljana; and ²Celica Biomedical Sciences Center, Ljubljana, Slovenia

Submitted 5 July 2005 ; accepted in final form 30 September 2005

	ABSTRACT

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A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells.

cytochrome c; caspase-9; apoptosis; apoptosome complex

Generally, apoptosis can be triggered by various stimuli, such as direct ligation of receptors to the cell surface, leading to the activation of initiator caspases (caspase-8 or -10) or by intracellular stress, which causes damage to mitochondria (15, 24, 31). Stimuli that damage mitochondria trigger the activation of the initiator caspase-9 (14, 18), which is synthesized as an inactive proenzyme (7, 25). The main mode of caspase-9 activation takes place in the apoptosome complex, where the caspase-9 proenzyme binds to the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) in a cytochrome c- and dATP-dependent fashion (15). Upon cell damage, cytochrome c, a protein of the mitochondrial intermembrane space (17, 19), is released into the cytoplasm (17) and promotes apoptotic activity through binding to Apaf-1 (34). Binding of procaspase-9 to Apaf-1 leads to the cleavage of procaspase-9, converting it to an active caspase-9 (15). In pituitary somatotrophs, it appears that the caspase-9-proenzyme translocates to the proximity of mitochondria upon the activation of apoptosis (21), suggesting a mechanism of recruitment of apoptosome complex elements to the proximity of mitochondria, including Apaf-1.

To monitor the proteins of the apoptosome complex, we studied the subcellular localization of Apaf-1 compared with the localization of cytochrome c, caspase-9 tagged with enhanced green fluorescent protein (EGFP) (Casp9E), and mitochondria before and after triggering apoptosis in rat pituitary somatotrophs. The subcellular localization was studied by immunocytochemistry, confocal microscopy, subcellular fractionation, and quantitative image analysis. Our results show that 2-h treatment with rotenone induced the subcellular redistribution of Apaf-1 from the cytoplasm to the proximity of mitochondria. A similar phenomenon was shown previously for the redistribution of procaspase-9 (21). Interestingly, the degree of subcellular colocalization between Apaf-1 and Casp9E was maximal 2 h after rotenone treatment. Therefore, the mechanisms that regulate the redistribution of Apaf-1 and Casp9E to the proximity of mitochondria seem to operate in the same time scale.

Furthermore, the colocalization between cytochrome c and Apaf-1 increased transiently, which indicates that in the apoptosome complex, a stable, long-lasting interaction between cytochrome c and Apaf-1 is not required. Moreover, we demonstrate the time window for interactions between cytochrome c and Apaf-1 in a single cell.

	MATERIALS AND METHODS

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Cell cultures. Primary cell cultures enriched for somatotrophs were prepared from the anterior lobe of pituitary glands of adult male rats (Wistar, 200–300 g) as described previously (3). Cells grown in a nutritive medium composed of 27% DMEM (Sigma), 54% nutrient mixture Ham's F-12 medium (Sigma), 9% -MEM (Sigma), 25 mM D-glucose (Sigma), 25 mM HEPES (Sigma), 2 mM L-glutamine, and 3% UltroserG (Life Technologies) at 37°C in a 5% CO₂ atmosphere were placed onto poly-L-lysine-coated coverslips before experiments. Cells from three different experiments were used for immunocytochemistry or DNA manipulation 24 h after cells were plated.

The animals were euthanized in accordance with the following ethical codes and directives: International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and the Directive on Conditions for Issue of License for Animal Experiments for Scientific Research Purposes (Official Gazette of the Republic of Slovenia 40/85 and 22/87).

DNA, immunocytochemistry, and subcellular fractionation. Cells were transiently transfected with a plasmid containing a cloned sequence of the fusion protein Casp9E (21). The DNA was introduced into cells using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. For immunocytochemistry, the following primary antibodies were used: anti-cytochrome c and anti-Tim23 (BD Biosciences) and anti-Apaf-1 (Abcam) as used in previous studies (5, 9, 15, 35). First, cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 15 min, and then incubated in 0.1% Triton X-100 for 10 min at room temperature. Nonspecific staining was reduced by incubating cells in blocking buffer containing 3% BSA and 10% goat serum in PBS at 37°C for 1 h. The cells were stained with primary and secondary antibodies diluted in 3% BSA in PBS and incubated at 37°C. Subsequently, they were mounted using the Light Antifade kit (Molecular Probes). Double staining was performed in the same way as staining with a single antibody, except that the two primary antibodies (raised in different species) were added sequentially. Secondary antibodies conjugated to fluorescent dyes Alexa Fluor 546 (red; Molecular Probes) or Alexa Fluor 488 (green; Molecular Probes) were used. Subcellular fractionation was performed using the Cytosol/Mitochondria Fractionation kit (Oncogene). Briefly, after isolation, cells were collected by being centrifuged at 600 g for 5 min at 4°C, washed with ice-cold PBS, and centrifuged again at 600 g for 5 min at 4°C. The supernatant was removed, and cells were resuspended with 1x cytosol extraction buffer mix containing protease inhibitors. Cells were homogenized at 4°C with 30–50 passes using a pestle in a 1.5-ml microcentrifuge tube. The homogenate was centrifuged at 700 g for 10 min at 4°C. The supernatant was transferred to a fresh microcentrifuge tube and centrifuged at 10,000 g for 30 min at 4°C. At the end, the supernatant was collected as the cytosolic fraction and the pellet was resuspended in the mitochondrial extraction buffer mix. Primary antibodies used for Western blot analysis were anti-cytochrome c (BD Biosciences) and anti-Apaf-1 (Abcam; Stressgen) diluted according to the manufacturer's instructions. Western blot analysis was performed with the BM chemiluminescence Western blotting kit (mouse/rabbit) (Roche), in which the concentration of horseradish peroxidase-labeled secondary antibodies was 60 mU/ml.

Induction of apoptosis. Apoptosis was triggered with rotenone, an inhibitor of complex I in the mitochondrial respiratory chain (10, 16). Rotenone is widely used in studies of the activation of mitochondria-dependent caspase pathways (20, 21, 29, 30). Unless stated otherwise, cells were incubated with 300 µM rotenone for the following periods (in min): 1, 5, 10, 15, 30 and 120. Apoptotic cells were demonstrated by labeling with annexin V-Alexa Fluor 568 conjugate (Molecular Probes), which stains the plasma membrane of apoptotic cells. To detect possible necrotic cells, we used Sytox Green nucleic acid stain (Molecular Probes), which does not permeate intact plasma membrane. Cells were stained under the confocal microscope according to the manufacturer's instructions, and the fractions of apoptotic and necrotic cells, respectively, were determined by counting all cells on a glass coverslip onto which 50,000 cells had been seeded. The experiment was repeated in two culture preparations.

Confocal microscopy and image analysis. The fluorescent images were collected from equatorial planes of cells with optical thickness of 2 µm using an inverted Zeiss LSM 510 confocal microscope (x40 oil-immersion lens objective, numerical aperture 1.3). To obtain intensity profiles, lines were drawn through the middle of the cell images. EGFP and the conjugate Alexa Fluor 488 were excited using an argon laser (488 nm). Fluorescence was collected through the band-pass filter (505–530 nm). For excitation of the conjugate Alexa Fluor 546, the HeNe laser (543 nm) was used in combination with a long-pass filter with the cutoff set at <560 nm. The green and red signals were acquired sequentially.

Confocal images were analyzed using custom-made MatLab software as described previously (13, 21). Briefly, the images obtained were exported as eight-bit tagged image file format, or TIFF, files and analyzed using the MatLab software (13) by counting the number of green, red, and colocalized pixels in each image. The degree of colocalization was used to compare two fluorescently tagged proteins and expressed as the percentage of colocalized pixels in the cell among all pixels in the cell. Overlaid green and red signals, indicating colocalization between the two proteins, were indicated by yellow coloration in the images. Data were analyzed using a two-tailed Student's t-test for equal variance.

	RESULTS

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Apaf-1 redistributes to mitochondria after triggering apoptosis with rotenone. First, we checked the distribution of Apaf-1 compared with the distribution of mitochondria before and after triggering apoptosis. Control cells and cells incubated with rotenone for 1, 5, 10, 15, and 30 min had a similar, relatively low degree of colocalization between the labeled proteins Apaf-1 and Tim23, a protein of the internal mitochondrial membrane (2) (Fig. 1C). However, after applying 2-h treatment with rotenone, we noticed a significant increase in the degree of colocalization between Apaf-1 and mitochondria (Fig. 1C). The change in the relative subcellular distribution of Apaf-1 to the proximity of mitochondria, shown in Fig. 1A, was similar to one found previously in the case of procaspase-9 (21). Furthermore, the line intensity profiles confirmed the distinct distribution of both labeled proteins in control cells (Fig. 1B). However, in apoptotic cells treated for 2 h with rotenone, a noticeable colocalization between the two signals was observed (Fig. 1B). The level of fluorescent signal colocalization in control cells was, on average, 23 ± 2% (n = 33), and in cells treated for 2 h with rotenone, it was 44 ± 3% (n = 34), a statistically significant difference (P < 0.001) (Fig. 1C).

View larger version (39K): [in this window] [in a new window]   Fig. 1. The apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) redistributes to mitochondria after triggering apoptosis. A: mitochondria were labeled with antibody against internal mitochondrial protein Tim23 (anti-Tim23, green), and the cytoplasmic protein Apaf-1 was labeled with antibody against Apaf-1 (anti-Apaf-1, red). In control cells (Control), Apaf-1 and mitochondria were poorly colocalized (overlay) but rotenone induced the redistribution of Apaf-1 to mitochondria (+2 h rotenone, overlay). Cells were treated with 300 µM rotenone. Bars, 5 µm. B: profiles of fluorescence signals (expressed as arbitrary units, A.U.) from the control cell confirm different distribution of Apaf-1 and mitochondria (left), whereas profiles from the rotenone-treated cell confirm a similar distribution pattern between Apaf-1 and mitochondria (right). C: graph representing average degree of colocalized signals of fluorescently labeled Apaf-1 and Tim23. No significant difference was detected between the controls and rotenone-treated cells for 1, 5, 10, 15, and 30 min. However, a significant increase in the mean degree of colocalization between Apaf-1 and mitochondria (Tim23) was detected after 2-h incubation [increase from 23 ± 2% in control (n = 33) to 44 ± 3% in rotenone-treated cells for 2 h (n = 34); *P < 0.001]. The degrees of colocalization were 20% in the control cell (Control) shown in A, left, and 44% in rotenone-treated cell for 2 h shown in A, right (+2 h rotenone, overlay). Numbers above columns represent number of cells analyzed for each condition.

View larger version (48K): [in this window] [in a new window]   Fig. 2. The degree of colocalization between cytochrome c (Cyt c) and Apaf-1 was transiently increased after rotenone application. A: control (Control) and rotenone-treated cells for 15 min (+15 min rotenone) and 2 h (+2 h rotenone) immunolabeled with antibody against cytochrome c (anti-Cyt c, green) and antibody against Apaf-1 (anti-Apaf-1, red). Overlaid signals (Overlay) of both antibodies show that in the control cell, the two proteins were distributed in a granular fashion and were intertwined. The cells treated with rotenone for 15 min showed an increase in colocalization between cytochrome c and Apaf-1 (overlay, yellow). The same pattern observed in control cells was demonstrated in cells treated with rotenone for 2 h, in which the degree of the colocalization of the 2 proteins compared with control remained unchanged. Bars, 5 µm. B: profiles of fluorescence intensity emitted by antibodies against cytochrome c and Apaf-1 confirmed different distributions of both proteins in the control as well as in the rotenone-treated cell for 2 h, whereas in the cell incubated with rotenone for 15 min, the profiles of the 2 antibodies showed a similar distribution pattern. C: mean degree of overlaid signals between Apaf-1 and cytochrome c showed a transient increase in the degree of colocalization 15 min after rotenone application, whereas in cells treated with rotenone for different time intervals (1, 5, 10, 30, and 120 min), the degree of colocalization was similar to that of controls. The mean degree of colocalization in control cells was 27 ± 2% (31% in cell shown in A), whereas in cells treated for 15 min with rotenone, the mean colocalization was significantly higher at 43 ± 3%. *P < 0.01 (46% in cell shown in A). Mean colocalization 2 h after triggering apoptosis with rotenone was 31 ± 3%, which was similar to controls (29% in cell shown). Numbers above columns represent number of cells analyzed. Cells were treated with 300 µM rotenone.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Apaf-1 becomes colocalized with caspase-9 tagged with EGFP (Casp9E) after triggering apoptosis with rotenone. A: somatotroph transfected with Casp9E (green) and stained with antibody against Apaf-1 (anti-Apaf-1, red). In control cells, Casp9E and Apaf-1 have distinct subcellular distributions (overlay). Rotenone-triggered apoptosis resulted in Casp9E and Apaf-1 redistribution to the same subcellular location (+2 h rotenone, overlay). Bar, 5 µm. B: intensity profiles of both fluorescent markers confirm distinct distributions of Casp9E and Apaf-1 in control conditions (left) and their redistribution to the same location after application of rotenone (right). C: average degree of colocalization between Casp9E and Apaf-1 in controls and in cells treated with rotenone for 1, 5, 10, 15, and 30 min was similar. However, after 2-h incubation with rotenone, a significant increase in the degree of colocalization was detected compared with controls [from 23 ± 2% (n = 25) in control cells to 47 ± 3% in rotenone-treated cells for 2 h (n = 30); *P < 0.01]. The degree of colocalized signals in the control cell shown in A, left, was 31% and it was 54% in the rotenone-treated cell shown in A, right. Cells were treated with 300 µM rotenone.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Western blot analysis of Apaf-1 and cytochrome c in subcellular fractions. Left: Apaf-1 (130 kDa) was detected mainly in the cytosolic fractions before and after triggering apoptosis (Cytosol). Apaf-1 was not detected in the mitochondrial fractions (Mitoch.) when the same amount of proteins used in the cytosolic fractions (1 µg) was loaded onto the gel. When proteins were loaded onto the gel in a 15 times larger amount (15 µg), a small increase in the amount of Apaf-1 was detected in the mitochondrial fractions of cells 15 min after triggering apoptosis (15 µg, 15 min). Right: cytochrome c (15 kDa) was increased in the cytosolic fraction after apoptosis was triggered with rotenone, along with a concomitant decrease in mitochondrial fractions. Cells were treated with 300 µM rotenone.

Rotenone triggered apoptosis but not necrosis. We used a relatively high concentration of rotenone (300 µM) to trigger apoptosis. A 10-fold lower concentration of rotenone (30 µM) was also effective in inducing apoptosis, consistent with the findings of a previous study (21). To verify that rotenone induced apoptosis and not necrosis, we labeled cells with annexin V. Figure 5 shows that annexin V-positive cells were also present in control cells but that their percentage was rather low (0.5%). After rotenone application, the extent of annexin V-positive cells increased by at least one order of magnitude (Fig. 5). A few necrotic cells were present in control cultures, but their number was 50-fold less than the level of apoptotic cells. Although rotenone application elicited a small but not significant increase in necrotic cell count in the culture (from 0.01 to 0.04%), the fraction of apoptotic cells increased significantly by at least one order of magnitude as confirmed using Student's t-test (P < 0.01) (Fig. 5).

View larger version (69K): [in this window] [in a new window]   Fig. 5. Rotenone induced apoptosis but not necrosis. A: rotenone-triggered apoptotic cell labeled with annexin V-Alexa Fluor 568 conjugate (red). Bar, 5 µm. B: transmission image of the cell shown in A. C: number of apoptotic cells significantly increased after treatment with rotenone (+R, Ap.) compared with control cells (–R, Ap.). *P < 0.01. A few necrotic cells were found among control cells (–R, Ne.) and among rotenone-treated cells (+R, Ne.). The percentage of necrotic cells in conditions with and without rotenone was similar. Cells were treated with 300 µM rotenone for 2 h.

	DISCUSSION

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On the basis of biochemical results, it was previously shown that the formation of the apoptosome complex is a multistep process (11, 15). During the early steps, cytochrome c released into the cytoplasm binds to Apaf-1 and induces oligomerization of Apaf-1 molecules (15, 23, 26, 35). First, we confirmed that after triggering apoptosis with rotenone, cytochrome c is released from the mitochondria (Fig. 3). Therefore, using immunocytochemistry, we expected to observe increased colocalization between cytochrome c and Apaf-1 in rotenone-treated cells. Interestingly, we have shown herein that the degree of colocalization between the two proteins increased only transiently (n = 15; P < 0.01) (Fig. 2C). Therefore, it appears that the potential for interaction between cytochrome c and Apaf-1 has a limited life span. On the other hand, a significant increase in the degree of colocalization between Apaf-1/Tim23 and Apaf-1/Casp9E was detected after 2-h incubation with rotenone and not at earlier time points investigated (Figs. 1C and 4C). These data are in agreement with observations obtained from cell lysates (22, 28). In cell lysates, the interacting proteins may bind less tightly once the apoptosome holoenzyme complex is formed and may be lost during purification (28). Therefore, until the present report, whether the lack of detection of cytochrome c in fractions, together with Apaf-1 and caspase-9, is due to inadequate detection techniques remained unexplained. We have shown for the first time the transient colocalization between cytochrome c and Apaf-1 in single cells, which supports the hypothesis that cytochrome c may be needed only for Apaf-1 oligomerization and that once the Apaf-1 oligomer is formed, cytochrome c unbinds from Apaf-1 (22). On the basis of the nonsynchronous rise in the degree of colocalization between Apaf-1/cytochrome c and Apaf-1/Casp9E, we assume that there is a significant delay between the binding of cytochrome c and caspase-9 to Apaf-1. Details of this delay need to be investigated in further studies. Moreover, we have shown for the first time the time window within which the interactions between cytochrome c and Apaf-1 occur.

The colocalization of Casp9E and Apaf-1 in apoptotic cells (Fig. 4) is consistent with the Apaf-1-facilitating function of caspase-9 activation by promoting the clustering of caspase-9 molecules (4). We cannot exclude nuclear localization of Casp9E in rat somatotrophs completely (21). However, 2-h treatment with rotenone induced a significant increase in the degree of colocalization between Casp9E and Apaf-1 (Fig. 4). Both proteins translocated to the proximity of mitochondria (Figs. 1 and 4) (21), which implies that the potential for both proteins to interact is greatly augmented under these conditions. The mechanisms that trigger the redistribution of both proteins seem to operate within the same time frame but remain to be determined.

	GRANTS

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This work was supported by Grants P3 521 0381 and P3 0310 0381 from the Ministry of Education, Sciences, and Sports of the Republic of Slovenia; EC Contracts DECG QLG3-CT-2001-02004 and GROWBETA QLG1-CT-2001-02233; and National Institutes of Health Grant R01 NS-36665-05.

	ACKNOWLEDGMENTS

 
The involvement of Dr. I. Milisav-Ribaric during an early stage of this work is acknowledged.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Zorec, Institute of Pathophysiology, Medical Faculty, Univ. of Ljubljana, Zaloka 4, SI-1000 Ljubljana, Slovenia (e-mail: Robert.Zorec{at}mf.uni-lj.si)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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This Article Abstract Full Text (PDF) All Versions of this Article: 290/3/C672    most recent 00331.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Potokar, M. Articles by Zorec, R. Search for Related Content PubMed PubMed Citation Articles by Potokar, M. Articles by Zorec, R.