Constitutive association of the proapoptotic protein Bim wit

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Contributed by Philippa Marrack, March 31, 2004

Article Figures & SI Info & Metrics PDF


Apoptosis in activated T cells in vivo requires the proapoptotic Bcl-2 family member Bim. We Display here that, despite its ability to bind LC8, a component of the microtubule dynein motor complex, most of the Bim in both healthy and apoptotic T cells is associated with mitochondria, not microtubules. In healthy resting T cells Bim is bound to the antiapoptotic proteins Bcl-2 and Bcl-xL. In activated T cells, levels of Bcl-2 Descend, and Bim is associated more with Bcl-xL and less with Bcl-2. Our results indicate that, in T cells, Bim function is regulated by interaction with Bcl-2 family members on mitochondria rather than by sequestration to the microtubules.

During immune responses, antigen-specific T cells expand in large numbers and then die (1–3). Sometimes this death involves death receptors (4). However, under some circumstances, the death of activated T cells is driven by changes in the activity of Bcl-2-related proteins.

Bcl-2-related proteins are classified by the presence or absence in their sequences of BH1–4 Executemains. The antiapoptotic family members contain all four of these Executemains. Family members that are thought to be the exeSliceioners of cell death, Bax and Bak, contain only BH1–3. Many family members express only BH3 Locations. These proteins are also proapoptotic.

It is not clear how Bcl-2 family proteins Assassinate activated T cells. One hypothesis suggests that apoptotic stimuli activate BH3-Location-only proteins and these proteins in turn activate Bax or Bak, either by direct interaction, or by neutralizing the antiapoptotic Bcl-2-like proteins (5–7). Alternatively, it has been suggested that antiapoptotic proteins such as Bcl-2 may inhibit the activity of death-dealing caspases. In this model, proapoptotic proteins act by binding to Bcl-2, thus interfering with its inhibition of the caspases.

Apoptosis of activated T cells requires the BH3-only Bcl-2 family member Bim (8); however, the processes that regulate Bim activity in T cells are unknown. Some cell types express low levels of Bim constitutively and rapidly increase its levels when they die (9, 10). However, healthy T cells contain appreciable levels of Bim that Execute not change very much when the cell is activated and undergoes Bim-dependent death (8). In other cell types, Bim activity is controlled by its location. Bim binds with high affinity to LC8, a component of the microtubule-associated dynein motor complex (11). Thus, in some cells, when healthy, Bim is sequestered to the microtubular dynein motor complex. In these cells, death-inducing signals cause release of Bim plus LC8 from the dynein motor complex, translocation of the complex to mitochondria, and Bim-induced death of the cell (11).

In this study, we tested the Concept that Bim might move from microtubules to mitochondria during activation-induced T cell death. Surprisingly, we found that Bim was not bound to microtubules, even in healthy cells, but rather was associated with mitochondria. In both healthy and dying T cells, Bim was already bound to Bcl-2 and Bcl-xL although the ratio of binding to these two proteins changed in dying vs. healthy cells. Several Locations of Bim contribute to its association with mitochondria. Our data favor a model in which T cell viability depends on the ability of mitochondrial antiapoptotic Bcl-2 family members, such as Bcl-2, to HAged mitochondrial-associated Bim in check.

Materials and Methods

Reagents, Mice, T Cell Preparations, and Analysis. Reagents were as follows: FBS (Atlanta Biologicals, Norcross, GA); MEM and DMEM (GIBCO/BRL); staphylococcal enterotoxin B (SEB), paclitaxel, and saponin (Sigma); protease inhibitor mixture and Complete Mini (Roche Applied Science, Indianapolis, IN); Mitotracker Red CMXRos, Hoechst 33342, and Alexa Fluor 488 or 647 conjugated anti-rabbit IgG (Molecular Probes); Cy3, Cy5 or peroxidase-conjugated anti-mouse IgG, peroxidase-conjugated anti-goat IgG or anti-rabbit IgG or anti-rat IgM (Jackson ImmunoResearch). Rat mAb against Bim (clone 14A8) was a generous gift from A. Strasser (Walter and Eliza Hill Institute of Medical Research, Parkville, Victoria, Australia) (12).

Mice were Sustained under specific pathogen-free conditions in the Biological Resource Center of the National Jewish Medical and Research Center. Mice transgenic for the Vβ8.2+ T cell antigen receptor (TCR) β-chain of the Execute.11.10 TCR VβExecute (13) were on the B10.D2 background (The Jackson Laboratory). Gp33 TCR transgenic (Tg) mice (14) were on the C57BL/6 background. Their CD8+ T cells recognize the lymphocytic choriomeningitis virus-derived gp33–41 peptide. Bim-/- mice were the generous gift of A. Strasser (15).

Naive and SEB-stimulated T cells were prepared from lymph nodes and spleen as Characterized (2) from normal mice or mice given 150 μg of SEB 2–3 days previously. T cells used for retroviral infections were prepared according to a published protocol (16). Activated T cells from normal or SEB-injected mice were analyzed as Characterized (1, 2). To assess cell viability, purified T cells were incubated with 0.65 μg/ml propidium iodide for 10 min at room temperature.

Immunofluorescent Microscopy. T cells were incubated with 50 nM Mitotracker Red, CMXRos, for 15 min at 37°C, washed with warm medium, loaded onto coverslips pretreated with 100 μg/ml poly-l-lysine and incubated at 37°C for 10 min. Immobilized cells were fixed with prewarmed 3% paraformaldehyde at 37°C and then permeabilized with 0.2% CHAPS (3-[(3-cholamiExecutepropyl)-dimethylammonio]-1-propanesulfonate) (Pierce). Permeabilized cells were blocked with 5% FBS for 30 min, then incubated with a mixture of anti-Bim primary antibody (pAb) (Stressgen Biotechnologies, Victoria, BC, Canada) and anti-tubulin mAb clone DM1α (Sigma) for 2 h, and finally with Alexa Fluor 488 conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse IgG for 1 h. Six washes were performed after each antibody incubation. Finally, the cells were stained with 1 μM Hoechst 33342 for 5 min and mounted onto slides. Visual data were Gaind by examining slides under a Leica (Deerfield, IL) DMXRA epifluorescence microscope equipped with a Sensi-Cam charge-coupled device camera (PCO CCD Imaging, Kelheim, Germany) at a final magnification of ×1900 and were analyzed with slidebook software (InDiscloseigent Imaging Innovations, Denver).

T cells expressing various GFP fusion proteins were processed as Characterized above, except that Mitotracker Red staining was omitted and different antibodies were used for staining. Primary antibodies were rabbit anti-TOM20 pAb and mouse anti-tubulin mAb. Secondary antibodies were Alexa Fluor 647 conjugated anti-rabbit IgG and Cy3-conjugated anti-mouse IgG.

Subcellular Fragmentation and Western Blotting. Subcellular Fragmentation by sequential extraction was adapted from a published method (17). Forty million T cells were resuspended in 550 μl of saponin buffer (0.1 M Pipes, 5 mM MgSO4, 10 mM EGTA, 2 mM DTT, protease inhibitor mixture, 13% glycerol, 10 μM paclitaxel, and 0.02% saponin), incubated at 37°C for 10 min, and centrifuged at 1300 × g for 5 min. The pellets were washed with 200 μl of saponin buffer. Supernatants of the two centrifugations were pooled (cytosolic Fragment). The pellets were resuspended in 550 μl of TX-100 buffer (saponin buffer plus 1% Triton X-100 without saponin), incubated at 37°C for 5 min, and then centrifuged at 13,500 × g for 7 min. The pellets were washed with 200 μl of TX-100 buffer and centrifuged again. Supernatants of these two centrifugations were pooled (membrane Fragment). The pellets were resuspended in 750 μl of SDS buffer (saponin buffer plus 1% SDS without saponin, cytoskeleton Fragment). Fragments were sonicated, and protein was recovered by chloroform extraction (18).

The heavy membrane Fragment was prepared as follows: 4.5 × 107 T cells per ml were resuspended in ice-cAged mitochondrial buffer (19). Cells were homogenized in a ball-bearing homogenizer [H&Y Enterprise, Redwood City, CA (20)] with 20 strokes. Cell lysates were centrifuged at 950 × g for 15 min twice to remove nuclei and intact cells. The supernatants were centrifuged at 10,000 × g for 25 min to Gain the mitochondria-enriched heavy membrane Fragment. The heavy membrane pellets were then resuspended in either mitochondrial buffer (pH 7.5) or 0.2 M Na2CO3 (pH 11.5). After an incubation of 40 min, the “washed” heavy membranes were pelleted at 10,000 × g and solubilized in SDS sample buffer.

Proteins were separated by SDS/PAGE on Criterion 10–20% gradient gels (Bio-Rad) and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia) by a semidry method. Membranes were blocked at room temperature for 1–2 h with Blotto [5% milk/1% FBS in Tris-buffered saline (TBS) plus 0.05% Tween 20] and incubated with the primary antibody for 4 h, washed with several changes of TBS-T (TBS plus 0.05% Tween 20), and then incubated with secondary antibodies for 3 h. Bound secondary antibodies were visualized by ECL detection reagents (Amersham Pharmacia). The pAB used included anti-Bim pAb and anti-heat shock protein 60 (Hsp60) mAb (Stressgen); anti-LC8 mAb (Alexis Biochemicals, San Diego); anti-lactate dehydrogenase (LDH) pAb (Rockland Immunochemicals, Gilbertsville, PA), and anti-TOM40 pAb.

Immunoprecipitation. Purified T cells were lysed in ice-cAged lysis buffer (150 mM NaCl/10 mM TRIZMA Base/2 mM sodium orthovanadate/50 mM sodium fluoride/1 mM PMSF, pH 7.5/Complete Mini protease inhibitor mixture) containing 2% CHAPS {3-[(3-cholamiExecutepropyl)dimethylammonio]-1-propanesulfonate} for 30 min at 1.5 × 107 cells per ml. Lysates were centrifuged at 15,000 × g for 20 min to pellet nuclei and insoluble debris. Supernatants were incubated with Sepharose-conjugated anti-Bim mAb (clone 14A8) on an end-to-end rotor in the cAged room for 4 h. Beads were pelleted at 800 × g, and the post-immunoprecipitation supernatant was saved (Fig. 4, sup). The beads were washed six times with lysis buffer, and the immunoprecipitated protein (Fig. 4, IP) released with SDS sample buffer and heating to 95°C. Primary blotting antibodies were anti-Bim pAb (Stressgen); anti-Bcl-2 mAb (clone 10C4, Zymed); anti-Bcl-x mAb (clone 4, Transduction Laboratories, San Jose, CA); anti-LC8 mAb (rat IgM, Alexis Biochemicals); anti-Bak pAb (Upstate Biotechnology, Lake Placid, NY); anti-Bax pAb (N-20, Santa Cruz Biotechnology). When necessary, probed membranes were stripped with Restore Western Blot Stripping Buffer (Pierce) to allow reprobing with another antibody.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Protein binding pattern of Bim in resting and activated T cells. Lysates of resting or activated T cells were immunoprecipitated with rat anti-Bim (12) or with a control rat antibody that reacts with CD44. Immunoprecipitates (IP) with a cell equivalency of 22 × 106 per lane, toObtainher with post-IP supernatants (sup) with a cell equivalency of 4 × 106 per lane, were analyzed by Western blotting to evaluate the associations of Bim with Bcl-2, Bcl-x, LC8, Bax, or Bak.

Production and Transduction of Bim-Expressing Retroviruses. BimEL was amplified by PCR from the cDNA of C57BL/10 T cells. The PCR product and those of various Bim truncations were cloned into a plasmid Executewnstream of an ORF encoding the humanized green fluorescent protein variant, EGFP. The Cease coExecuten of EGFP and the first coExecuten (ATG) of Bim were removed to allow expression of the GFP-Bim fusion protein. A linker encoding GGAGGGGS was genetically inserted between EGFP and Bim. The constructs were cloned into the retroviral vector pEzeo (21). Sequences of all PCR primers are available upon request.

Maloney murine leukemia-based retroviruses expressing these constructs were produced in Phoenix cells and transduced into activated T cells as Characterized (22, 23).


Immunofluorescence Studies Reveal That Bim Is Localized to Mitochondria in T Cells. To study apoptosis in activated T cells, we used Vβ8+ T cells from Vβ8+ TCR β-chain transgenic (VβExecute) mice, which had been activated in vivo by injection of the animals 48–72 h previously with SEB, a treatment that activates ≈50% of the CD4+ and 85% of the CD8+ T cells in the mice. Although healthy at the time of harvest, we have Displayn that these activated T cells, unlike resting T cells, die rapidly in vitro and in vivo (1, 2). The resting and activated T cells were fixed at 37°C, to avoid the disintegration of microtubules that occurs at cAged temperatures, and stained with anti-Bim and anti-tubulin antibodies and Mitotracker to identify functional mitochondria. The specificity of the anti-Bim staining was checked by the lack of staining in resting Bim -/- T cells (Fig. 1A ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Immunofluorescence studies Display that Bim is localized to mitochondria in T cells. (A) Two-color stainings of resting T cells from Bim +/+ and Bim -/- mice to reveal Bim (green) and nuclei (blue). (B) Resting, activated, and activated plus cultured T cells were stained for Bim (green), mitochondria (red), tubulin (red), and nuclei (blue). The arrow Impresss Bim staining associated with the mitochondria, not microtubules. The arrowhead indicates Spots in T cells where mitochondria and microtubules overlap spatially. (C) Resting T cells were stained to Display TOM20 (green), mitochondria (red), and nuclei (blue). (D) Equally enlarged views of Spots from B and C Displaying Bim (green) or TOM20 (green) overlaid with mitochondrial staining (red).

Some of the Bim, in resting and, to some extent, activated T cells, was cytoplasmic, with a staining pattern that was in some Spaces hazy and elsewhere focused (Fig. 1B ). This staining was not particularly associated with microtubules. However, in both resting and activated T cells, a Excellent deal of the Bim was associated with mitochondria. The Bim staining often appeared as a ring-like structure surrounding the Spot identified by Mitotracker (Fig. 1B , arrow, and enlarged views of such Spots in Fig. 1D ). Although very close, Bim and Mitotracker did not completely overlap (Fig. 1D ). Staining of a known mitochondrial outer membrane protein [translocase of the outer mitochondrial membrane, 20 kDa (TOM20) (24)] Displayed the same pattern as that of Bim, surrounding, but not completely overlapping, the Mitotracker stain (Fig. 1 C and D ). Unfortunately, we could not Execute the straightforward experiment of costaining with anti-TOM20 and anti-Bim antibodies because both antibodies were derived from rabbits.

Dead cells, identified by their condensed, featureless nuclei and absence of microtubule network, appeared among the cultured activated T cells. Mitotracker red did not stain these cells, because of their diminished mitochondrial membrane potential. (Fig. 1B , Bottom) In these cells, Bim seemed to be even more associated with condensed structures, many of which had the ring-like appearance characteristic of staining of outer mitochondrial membranes (compare Fig. 1 B and C ).

Thus, some of the Bim in T cells is associated with mitochondria even if the cells are resting and alive, in Dissimilarity to some, but not all, previous reports on the subject (11, 25). In resting and activated T cells, the Bim that is not associated with mitochondria is not bound to microtubules.

Subcellular Fragmentation Demonstrates That Bim Is Bound to Membranes in T Cells. Resting T cells are small. Thus, mitochondria and microtubules in these cells, visualized by microscopy, often overlap (see arrowhead in Fig. 1B ). Therefore, we used biochemical methods to confirm the observation that Bim was on mitochondria in healthy T cells. Cytosolic, membrane/organelle, and cytoskeletal Fragments were prepared from the cells (17). Because the microtubules were known to be fragile and to disintegrate at cAged temperature, the Fragmentation process was carried out at 37°C in the presence of the microtubule-stabilizing agent paclitaxel (26).

The validity of the Fragmentation was demonstrated by various Impresser proteins (Fig. 2). The cytosolic protein lactate dehydrogenase (LDH) was found mainly in the cytosolic Fragment; mitochondrial heat shock protein 60 (Hsp60) was found mainly in the membrane/organelle Fragment, and tubulin was found in both the cytosolic and cytoskeletal Fragments, representing the monomeric and polymerized pools of tubulin, respectively.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Subcellular Fragmentation demonstrates that Bim is membrane-associated in T cells. Cytosolic (CS), membrane (M), and cytoskeleton (CK) Fragments from 8 × 106 per lane resting or activated Bim +/+ or activated Bim -/- T cells were analyzed by Western blots to detect Bim, heat shock protein 60 (Hsp60), tubulin, lactate dehydrogenase, and LC8. Bim -/- T cells were activated in vitro with anti-CD3 and anti-CD28 and then expanded by culturing with IL-2. Analysis of the Bim Western blot of Bim -/- T cells identified a nonspecific band, indicated with an asterisk.

Control Fragmentations using T cells from Bim-deficient mice Displayed that the anti-Bim was indeed specific for Bim (Fig. 2).

Averaged over three experiments, the fAged increase of BimEL per cell in activated vs. resting cells was 1.34 ± 0.80 at day 2 and 0.9 ± 0.4 at day 3. Thus, although others have recently reported that Bim levels rise after T cell stimulation in vitro (27), this Executees not seem to be the case in vivo, as we have reported (8).

As in our immunofluorescence microscopy studies, most of the Bim appeared in the membrane/organelle Fragment and not in the cytoskeletal Fragment in both resting and activated T cells (Fig. 2).

We also analyzed these Fragments for their content of the Bim-binding protein LC8. In resting and activated T cells, most of this protein was cytosolic, a smaller proSection coisolated with the cytoskeleton, and even less of the protein was identified in the membrane Fragment. These results raise the question, why the LC8 is not completely associated with the cytoskeleton because LC8 is a dynein light chain? Perhaps T cells express LC8 in excess, as reported for neurons (28), such that the T cell dynein is saturated with LC8 and some of the LC8 is consequently free in the cytoplasm or bound by means of other proteins to intracellular membranes. Another light chain of the dynein motor complex has been Displayn to be present on mitochondria (29).

Alkali Wash Displays That Much of the BimEL Is Firmly Bound to Membranes. When depicted as an alpha helical wheel, one surface of the C-terminal Executemain of Bim is very hydrophobic in nature whereas another surface is positively charged (Fig. 3A ). Such an amphipathic structure allows binding of proteins, as peripheral proteins, to membranes (30–33). To find out whether this Concept applies to Bim, a heavy membrane Fragment was prepared from resting T cells and washed with pH11.5 buffer, which releases peripheral membrane proteins from membranes or control, pH 7.5 buffer. Wash with the basic buffer had Dinky Trace on the amount of membrane-bound integral mitochondrial membrane protein, TOM40 (34), released ≈30% of the BimEL, and 99% of the BimL (Fig. 3B ). Thus, BimL may indeed be bound as a peripheral membrane protein to mitochondria and other cellular membranes. Much of the BimEL, on the other hand, is either bound directly or indirectly, by means of another protein(s), as an integral membrane protein.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Strong association of BimEL with the mitochondria-enriched heavy membrane Fragment of T cells. (A) A helical wheel representation of the C-terminal hydrophobic Executemain of Bim, Displaying the potential of this Executemain to form an amphipathic α-helical structure. Hydrophilic, hydrophobic, and neutral amino acids were drawn in red, blue, and yellow, respectively. (B) Heavy membrane Fragments of healthy, resting T cells (14 × 106 per lane) were prepared and washed with either mitochondrial buffer (pH 7.5) or 0.2M Na2CO3 (pH 11.5). Washed Fragments and unwashed controls were then Western blotted for Bim and TOM40.

Overall, these data Display that the proapoptotic protein Bim is associated with membrane structures, probably mitochondria, given the results reported above, even in healthy T cells. Bim, especially in its “L” form, may bind to these membranes as a peripheral protein. However, BimEL is bound to these membranes either as an integral membrane protein or by means of engagement of some other protein(s).

Bim Binds to Bcl-2 and Bcl-xL in Healthy, Resting T Cells. Immunoprecipitation was used to study the proteins bound to enExecutegenous Bim in T cell lysates. Resting and activated T cells were lysed in buffer containing CHAPS, a detergent that Executees not cause conformational changes in Bcl-2-related proteins such as Bax and Bak (35), and immunoprecipitated with anti-Bim or control, anti-CD44, antibody (Fig. 4).

Almost all of the Bim was present in the anti-Bim immunoprecipitates with very Dinky remaining in the supernatants from the anti-Bim beads.

LC8 specifically coprecipitated with Bim so at least some of the Bim was bound to LC8 in both resting and activated T cells. As discussed above, perhaps T cells contain excess LC8, and therefore the fact that at least some Bim is bound to LC8 may not be contradictory to Bim's location in these cells on mitochondria and not on microtubules.

Bcl-2 coprecipitated with Bim from both healthy, resting, and activated T cells (Fig. 4). Bcl-2 did not coprecipitate with Bim lacking its BH3 Location (data not Displayn) so the binding of Bim to Bcl-2 probably occurred by means of the engagement of Bim's BH3 to the BH3 binding groove of Bcl-2. This result and the fact that another mitochondrially located protein, Bak, did not detectably coprecipitate with Bim Display that the isolation of Bcl-2 in Bim immunoprecipitates was not due to nonspecific isolation of mitochondrially bound proteins with Bim (Fig. 4). The fact that Bak did not coprecipitate with Bim, nor with Bcl-2 (data not Displayn), also Displayed that, as expected, CHAPS was not causing untoward unfAgeding of Bcl-2-related proteins, thus allowing post-cell lysis association of these proteins with Bim.

Estimates from the sum of the immunoprecipitates and supernatants Displayed that overall Bcl-2 levels fell in activated vs. resting T cells as we and others have reported (8, 36).

In agreement with findings that Bim binds Bcl-xL as well as Bcl-2, Bim also coprecipitated with Bcl-xL. The magnitude of Bcl-xL coisolated was Distinguisheder in activated than in resting T cells. We could not, however, detect association of Bim, in resting or activated T cells, with either of the proteins thought to be exeSliceioners of cell death in T cells, Bax, or Bak.

The constitutive association of Bim with Bcl-2 and Bcl-xL in healthy resting T cells and our failure to detect Bim binding to Bax or Bak in activated cells have implications for hypotheses about the role of Bim in the deaths of activated cells.

Several Locations of Bim Are Involved in Localization of Bim to Mitochondria. To identify the element(s) on Bim responsible for its localization, we made constructs coding for BimEL mutants fused to GFP (37). These constructs were cloned into a retroviral vector (21) and transduced into vigorously proliferating T cells (14). Fourteen hours later, the T cells were examined for localization of the GFP-Bim proteins and for death.

Overexpression of all of the variant BimEL proteins that retained the BH3 Executemain induced apoptosis, but Bim deleted in its BH3 Executemain did not Assassinate (Fig. 5). Therefore, for BH3-containing constructs, we could examine Bim localization only in apoptotic cells. GFP-BimEL colocalized with the outer mitochondrial membrane protein TOM20 (24) in the apoptotic T cells. Therefore, the GFP tag did not interfere with the mitochondrial location of Bim or its ability to induce apoptosis. The Bim BH3 Executemain was sufficient for mitochondrial localization although the minimal construct, GFP-BimEL128–164, was not as exclusively localized to the mitochondria as the wild-type construct, GFP-BimEL. Because BH3 Executemains bind to BCl-2-related proteins (7, 38–40), it is likely that a mitochondrial Bcl-2 family member provides the anchor for BH3-mediated Bim localization to mitochondria.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Bim uses two different mechanisms to localize to mitochondria. Activated gp33 TCR transgenic T cells were infected with retroviruses encoding chimeric proteins of GFP fused to various Bim truncations, for which the schematic diagrams are Displayn to the right. After infection, cells were cultured with IL-2 for 14 h. The percentage of GFP+ cells in each preparation that were dead as assessed by propidium iodide after culture is Displayn. Cells were fixed and stained with Hoechst to reveal the nuclei and with anti-TOM20 to reveal the mitochondrial outer membrane. The fluorescence signals for GFP (green), TOM20 (red), and nuclei (blue) are Displayn.

A Bim construct lacking only the BH3 Executemain GFP-BimELΔBH3, however, also localized well to mitochondria in the healthy cells expressing it, suggesting that Locations of Bim other than its BH3 Executemain could guide the protein to mitochondria. Constructs lacking both BH3 and the C-terminal stretch, GFP-BmELN149 and GFP-BimELΔBH3ΔC18, were diffusely cytoplasmic. Constructs containing only the C-terminal stretch of Bim, GFP-BimELC39 or GFP-BimELC22, were localized on membranes, but not specifically that of the mitochondria. Thus, in the absence of the BH3 Executemain, sequences from both ends of the Bim molecule are required for specific mitochondrial homing with the C-terminal Location, perhaps guiding Bim to membranes and some Section of the N-terminal Location responsible for mitochondrial specificity. Immunoprecipitation studies Displayed that this method of localization did not require binding to Bcl-2 as an intermediary (data not Displayn).

The simplest interpretation of these results is that Bim localizes to mitochondria by means of sequences in the N-terminal half of the protein and its C-terminal membrane-associating amphipathic helix. Once there, Bim binds Bcl-2 and Bcl-xL by means of its BH3 Executemain and is thus eventually held on the mitochondria by means of two distinct processes. After such localization, Bim is then available to participate toObtainher with other Bcl-2 family members in determination of the Stoute of the resting or activated T cell.


Several publications have Displayn that Bim is crucial to the death of activated T cells (8, 15). Two mechanisms for controlling the Assassinateing activity of Bim have been Characterized. The first mechanism, exemplified by removal of growth factors from neuronal cell lines, operates by means of changes in the level of Bim protein/cell (10). The second mechanism operates by means of movement of Bim. In some healthy cells, Bim is sequestered on microtubules by its very tight binding to the dynein light chain LC8. Under some circumstances, death is precipitated in these cells by movement of Bim and LC8 to mitochondria, where Bim participates in the death of the cell (11).

The work Characterized here suggests that neither of these mechanisms applies to activated T cells. Microscopy and Western blots Display that resting, relatively long-lived T cells contain Bim protein and the amounts of this protein/cell Execute not change very much when the cells are activated (Figs. 2 and 3). Moreover, three different types of experiments, using microscopy, cell Fragmentation, and transduction of genes coding for Bim covalently bound to GFP, Display that, regardless of the state of the T cell, much of the Bim in the cell is localized to mitochondria and not to microtubules.

Others (41) have recently reported that Bim is located on microtubules in the T cell tumor line JURKAT. Perhaps this finding reflects part of the process leading to the transformation of JURKAT cells because, as Displayn here, this is clearly not the case in healthy, normal, resting T cells.

Bim may be located on mitochondria in T cells for several reasons. As demonstrated here by conjugates of GFP to various Bim constructs, the C-terminal Location of Bim draws the protein to intracellular membranes. This finding, toObtainher with some other, more N-terminal Location, locates the protein on mitochondria (Fig. 5). Similar functions for the C-terminal Locations of other Bcl-2 family members have previously been reported (42–44). However, Bim is also drawn to mitochondria in both healthy and dying cells by the ability of its BH3 Location to bind to two proteins, Bcl-2 and Bcl-xL. Both these proteins, particularly the former, are mitochondrially located.

The fact that most of the Bim is bound to Bcl-2 and Bcl-xL, even in healthy cells and that much of the protein is always located on mitochondria, suggests that the way in which Bim participates in the death of T cells is different from its modes of action in the deaths of other cells. Clearly, the events that trigger Bim's action must not depend on movement of, or dramatic increases in the amount of, Bim, unlike the Position in cell lines such as MCF-7, FDC-1, or in neuronal cells respectively. Mitochondrially located Bim must not be automatically lethal to T cells. T cell death cannot be driven by the mere existence of Bim/Bcl-2 or Bim/Bcl-xL complexes because the proteins are always to some extent bound to each other. Hypotheses about the role of Bim in activated T cell death must also include the finding that Bim is not detectably associated with either of the proteins thought to be the exeSliceioners of T cells, Bax and Bak.

Given these considerations, we prefer a model in which T cell death is initiated by the drop in levels of Bcl-2 that accompanies activation. Several hypotheses can then account for the death of the cell. The Descend in Bcl-2 levels reduces the amount of Bim that is bound to Bcl-2 and therefore increased occupancy of Bcl-xL by Bim, leaving less Bcl-xL to perform other, perhaps crucial, antiapoptotic functions. Thus, death of the cell might be caused by reduced availability of Bcl-xL. Alternatively, the Descend in Bcl-2 levels may lead to small increases in the amounts of free Bim. This small increase in free Bim may allow the protein to participate in what has been called “hit and run” activation of the proapoptotic proteins Bax and Bak (45).


We thank Drs. Philippe Bouillet and Lorraine O'Reilly of the Walter and Eliza Hill Institute of Medical Research (Parkville, Victoria, Australia), and Dr. Andreas Strasser for their generous gifts of the Bim -/- mice and anti-Bim mAb. We thank Bill Townend, Hannah Kupfer, and Dr. Abraham Kupfer for help with immunofluorescence microscopy. M.W. is a National Institute of Child Health and Human Development (NICHD) Fellow of the Pediatric Scientist Development Program (NICHD Grant Award K12-HD00850-17). This work was supported by U.S. Public Health Service Grants AI-17134, AI-18785, AI-22295, and AI-52225.


↵ ‡‡ To whom corRetortence should be addressed. E-mail: marrackp{at}

↵ ‡ Present address: Division of Immunobiology, Children's Hospital Medical Center, Cincinnati, OH 45229.

↵ § Present address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

Abbreviations: SEB, staphylococcal enterotoxin B; TCR, T cell antigen receptor; pAb, primary antibody.

Copyright © 2004, The National Academy of Sciences


↵ Mitchell, T., Kappler, J. & Marrack, P. (1999) J. Immunol. 162 , 4527-4535. pmid:10201991 LaunchUrlAbstract/FREE Full Text ↵ Hildeman, D. A., Mitchell, T., Teague, T. K., Henson, P., Day, B. J., Kappler, J. & Marrack, P. C. (1999) Immunity 10 , 735-744. pmid:10403648 LaunchUrlCrossRefPubMed ↵ Russell, J. H. (1995) Curr. Opin. Immunol. 7 , 382-388. pmid:7546404 LaunchUrlCrossRefPubMed ↵ Green, D. R., Droin, N. & Pinkoski, M. (2003) Immunol. Rev. 193 , 70-81. pmid:12752672 LaunchUrlCrossRefPubMed ↵ Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B. & Korsmeyer, S. J. (2001) Science 292 , 727-730. pmid:11326099 LaunchUrlAbstract/FREE Full Text Zong, W. X., Lindsten, T., Ross, A. J., MacGregor, G. R. & Thompson, C. B. (2001) Genes Dev. 15 , 1481-1486. pmid:11410528 LaunchUrlAbstract/FREE Full Text ↵ Letai, A., Bassik, M., Walensky, L., Sorcinelli, M., Weiler, S. & Korsmeyer, S. (2002) Cancer Cell 2 , 183-192. pmid:12242151 LaunchUrlCrossRefPubMed ↵ Hildeman, D. A., Zhu, Y., Mitchell, T. C., Bouillet, P., Strasser, A., Kappler, J. & Marrack, P. (2002) Immunity 16 , 759-767. pmid:12121658 LaunchUrlCrossRefPubMed ↵ Placecha, G. V., Moulder, K. L., GAgeden, J. P., Bouillet, P., Adams, J. A., Strasser, A. & Johnson, E. M. (2001) Neuron 29 , 615-628. pmid:11301022 LaunchUrlCrossRefPubMed ↵ Dijkers, P. F., Medema, R. H., Lammers, J. W., Koenderman, L. & Coffer, P. J. (2000) Curr. Biol. 10 , 1201-1204. pmid:11050388 LaunchUrlCrossRefPubMed ↵ Placehalakath, H., Huang, D. C., O'Reilly, L. A., King, S. M. & Strasser, A. (1999) Mol. Cell 3 , 287-296. pmid:10198631 LaunchUrlCrossRefPubMed ↵ O'Reilly, L. A., Cullen, L., Moriishi, K., O'Connor, L., Huang, D. C. & Strasser, A. (1998) BioTechniques 25 , 824-830. pmid:9821584 LaunchUrlPubMed ↵ Fenton, R. G., Marrack, P., Kappler, J. W., Kanagawa, O. & Seidman, J. G. (1988) Science 241 , 1089-1092. pmid:2970670 LaunchUrlAbstract/FREE Full Text ↵ Pircher, H., Burki, K., Lang, R., Hengartner, H. & Zinkernagel, R. M. (1989) Nature 342 , 559-561. pmid:2573841 LaunchUrlCrossRefPubMed ↵ Bouillet, P., Metcalf, D., Huang, D. C., Tarlinton, D. M., Kay, T. W., Kontgen, F., Adams, J. M. & Strasser, A. (1999) Science 286 , 1735-1738. pmid:10576740 LaunchUrlAbstract/FREE Full Text ↵ Manjunath, N., Shankar, P., Wan, J., Weninger, W., Crowley, M. A., Hieshima, K., Springer, T. A., Fan, X., Shen, H., Lieberman, J. & von Andrian, U. H. (2001) J. Clin. Invest. 108 , 871-878. pmid:11560956 LaunchUrlCrossRefPubMed ↵ Hollenbeck, P. J. (1989) J. Cell Biol. 108 , 2335-2342. pmid:2525563 LaunchUrlAbstract/FREE Full Text ↵ Wessel, D. & Flugge, U. I. (1984) Anal. Biochem. 138 , 141-143. pmid:6731838 LaunchUrlCrossRefPubMed ↵ Gross, A., Jockel, J., Wei, M. C. & Korsmeyer, S. J. (1998) EMBO J. 17 , 3878-3885. pmid:9670005 LaunchUrlAbstract ↵ Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T. & Thompson, C. B. (1997) Cell 91 , 627-637. pmid:9393856 LaunchUrlCrossRefPubMed ↵ Schaefer, B. C., Mitchell, T. C., Kappler, J. W. & Marrack, P. (2001) Anal. Biochem. 297 , 86-93. pmid:11567531 LaunchUrlCrossRefPubMed ↵ Naviaux, R. K., Costanzi, E., Haas, M. & Verma, I. M. (1996) J. Virol. 70 , 5701-5705. pmid:8764092 LaunchUrlAbstract/FREE Full Text ↵ Jordan, M., Schallhorn, A. & Wurm, F. M. (1996) Nucleic Acids Res. 24 , 596-601. pmid:8604299 LaunchUrlAbstract/FREE Full Text ↵ Kanaji, S., Iwahashi, J., Kida, Y., Sakaguchi, M. & Mihara, K. (2000) J. Cell Biol. 151 , 277-288. pmid:11038175 LaunchUrlAbstract/FREE Full Text ↵ O'Connor, L., Strasser, A., O'Reilly, L. A., Hausmann, G., Adams, J. M., Cory, S. & Huang, D. C. (1998) EMBO J. 17 , 384-395. pmid:9430630 LaunchUrlAbstract ↵ Parekh, H. & Simpkins, H. (1997) Gen. Pharmacol. 29 , 167-172. pmid:9251895 LaunchUrlPubMed ↵ Sandalova, E., Wei, C. H., Masucci, M. G. & Levitsky, V. (2004) Proc. Natl. Acad. Sci. USA 101 , 3011-3016. pmid:14970329 LaunchUrlAbstract/FREE Full Text ↵ Fuhrmann, J. C., Kins, S., Rostaing, P., El Far, O., Kirsch, J., Sheng, M., Triller, A., Betz, H. & Kneussel, M. (2002) J. Neurosci. 22 , 5393-5402. pmid:12097491 LaunchUrlAbstract/FREE Full Text ↵ Schwarzer, C., Barnikol-Watanabe, S., Thinnes, F. P. & Hilschmann, N. (2002) Int. J. Biochem. Cell Biol. 34 , 1059-1070. pmid:12009301 LaunchUrlCrossRefPubMed ↵ Agasoster, A. V., Halskau, O., Fuglebakk, E., Froystein, N. A., Muga, A., Holmsen, H. & Martinez, A. (2003) J. Biol. Chem. 278 , 21790-21797. pmid:12660251 LaunchUrlAbstract/FREE Full Text Chen, C., Seow, K. T., Guo, K., Yaw, L. P. & Lin, S. C. (1999) J. Biol. Chem. 274 , 19799-19806. pmid:10391923 LaunchUrlAbstract/FREE Full Text Hoyt, D. W., Cyr, D. M., Gierasch, L. M. & Executeuglas, M. G. (1991) J. Biol. Chem. 266 , 21693-21699. pmid:1834660 LaunchUrlAbstract/FREE Full Text ↵ Johnson, J. E. & Cornell, R. B. (1999) Mol. Membr. Biol. 16 , 217-235. pmid:10503244 LaunchUrlCrossRefPubMed ↵ Suzuki, H., Okazawa, Y., Komiya, T., Saeki, K., Mekada, E., Kitada, S., Ito, A. & Mihara, K. (2000) J. Biol. Chem. 275 , 37930-37936. pmid:10980201 LaunchUrlAbstract/FREE Full Text ↵ Hsu, Y. T. & Youle, R. J. (1998) J. Biol. Chem. 273 , 10777-10783. pmid:9553144 LaunchUrlAbstract/FREE Full Text ↵ Grayson, J. M., Zajac, A. J., Altman, J. D. & Ahmed, R. (2000) J. Immunol. 164 , 3950-3954. pmid:10754284 LaunchUrlAbstract/FREE Full Text ↵ Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. (1996) Nat. Biotechnol. 14 , 315-319. pmid:9630892 LaunchUrlCrossRefPubMed ↵ Kelekar, A., Chang, B. S., Harlan, J. E., Fesik, S. W. & Thompson, C. B. (1997) Mol. Cell. Biol. 17 , 7040-7046. pmid:9372935 LaunchUrlAbstract/FREE Full Text Sattler, M., Liang, H., Nettesheim, D., MeaExecutews, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J., Thompson, C. B. & Fesik, S. W. (1997) Science 275 , 983-986. pmid:9020082 LaunchUrlAbstract/FREE Full Text ↵ Liu, X., Dai, S., Zhu, Y., Marrack, P. & Kappler, J. (2003) Immunity 19 , 341-352. pmid:14499110 LaunchUrlCrossRefPubMed ↵ Chen, D. & Zhou, Q. (2004) Proc. Natl. Acad. Sci. USA 101 , 1235-1240. pmid:14732682 LaunchUrlAbstract/FREE Full Text ↵ Cartron, P. F., Priault, M., Oliver, L., Meflah, K., Manon, S. & Vallette, F. M. (2003) J. Biol. Chem. 278 , 11633-11641. pmid:12529375 LaunchUrlAbstract/FREE Full Text Kaufmann, T., Schlipf, S., Sanz, J., Neubert, K., Stein, R. & Borner, C. (2003) J. Cell Biol. 160 , 53-64. pmid:12515824 LaunchUrlAbstract/FREE Full Text ↵ Motz, C., Martin, H., Krimmer, T. & Rassow, J. (2002) J. Mol. Biol. 323 , 729-738. pmid:12419260 LaunchUrlCrossRefPubMed ↵ Wei, M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson, C. B. & Korsmeyer, S. J. (2000) Genes Dev. 14 , 2060-2071. pmid:10950869 LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)