Article |
Address correspondence to A.P. Gilmore, Wellcome Trust Centre for Cell Matrix Research, School of Biological Sciences, University of Manchester, 3.35 Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel.: 44-161-275-3892. Fax: 44-161-275-1505. email: agilmore{at}man.ac.uk
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Abstract |
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Key Words: anoikis; apoptosis; Bax; mitochondria; caspases
Abbreviations used in this paper: MMP, mitochondrial membrane permeabilization; OMM, outer mitochondrial membrane.
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Introduction |
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Proapoptotic Bax and Bak are essential regulators of the mitochondrial pathway of apoptosis (Lindsten et al., 2000; Ranger et al., 2001; Wei et al., 2001). Bak resides permanently on the outer mitochondrial membrane (OMM), whereas Bax is normally found in the cytosol of healthy cells and translocates to the OMM during apoptosis (Hsu et al., 1997; Wolter et al., 1997; Goping et al., 1998; Gross et al., 1998). Both undergo a conformational change, which may be an obligatory step in their proapoptotic function (Desagher et al., 1999; Griffiths et al., 1999; Nechushtan et al., 1999). In the case of Bax, transition to this alternative conformation correlates with translocation to mitochondria.
Bax and/or Bak are required for mitochondrial membrane permeabilization (MMP; Wei et al., 2001). How MMP occurs is controversial, and a number of possible models have been proposed (Martinou and Green, 2001; Zamzami and Kroemer, 2001). During apoptosis, mitochondrial Bax forms large complexes, whereas cytosolic Bax is a monomeric 21-kD protein (Antonsson et al., 2000, 2001; Eskes et al., 2000; Nechushtan et al., 2001). The role of these complexes is unclear, but current models suggest that they regulate mitochondrial morphology and permeability via existing OMM proteins, or by forming de novo channels (Jurgensmeier et al., 1998; Narita et al., 1998; Martinou and Green, 2001; Zamzami and Kroemer, 2001). Ultimately, they cause mitochondria to release a host of factors from the intermembrane space, including cytochrome c, SMAC/Diablo, and apoptosis-inducing factor, leading to cell death (Degterev et al., 2001; Waterhouse et al., 2002). Mitochondrial fission also occurs at this time, in which Bax has also been implicated (Frank et al., 2001; Karbowski et al., 2002).
Inhibition of adhesion in epithelial cells leads to apoptosis (anoikis; Frisch and Ruoslahti, 1997; Gilmore et al., 2000). Bax translocates to mitochondria within 15 min of removal of cells from ECM, but they do not die immediately, and it is a number of hours before MMP occurs. Furthermore, if cells are replated, Bax exits the mitochondria and cells survive, suggesting that Bax translocation does not, per se, commit cells to apoptosis (Gilmore et al., 2000). Here, we examine spatial and temporal changes in Bax localization, conformation, and oligomerization during anoikis. Bax initially translocates to mitochondria as an inactive monomer. A fraction of this Bax undergoes a conformational change after translocation, but this precedes MMP and apoptosis by several hours. The formation of prominent Bax clusters is a late event coinciding with activation of all mitochondrial Bax, MMP, and apoptosis. We have also investigated the time window during which epithelial cells can be rescued from anoikis. We find that the point beyond which cells are committed is before Bax forms large perimitochondrial clusters and cytochrome c is released. These results suggest that critical events committing a cell to die occur before the detectable changes in mitochondrial permeability.
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Results |
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First, we asked if the conformational change occurs before or subsequent to Bax translocation. FSK-7 mammary epithelial cells were maintained as an adherent monolayer or detached and cultured on a nonadhesive substrate (poly-HEMA) for 15 min or 4 h. Cytosol and membrane fractions were prepared and assayed for the presence of Bax (Fig. 1 a, top). In adherent cells, Bax is predominantly cytosolic. Detaching cells for 15 min results in a significant amount of Bax translocating to the membrane fraction, which persists after longer times in suspension. To determine the conformational state of Bax, we immunoprecipitated from these fractions (in the presence of 0.25% CHAPS) using anti-Bax pAb 62M, which recognizes a BH3 domainproximal epitope. This antibody only recognizes the proapoptotic conformation of Bax (Pullan et al., 1996; Gilmore et al., 2000). Only Bax from the membrane fraction of detached cells was precipitated by 62M (Fig. 1 a, bottom). 62M-reactive Bax was detected in the membrane fraction of cells detached from ECM for 15 min. No Bax was immunoprecipitated from the cytosolic fraction of detached cells, or from the cytosolic and membrane fractions of adherent cells. This change in reactivity with 62M was not due to covalent modification, as addition of Triton X-100 allowed Bax to be immunoprecipitated from all fractions (unpublished data). These data demonstrate that the Bax molecules that have undergone a conformational change can only be found in the membrane fraction of cells undergoing anoikis.
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Staurosporine treatment of HeLa cells results in mitochondrial Bax forming high mol wt complexes (Antonsson et al., 2001). Perimitochondrial clusters seen with GFP-Bax (Nechushtan et al., 2001) and here with anti-Bax 62M may represent these complexes. As we observed 62M-reactive Bax only in perimitochondrial clusters, we asked if, during anoikis, membrane-associated Bax was exclusively in large complexes, or whether it initially translocated as a monomer. Cytosolic and membrane fractions were prepared in CHAPS and separated by size-exclusion chromatography. In adherent cells, Bax is cytosolic, and elutes as a single peak consistent with it being a monomer (Fig. 1 c). Cytosolic Bax from cells on poly-HEMA for 4 h is also a single species of monomer, suggesting that, as with the conformational change, oligomerization does not precede translocation. Membrane-associated Bax in detached cells elutes as two peaks, both low and high mol wts. The latter elutes 200 kD in size, consistent with previous data (Antonsson et al., 2001).
Next, these isolated S100 chromatography fractions were cross-linked using the homo-bifunctional reagent BS3. By SDS-PAGE, Bax from adherent cells runs as a single band (Fig. 1 d, band 1). Bax in both the high and low mol wt S100 fractions of detached cells also run as a single bands under denaturing conditions (Fig. 1 d, band 1). On cross-linking, cytosolic Bax is still a discrete band by SDS-PAGE, but migrates slightly faster (Fig. 1 d, band 2), suggesting internal cross-links, consistent with its folded structure (Suzuki et al., 2000). Addition of Triton X-100 before cross-linking produces a band indicative of Bax homodimers (Fig. 1 d, band 3), also consistent with previous data (Hsu and Youle, 1998). The low mol wt fraction from membranes isolated from detached cells behaves identically to cytosolic Bax when cross-linked with BS3. However, the high mol wt fraction does not show any immunoreactive bands after cross-linking, although some immunoreactive material was seen in the stacking gel (unpublished data). Identical results were seen with a number of anti-Bax antibodies (unpublished data). Furthermore, this fraction was insensitive to the addition of Triton X-100, and no dimer-sized band was induced. These data show that monomeric and oligomeric forms of Bax are both associated with the membrane fraction of detached cells.
We examined the kinetics of complex formation during anoikis. Membrane fractions isolated from adherent cells or cells on poly-HEMA for various times were extracted in CHAPS and separated by S100 chromatography. Very little Bax is seen in the membrane fraction of adherent cells (Fig. 1 e). After detachment for 15 min, the majority of Bax in the membrane fraction is in the low mol wt, monomeric form. With detachment for longer periods, increasing amounts of Bax are found in the high mol wt complex. Membrane fractions isolated from detached cells were also cross-linked with BS3. Consistent with the gel filtration data, significant amounts of Bax on the membrane after detachment for 15 min is a monomer (Fig. 1 f). In the 15-min sample, Bax was cross-linked into a discrete, faster migrating band (Fig. 1 f, band 2). Triton X-100 produced a cross-linked dimer-sized band (Fig. 1 f, band 3), identical to cytosolic Bax from adherent cells (Fig. 1 d). Bax from cells on poly-HEMA for 1 h was also largely monomeric. However, membrane fractions from cells detached for 4 h showed a significant decrease in the amount of monomeric Bax, as judged by absence of the faster migrating cross-linked band (Fig. 1 f, band 2) and the Triton X-100induced dimer (Fig. 1 f, band 3).
If cross-linking with BS3 resulted in no 62M immunoreactive bands by Western blotting, why were 62M-positive clusters seen associated with mitochondria by immunofluorescence? It was estimated that GFP-Bax clusters contain thousands of molecules, suggesting a mol wt much greater than 200 kD (Nechushtan et al., 2001). Complexes of this size may be largely insoluble with extraction conditions required to isolate native Bax. To examine this, we separated cells into cytosolic and membrane fractions as before. The membrane fraction was extracted with 0.25% CHAPS, and the CHAPS-insoluble material was solubilized in SDS-PAGE sample buffer. We compared all three fractions by immunoblotting for Bax (Fig. 2 a). In adherent cells, the majority of Bax was found in the cytosolic fraction. All detached cell samples had a proportion of Bax present in the CHAPS-insoluble membrane fraction. However, in cells detached for 4 h, the majority of Bax was associated with the CHAPS-insoluble fraction. The amount of Bax extracted was not increased with higher concentrations of CHAPS (unpublished data). Next, we asked if perimitochondrial Bax clusters represented this CHAPS-insoluble material. Cells expressing YFP-Bax were maintained on poly-HEMA for 5 h, and then either fixed in 4% formaldehyde or extracted with 0.25% CHAPS before fixation. They were immunostained for cytochrome c and the mitochondrial membrane protein TOM-20 (Fig. 2 b). TOM-20 was not extracted by CHAPS, whereas cytochrome c staining was diminished. However, cells expressing YFP-Bax showed identical perimitochondrial clusters with or without CHAPS extraction. Adherent cells extracted with CHAPS did not have any detectable cytosolic YFP-Bax (unpublished data).
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Spatial and temporal relationship between Bax activation complex formation and apoptosis
The experiments just mentioned raised a number of questions regarding the spatial and temporal relationship between Bax translocation, the formation of large complexes, and MMP. Furthermore, the requirement of any of these events for anoikis was unclear. To address this, we isolated primary epithelial cells from mid-pregnant Bax-null mice and wild-type counterparts. These were detached from ECM for 5 h, and apoptosis was quantified (Fig. 3 a). Bax -/- cells showed a marked delay in the onset of apoptosis, as well as having a lower baseline level of cell death in monolayer culture.
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To assess the spatial and temporal events after Bax translocation, cells were cotransfected with YFP-Bax and a fluorescent marker for the OMM (HcRed-XT). In adherent cells, YFP-Bax showed a diffuse, cytosolic distribution (Fig. 4 a). However, 15 min after detachment, YFP-Bax was localized over the entire surface of the mitochondria, in contrast to the distribution seen with anti-Bax 62M. This distribution changed over time, and after 5 h in suspension, the majority of cells showed YFP-Bax in perimitochondrial clusters. It was striking that all cells that showed this perimitochondrial distribution also had apoptotic nuclei, shown by Hoechst staining.
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Release of cytochrome c is a rapid and complete event, occurring over a 5-min period once initiated (Goldstein et al., 2000). These kinetics are similar in response to a range of apoptotic stimuli. To determine the relationship between cytochrome c release and redistribution of Bax within mitochondria, YFP-Baxexpressing cells were detached for various times and immunostained with an antibody to cytochrome c and anti-Bax 62M. Only cells that had no mitochondrial cytochrome c showed complete colocalization of YFP-Bax with 62M immunoreactivity (Fig. 4 c). All cells with released cytochrome c also had apoptotic nuclei.
The fact that we did not see any intermediate cells that had Bax present in large clusters but still had mitochondrial cytochrome c suggested that the formation of clusters and MMP were rapid and coincident. Given the rapid kinetics of cytochrome c release (Goldstein et al., 2000), it was impossible from these experiments to determine if Bax clusters formed before or after MMP, caspase activation, and cell death. To address this, we followed cluster formation in detached cells in the presence or absence of zVAD-fmk. Cells were cytospun and fixed after various times of anoikis, and were immunostained for cytochrome c and TOM-20 (Fig. 5 a). As we have previously shown, inhibition of caspases did not prevent Bax association with mitochondria after 15 min (Gilmore et al., 2000). At later times, although nuclear condensation was inhibited by zVAD-fmk, cytochrome c release and the formation of YFP-Bax clusters were unaffected, indicating that they were independent of caspase activation.
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Together, these results demonstrate that during anoikis, cytosolic Bax translocates to mitochondria within 15 min and remains evenly distributed over the OMM until MMP. Redistribution of Bax into perimitochondrial clusters is followed after several minutes by the formation of apoptotic nuclei. The rapidity of this process agrees with our data obtained with detached, fixed cells, where we did not observe any cells in an intermediate state with Bax clusters, but with cells still showing mitochondrial cytochrome c. Thus, the formation of clusters occurs concomitant with, but independent of, caspase activation, suggesting that they form at the same time as MMP.
Epithelial cells undergoing anoikis commit to the apoptotic pathway before formation of large Bax clusters
It is unclear at what point a cell has become irreversibly committed to apoptosis, and whether this relates to the changes in Bax localization and complex formation. Previously, we have shown that if cells are replated after detachment for <1 h, endogenous Bax is lost from the mitochondria and redistributes to the cytosol (Gilmore et al., 2000). Now, we asked how long a cell needs to be kept in suspension before irreversibly committing to apoptosis, and whether this is related to the formation of Bax complexes. FSK-7 cells expressing YFP-Bax were used to assess complex formation and apoptosis commitment. Cells were detached from ECM for varying times. Cells were then cytospun onto slides, and Bax localization and apoptosis at that time were quantified. Virtually all cells showed mitochondrial YFP-Bax after 15 min detachment from ECM (Fig. 3 a). This did not change over increasing times in suspension. YFP-Bax cells undergo apoptosis only after several hours detachment, even though translocation to mitochondria was rapid. In cells maintained on poly-HEMA, an increase in apoptosis was not apparent until after 3 h in suspension, and occurred in the majority of cells by 5 h (Fig. 6 b).
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Discussion |
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In adhesion-dependent epithelial cells, Bax translocation occurs rapidly after detachment from ECM (Gilmore et al., 2000; Wang et al., 2003). Despite this, the release of cytochrome c and morphological apoptosis occur in a stochastic manner over several hours. This delay between apoptotic signal and death prompted us to investigate the kinetics of Bax translocation and oligomer assembly, and how these relate to mitochondrial permeabilization. We have shown that Bax translocates to mitochondria as an inactive monomer. Oligomerization occurs after association with the OMM. Although activated Bax is detectable on mitochondria immediately after detachment, this is a fraction of the total protein and is restricted to small foci. Only when mitochondria permeabilize is the majority of Bax found in perimitochondrial clusters. Time-lapse experiments show that formation of these clusters is concomitant with caspase activation, and before nuclear condensation. Both MMP and cluster formation occur in the presence of caspase inhibitors.
Translocation of Bax to mitochondria precedes both its conformational change and formation of large complexes
Our data clearly indicate that Bax translocation and conformational change into its "active" form are distinct molecular events, although they are often described as being one and the same. We only detected active Bax on the membrane fraction of detached cells, and the small proportion of Bax present in the membrane fraction of adherent cells was not immunoprecipitated with conformation-specific antibodies. Furthermore, cytosolic Bax could not be immunoprecipitated from detached cells, at either early (before MMP) or late stages of anoikis. These data indicate that after a physiological death stimulus, translocation must occur before, and not as a consequence of, the conformational change. This is supported by data from HeLa cells, where Bax can be constitutively associated with mitochondria, but not in the active conformation (Desagher et al., 1999). The COOH-terminal tail appears to drive mitochondrial targeting, and mutations within it can uncouple translocation from apoptosis (Wolter et al., 1997; Nechushtan et al., 1999). The three-dimensional structure of Bax shows remarkable similarity to anti-apoptotic Bcl-XL (Muchmore et al., 1996; Suzuki et al., 2000). Unlike Bcl-XL, the structure of Bax was solved with the COOH-terminal mitochondrial-targeting sequence (helix 9) present, lying within a hydrophobic groove along the surface of the molecule. The similarity between the structures of Bax and Bcl-XL suggests that occupation of this groove is not required for helices 18 to fold correctly. Therefore, one can envision that altering the orientation of helix 9 may regulate translocation without affecting the conformation of the NH2 terminus (Suzuki et al., 2000). Our data, showing that only a fraction of the YFP-Bax on mitochondria of detached cells colocalizes with 62M reactivity, supports this hypothesis.
Large oligomers of Bax are clearly associated with apoptosis, though their function and composition is far from clear (Antonsson et al., 2001). Conversely, cytosolic Bax is a monomer. Oligomerization and dimerization have been suggested as mechanisms that regulate Bax association with and insertion into the OMM (Antonsson et al., 2000; Korsmeyer et al., 2000; Suzuki et al., 2000; Kuwana et al., 2002). Forcing cytosolic Bax to dimerize induced translocation in FL5.12 cells (Gross et al., 1998). However, these enforced dimers did not result in release of cytochrome c, suggesting that they were not functioning in the same way as endogenous Bax. The BH3-only protein BID can induce the formation of Bax dimers and trimers, leading to insertion into isolated mitochondria (Eskes et al., 2000). We clearly saw that in the initial stages of anoikis, Bax had translocated primarily as a monomer. Our cross-linking data also indicated that dimers were not required for translocation, though we could see dimers induced by addition of Triton X-100. Interestingly, dimers, trimers, and tetramers were not seen in our cross-linking experiments with detached cells at any time point, suggesting that if they do occur and play a role in MMP, then they must exist very transiently in only those cells in which it is occurring. Larger complexes appear over time in suspension, being more apparent at 1 h and particularly at 4 h. However, the analysis of Bax complexes by gel filtration does come with caveats, as they represent an average of a cell population in which cell death occurs in a stochastic manner. This is seen with other death-inducing stimuli, where MMP appears to occur randomly within a population of cells that will all ultimately undergo apoptosis (Goldstein et al., 2000). Therefore, from these experiments, it is impossible to determine whether complexes are forming in all cells over time, or if they occur only in those cells that have undergone MMP and have become apoptotic. Our single-cell analysis of cells expressing fluorescently tagged Bax suggests that large clusters only form in cells that are undergoing MMP, and that their formation is rapidly followed by downstream apoptotic events.
Do large perimitochondrial clusters represent the functional Bax complex?
Bax has been shown to form a variety of different-sized complexes, and this oligomerization has been shown to be required for Bax to permeabilize membranes in cell-free systems (Antonsson et al., 2000). Formation of dimers and tetramers occur in a concentration-dependent manner in vitro, the latter of which can form cytochrome cconducting channels in isolated mitochondria and lipid vesicles (Saito et al., 2000; Pavlov et al., 2001). Yet larger Bax complexes (>200 kD) are seen by gel filtration in CHAPS-extracted mitochondria here and elsewhere (Antonsson et al., 2001). Analysis of YFP-Bax clusters has suggested much larger complexes, containing thousands of Bax molecules (Nechushtan et al., 2001). However, recent data suggests that very large Bax complexes do not have permeabilizing activity, whereas smaller complexes (100 kD) can release proteins up to megadalton sizes (Kuwana et al., 2002). We found that much of the Bax present in cells after several hours of anoikis was resistant to CHAPS extraction, as were the perimitochondrial clusters of YFP-Bax in cells with apoptotic nuclear morphology. Therefore, very large Bax clusters must be different from the 200-kD complexes seen by gel filtration of CHAPS-extracted mitochondria, and may represent the endpoint of mitochondrial permeabilization. Therefore, the active complex that releases intermembrane proteins may be extremely transient, and may result in large, insoluble aggregates of Bax after permeabilization. Time-lapse imaging of single cells undergoing apoptosis in response to staurosporine indicates that cluster formation is rapid, occurring over a few minutes exactly when caspase activity can be first detected at a single-cell level. MMP is also a sudden, complete, and dramatic event, releasing cytochrome c within 5 min of commencement (Goldstein et al., 2000). This is consistent with our inability to identify intermediate complexes by cross-linking. Together with our data, it appears that formation of Bax clusters occurs rapidly at the same time as MMP.
Do these large clusters have a role in apoptosis? It has been hypothesized that Bax may act as a protein translocase, rather than by forming a permanent membrane channel (Lazebnik, 2001). Some evidence for this exists. The transport of megadalton dextrans across the OMM has been shown to occur without the formation of permanent pores or membrane disruption (Kuwana et al., 2002). If this requires Bax to unfold and form transient pores, then it may subsequently form large aggregates adjacent to mitochondria. Indeed, unfolding purified Bax with detergents induces it to form large aggregates (Suzuki et al., 2000). Mitochondria can regulate protein unfolding, and this may have a role in apoptosis regulation (Matouschek et al., 2000; Thress et al., 2001). Therefore, large perimitochondrial clusters might represent a by-product of a process involving Bax unfolding during MMP. Clusters may have other roles, and several proteins have also been identified in them, including Drp-1 and Mfn-1 (Karbowski et al., 2002). These regulate mitochondrial fission during apoptosis, although inhibition of Drp-1 did not prevent Bax itself from forming large complexes (Frank et al., 2001). Interactions with these or other mitochondrial proteins may be required for mitochondrial fragmentation, but it is unclear if they are required for Bax apoptotic activity (Roucou et al., 2002).
Epithelial cells commit to apoptosis before the appearance of Bax oligomers
It has been presumed that the point at which a cell has irreversibly committed to apoptosis equates with release of cytochrome c, SMAC/Diablo, and other proapoptotic factors (Zamzami and Kroemer, 2003). Unlike many model systems used to study the kinetics of apoptosis, detached cells can be rescued if they are allowed to reattach to ECM (Gilmore et al., 2000). We have used this system to determine the temporal relationship between the molecular events associated with Bax activation and commitment to apoptosis. Our data suggest that, although the formation of large Bax clusters is associated with release of cytochrome c and apoptosis, cells actually pass the point of commitment before these events. The majority of epithelial cells kept in suspension for >1 h could not be rescued by replating them onto ECM, eventually showing apoptotic morphology. However, cells replated after only 15 min of anoikis were rescued. At the time of replating, YFP-Bax was present on the mitochondria of all cells, but had not yet formed large clusters or resulted in cytochrome c release. The conclusion must be that neither Bax translocation nor complex formation mark the point of commitment. This raises interesting questions regarding events occurring on the mitochondrial surface after an apoptotic stimulus but before MMP. The answers to these questions may explain the stochastic nature of this process in response to diverse cellular insults.
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Materials and methods |
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Expression constructs
The Bax cDNA was amplified from mouse embryo cDNA with Pfu polymerase (Promega) using oligonucleotide primers to the 5' and 3' ends of the coding sequence, cloned into pCRScript (Stratagene), and confirmed by sequencing. To generate YFP-Bax, PCR primers were used that incorporated restriction endonuclease sites and the Bax start and stop codons. The product was ligated into either pEYFP-C1 or pECFP-C1 (CLONTECH Laboratories, Inc.). The BH3 domain construct was generated using PCR primers based on the three-dimensional structure of Bax, and restriction sites were incorporated into each primer. This was cloned into pEYFP-C1. The inactive BH3 mutant was constructed using PCR primers to incorporate a double point mutation (L64 to E and G68 to E). pHcRed1-XT and pEYFP-XT were made by PCR from a Bcl-XL cDNA (a gift from Richard Youle, National Institutes of Health, Bethesda, MD), using primers that encompassed the coding sequence for the last 25 amino acids including the stop codon. pEYFP-caspase 3 sensor was purchased from CLONTECH Laboratories, Inc. All constructs were confirmed by DNA sequencing and immunoblotting.
Cell culture and transfection
FSK-7 cells (Kittrell et al., 1992) were grown in DME/Ham's F12 (2% FCS, 5 ng/ml EGF, and 880 nM insulin). Transfection of FSK-7 cells with LipofectAMINETM plus (Life Technologies) has been described previously (Gilmore et al., 2000). In brief, cells were plated at 105 cells/cm2 on glass coverslips 18 h before transfecting with 0.5 µg DNA in 12-well culture plates. For detachment assays, cells were trypsinized and replated on 35-mm dishes coated with poly-HEMA (Sigma-Aldrich). Cells on poly-HEMA were collected by centrifugation (5,000 g, 30 s). For immunostaining, cells were resuspended in culture medium and centrifuged onto polylysine slides (Merck) using a centrifuge (Cytospin; Shandon).
Cell fractionation, chromatography, cross-linking, and immunoprecipitation
Manipulations were performed on ice unless otherwise stated. Cells were resuspended in hypotonic buffer (10 mM Hepes, pH 7.6, 10 mM NaCl, 1.5 mM/MgCl2, 4 mM NaF, 100 µM sodium orthovanadate, and protease inhibitors) and were lysed with 20 strokes in a Dounce homogenizer (Wheaton), followed by addition of NaCl to 150 mM. Cytosol and membrane fractions were separated by centrifugation (100,000 g for 30 min). For size-exclusion chromatography, CHAPS was added to the cytosol fraction to 4 mM. To extract the membrane fraction, the 100,000-g pellet was resuspended in 10 mM Hepes/Cl, pH 7.6, 150 mM NaCl, 4 mM CHAPS, 4 mM NaF, 100 µM sodium orthovanadate, and protease inhibitors, and was sonicated. The solubilized membrane fraction was centrifuged at 100,000 g. The insoluble material left at this stage was solubilized by boiling in SDS-PAGE sample buffer. Lysates were concentrated in Centricon 10 concentrators (Amicon), and 5 mg protein in 0.5 ml was loaded onto a Sephacryl S100-HR column (1.5 x 25 cm, Amersham Biosciences; equilibrated in 10 mM Hepes/Cl, pH 7.6, 150 mM NaCl, and 4 mM CHAPS). The column was resolved at 4°C, flow rate 0.1 ml/minute. Calibration was with mol wt standards from Sigma-Aldrich (12.4 kD, cytochrome c; 29 kD, carbonic anhydrase; 66 kD, BSA; 200 kD, ß-amylase; void volume, blue dextran). Column fractions were concentrated with TCA, separated by reducing SDS-PAGE, transferred to nitrocellulose (Bio-Rad Laboratories), and immunoblotted. For cross-linking experiments, lysates were incubated with 5 mM BS3 for 30 min at RT. Where indicated, Triton X-100 or CHAPS was added 30 min before BS3. Reactions were stopped with SDS-PAGE sample buffer. Reactions were separated by SDS-PAGE. For immunoprecipitation of active Bax, soluble and membrane fractions isolated in 4 mM CHAPS buffer were clarified by centrifugation. 1 mg protein was precipitated with 1 µg/ml anti-Bax antibody 62M on protein ASepharose (Roche) at 4°C for 2 h, and was washed three times in CHAPS buffer. Proteins were recovered by boiling the beads in SDS-PAGE sample buffer before immunoblotting with anti-Bax 5B7. Immunoblots were visualized with SuperSignal® chemiluminescence (Pierce Chemical Co.).
Immunofluorescence microscopy and live-cell imaging
Cells fixed in 3.7% PFA/PBS were permeabilized in PBS/0.5% Triton X-100. Primary antibodies were diluted in PBS/0.1% Triton X-100/0.1% horse serum (37°C for 1 h). After washing in PBS, secondary goat antimouse or goat antirabbit Cy5 or Cy3 conjugates were incubated in above buffer (37°C for 30 min). Cells were counterstained with 1 µg/ml Hoescht 33258. Coverslips were mounted in ProLong® (Molecular Probes, Inc.). Images were collected on an inverted microscope (model IX70; Olympus) using a 100x Plan-Apo 1.4 NA objective, equipped with a DeltaVision imaging system. Images were processed by constrained iterative deconvolution on softWoRxTM v3.0 software (Applied Precision). For live imaging, cells expressing fluorescently tagged proteins were incubated with 1 µg/ml Hoescht 33258 and/or 5 µM Rhodamine 123 for 30 min before washing in growth medium, and 10 µM staurosporine was added. Images were captured using a Leica AS-MDW workstation (37°C, 5% CO2) with an HCX Plan-Apo 63x/1.3 NA glycerol objective.
Online supplemental material
Videos 13 show FSK-7 cells treated with staurosporine (images taken every 3 min), and are the complete sequence from which Fig. 5 b was compiled. Videos 4 and 5 show FSK-7 cells loaded with Rhodamine 123 and treated with staurosporine, and are the complete sequence from which Fig. 5 c was compiled. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200302154/DC1.
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Acknowledgments |
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This work was supported by the Wellcome Trust. A.P. Gilmore is a Wellcome Trust Research Career Development Fellow.
Submitted: 25 February 2003
Accepted: 8 July 2003
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