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The 55-kDa Tumor Necrosis Factor Receptor Induces Clustering of Mitochondria through Its Membrane-proximal Region

1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês

10.1074/jbc.273.16.9673

1083-351X

Kurt J. De Vos, Vera Goossens, Elke Boone, Dominique Vercammen, Katia Vancompernolle, Peter Vandenabeele, Guy Haegeman, Walter Fiers, Johan Grooten,

ATP Synthase and ATPases Research

The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria. The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria. Mitochondria are the energy-providing organelles in eukaryotic cells. However, accumulating evidence shows that these organelles also have an active function in cell death. Disruption of mitochondrial transmembrane potential (ΔΨm) 1The abbreviations used are: ΔΨm, mitochondrial transmembrane potential; Ab, antibody; CHX, cycloheximide; CLSM, confocal laser scanning microscopy; DD, death domain; FITC, fluorescein isothiocyanate; hTNF, human TNF; IL-6, interleukin-6; KHC, kinesin heavy chain; mAb, monoclonal antibody; MT, microtubule; mTNF, murine TNF; NF-κB, nuclear factor κB; PI, propidium iodide; R123, rhodamine 123; ROS, reactive oxygen species; TNF, tumor necrosis factor; TNF-R55, 55-kDa TNF receptor; TNF-R75, 75-kDa TNF receptor; MEM, minimal essential medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 1The abbreviations used are: ΔΨm, mitochondrial transmembrane potential; Ab, antibody; CHX, cycloheximide; CLSM, confocal laser scanning microscopy; DD, death domain; FITC, fluorescein isothiocyanate; hTNF, human TNF; IL-6, interleukin-6; KHC, kinesin heavy chain; mAb, monoclonal antibody; MT, microtubule; mTNF, murine TNF; NF-κB, nuclear factor κB; PI, propidium iodide; R123, rhodamine 123; ROS, reactive oxygen species; TNF, tumor necrosis factor; TNF-R55, 55-kDa TNF receptor; TNF-R75, 75-kDa TNF receptor; MEM, minimal essential medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. during apoptosis induced by various stimuli in diverse cell types represents an irreversible commitment to cell death, preceding the late characteristics of apoptosis, such as DNA condensation and degradation as well as formation of apoptotic bodies (1Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssière J.-L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1092) Google Scholar, 2Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsch T. Macho A. Haeffner A. Hirsch F. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (780) Google Scholar). A causative link between ΔΨm and nuclear apoptosis is supported by the release of a caspase-like, apoptosis-inducing factor from the mitochondrial intermembrane space after permeability transition, a condition leading to ΔΨm disruption (3Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). In addition to apoptosis-inducing factor, cytochrome c induces apoptosis in a cell-free system in the presence of dATP and cytosolic extracts (4Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4448) Google Scholar). The release of cytochrome c from mitochondria is independent of permeability transition and ΔΨm disruption, suggesting a possible role in apoptosis in cell types that do not exhibit disruption of ΔΨm (5Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4267) Google Scholar, 6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4397) Google Scholar). Moreover, Bcl-2 prevents release of apoptosis-inducing factor and cytochromec from the mitochondria (3Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 5Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4267) Google Scholar, 6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4397) Google Scholar), and Bcl-xLinhibits the accumulation of cytochrome c in the cytosol during apoptosis possibly by binding to it and thus blocking its availability (7Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (368) Google Scholar). Therefore, the antiapoptotic role of Bcl-2 and family members could be based on counteracting mitochondrial dysfunction and subsequent release of apoptogenic factors. Mitochondrial dysfunction also plays a crucial role in cell types that exhibit necrosis-like cell death after activation of their death program. Disruption of plasma membrane integrity by the proinflammatory cytokine tumor necrosis factor (TNF) represents an irreversible commitment to cell death in the murine fibrosarcoma cell line L929 (8Grooten J. Goossens V. Vanhaesebroeck B. Fiers W. Cytokine. 1993; 5: 546-555Crossref PubMed Scopus (87) Google Scholar) and depends on the production of reactive oxygen species (ROS) by the mitochondria (9Schulze-Osthoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Abstract Full Text PDF PubMed Google Scholar, 10Schulze-Osthoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 11Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (555) Google Scholar). Also, in drug-resistant leukemia cells, inhibition of the mitochondrial respiratory chain by TNF is, at least partially, responsible for cytotoxicity (12Jia L. Kelsey S.M. Grahn M.F. Jiang X.R. Newland A.C. Blood. 1996; 87: 2401-2410Crossref PubMed Google Scholar), while in human ovarian carcinoma cell lines there is evidence for an involvement of mitochondrial ROS in TNF-mediated cell death (13Uslu R. Bonavida B. Cancer. 1996; 77: 725-732Crossref PubMed Scopus (47) Google Scholar). The 55-kDa TNF receptor (TNF-R55) and Fas (APO-1 or CD95), both members of the TNF receptor family, can trigger the cell death program of several cell types. Additionally, TNF-R55 mediates gene induction by activation of the nuclear factor κB (NF-κB) and the transcription factors AP-1 and ATF-2 (14Beyaert R. Fiers W. FEBS Lett. 1994; 340: 9-16Crossref PubMed Scopus (243) Google Scholar) and triggers phosphorylation/dephosphorylation cascades by activation of p38/RK mitogen-activated protein kinase (15Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). The clustered intracellular domains of TNF-R55 bind, directly or indirectly, a variety of signaling molecules responsible for the multiple signals emanating from the activated receptor. Evidence for the role of TNF-R55-associated (but also of Fas-associated) proteins in the cytocidal function of both receptors emerged from the identification of the so-called "death domain" (DD) located in the C-terminal region of the intracellular part of the receptors (16Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1165) Google Scholar, 17Vandevoorde V. Haegeman G. Fiers W. J. Cell Biol. 1997; 137: 1627-1638Crossref PubMed Scopus (55) Google Scholar). The DD mediates protein-protein interactions of the receptor with other DD-containing proteins that act as initiating centers for different signaling cascades (18Varfolomeev E.E. Boldin M.P. Goncharov T.M. Wallach D. J. Exp. Med. 1996; 183: 1271-1275Crossref PubMed Scopus (107) Google Scholar). Thus, Fas-associated, recruited directly by Fas (19Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2155) Google Scholar, 20Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar) and indirectly by TNF-R55 through prior binding of TNF receptor-associated DD (21Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1739) Google Scholar), links the receptors to caspase-8 and the cell death program (22Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (966) Google Scholar, 23Greidinger E.L. Miller D.K. Yamin T.T. Casciola-Rosen L. Rosen A. FEBS Lett. 1996; 390: 299-303Crossref PubMed Scopus (107) Google Scholar). Also, receptor-interacting protein binds to TNF receptor-associated DD and may be involved in binding and/or function of TRAF2 (18Varfolomeev E.E. Boldin M.P. Goncharov T.M. Wallach D. J. Exp. Med. 1996; 183: 1271-1275Crossref PubMed Scopus (107) Google Scholar, 24Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (861) Google Scholar). Although it seems that the DD of TNF-R55 elicits most of the known TNF-induced phenomena, there is accumulating evidence for a role of the membrane-proximal region of the intracellular part of the receptor. Induction of nitric-oxide synthase requires this region in addition to the DD (16Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1165) Google Scholar). Activation of c-Raf-1 kinase and phospholipase A2 occurs through the membrane-proximal half of TNF-R55 (25Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 26Belka C. Wiegmann K. Adam D. Holland R. Neuloh M. Herrmann F. Krönke M. Brach M.A. EMBO J. 1995; 14: 1156-1165Crossref PubMed Scopus (91) Google Scholar). Furthermore, the activation of membrane-associated neutral sphingomyelinase is linked to this region by the protein FAN (27Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell. 1996; 86: 937-947Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar), while also phosphatidylinositol-4-phosphate 5-kinase can interact with TNF-R55 through its juxtamembrane region (28Castellino A.M. Parker G.J. Boronenkov I.V. Anderson R.A. Chao M.V. J. Biol. Chem. 1997; 272: 5861-5870Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In this report, we describe a novel activity of TNF, namely the induction of an altered spatial distribution of mitochondria in TNF-sensitive cells. This translocation of mitochondria depends on a signal from the membrane-proximal region of TNF-R55 and thus represents a novel function of this region. The driving force of this translocation was not the microtubule (MT) cytoskeleton, but rather a loss of outward directed movement of mitochondria caused by an impaired activity of the molecular motor kinesin. Functional studies as well as the kinetics of mitochondrial translocation and its correlation with the death-inducing activity of TNF, but not with gene induction, implicate this response and its apparent molecular counterpart, namely inactivation of mitochondria-associated kinesin, in the cell death response. L929, L929r2, L929hFas, L929hR55wt, and L929hR55ΔMPR cells (29Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar) 2D. Vercammen and E. Boone, unpublished data. were cultured in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) heat-inactivated fetal calf serum, 5% (v/v) heat-inactivated newborn calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mml-glutamine, at 37 °C in a humidified 5% CO2, 95% air incubator. U937 human histiocytic lymphoma cells were maintained in RPMI 1640, supplemented with 10% fetal calf serum, 0.4 mm sodium pyruvate, 50 μmβ-mercaptoethanol, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mml-glutamine. SUK4 and SUK5 hybridomas (30Ingold A.L. Cohn S.A. Scholey J.M. J. Cell Biol. 1988; 107: 2657-2667Crossref PubMed Scopus (139) Google Scholar) were purchased from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences (Johns Hopkins University School of Medicine, Baltimore, MD) and the Department of Biological Sciences (University of Iowa, Iowa City, IA) under contract N01-HD-6–2915 from the NICHD, National Institutes of Health; they were cultured in RPMI 1640 supplemented as above. All cell lines were mycoplasma-free as judged from an enzyme immunoassay (Boehringer Mannheim). Murine TNF (mTNF) was produced inEscherichia coli and purified to at least 99% homogeneity in our laboratory. It had a specific activity of 1.9 × 108 IU/mg of protein (National Institute for Biological Standards and Control, Potters Bar, UK), contained 4 ng of endotoxin/mg of protein, and was used at 1000 IU/ml. Propidium iodide (PI), cycloheximide (CHX), H2O2, menadione,tert-butyl hydroperoxide, and nocodazole (all from Sigma) were used at final concentrations of 30 μm, 50 μg/ml, 2.5 mm, 1 mm, 2.5 mm, and 10 μm, respectively. Rhodamine 123 (R123), MitoTracker CMTMRos, and paclitaxel (all from Molecular Probes, Eugene, OR) were used at final concentrations of 1 μm. Staurosporine (Boehringer Mannheim) was used at 10 μm. SUK4 antibody (Ab) and SUK5 Ab were purified by means of a Protein G column (Amersham Pharmacia Biotech) from ascites fluid and culture supernatant, respectively. Ab labeling with Cy5 fluorochrome was performed using an Ab labeling kit from Amersham Pharmacia Biotech according to the manufacturer's instructions. Cells were seeded in chambered coverslips (2 cm2, 105 cells/well) and preincubated overnight at 37 °C in a humidified 5% CO2, 95% air incubator. Mitochondria were stained with R123 or MitoTracker CMTMRos for 30 min at 37 °C before analysis. L929 cells and L929-derived cells were washed three times with Hepes-buffered MEM, supplemented with the same additives as Dulbecco's modified Eagle's medium (MEM-Hepes), to remove excess R123 or MitoTracker CMTMRos and kept in MEM-Hepes. MEM-Hepes was preferred over Dulbecco's modified Eagle's medium because of the CO2independence and the fluorescent background problems generated by Dulbecco's modified Eagle's medium. U937 cells were maintained in RPMI 1640 medium during the measurements. The distribution of mitochondria was analyzed with a Zeiss LSM 410 confocal microscope. R123 and MitoTracker CMTMRos were excited and detected as shown in Table I. The time point at which TNF was added is referred to as 0 h. At regular times after TNF administration, fluorescence confocal laser scanning microscopy (CLSM) images from four randomly chosen microscopic fields, each containing approximately 50 cells, were recorded. Translocation was represented as the percentage of viable cells exhibiting the clustered mitochondrial distribution.Table ISpectral properties of fluorochromes usedFluorochromeExcitation wavelengthEmission filtersnmnmFITC488BP 515–525PI488LP 610R123488BP 515–525CMTMRos543BP 570Cy5633LP 665 Open table in a new tab Apoptosis induced by TNF and anti-human Fas mAb (500 ng/ml; ImmunoTech, Alston, MA) in U937 cells was quantified in a flow cytometric assay based on DNA degradation using PI. After intercalation in DNA, PI generates a fluorescent emission light, the intensity of which correlates with the amount of DNA. In viable, permeabilized cells, this results in fluorescence intensity peaks corresponding to the G1 (2n), S and G2/M (4n) phases of the cell cycle. Cells dying by apoptosis contain degraded DNA, resulting in hypoploidy and hence reduced PI fluorescence. U937 cells were permeabilized by freezing/thawing in the presence of PI. The number of hypoploid cells was determined with an Epics 753 flow cytometer (Coulter, Hialeah, FL), using the same excitation and emission wavelengths as in CLSM (Table I), as a measure for TNF-induced apoptosis. Cell death of L929 and L929-derived cell lines was measured by quantification of PI-positive cells by CLSM and was induced by incubation with either TNF in L929, TNF and CHX in L929r2, htr-1 mAb (100 ng/ml; a gift of Dr. M. Brockhaus, F. Hoffmann-La Roche, Basel, Switzerland), or anti-mTNF-R55 (500 ng/ml; Genzyme Corporation, Boston, MA) in L929hR55wt and L929hR55ΔMPR, and anti-human Fas mAb (500 ng/ml) in L929hFas cells. Cell death is presented as the percentage of PI-positive cells in the whole cell population. Dihydrorhodamine 123 was added to suspension cultures at the same time as TNF. Cell samples were taken at regular time intervals and analyzed on an Epics 753 flow cytometer. R123 fluorescence resulting from dihydrorhodamine 123 oxidation was excited and detected (Table I). 3000 viable cells were measured per sample. Cell debris and multicell aggregates were gated out electronically. The variation observed in ROS measurements was <10%. The presence of IL-6 bioactivity in the culture supernatant of 3 × 105cells/ml was determined after 5 h of TNF stimulation, using the proliferative response of the IL-6-responsive murine plasmacytoma cell line 7TD1 (31Van Snick J. Cayphas S. Vink A. Uyttenhove C. Coulie P.G. Rubira M.R. Simpson R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9679-9683Crossref PubMed Scopus (677) Google Scholar). L929 cells were grown on glass coverslips. Where necessary, MitoTracker CMTMRos was added to the culture medium for 30 min to stain mitochondria before fixation. Coverslips, fixed at −20 °C with methanol, were stained for MTs by incubation with 10-fold diluted rat anti-tubulin hybridoma YOL1/34 supernatant (Harlan Sera-Lab, Crawley Down, UK) at room temperature for 1 h, followed by extensive washing with phosphate-buffered saline containing 1% bovine serum albumin and a 30-min incubation with FITC-conjugated goat anti-rat IgG Ab (1:100; Harlan Sera-Lab). Staining of the kinesin heavy chain (KHC) was performed by incubation of coverslips with 10 μg/ml Cy5-labeled SUK4 mAb. Coverslips were mounted in VectaShield (Vector Laboratories, Burlingame, CA). Cells were observed with a Zeiss LSM410 confocal microscope, typically with a scanning time of 1 s and 8× line averaging. The excitation wavelengths and emission filters used for the different fluorochromes are shown in Table I. Syringe loading of L929 cells with mAb was performed as described previously (32Clarke M.S.F. McNeil P.L. J. Cell Sci. 1992; 102: 533-541PubMed Google Scholar). Briefly, L929 cells were harvested from adherent, subconfluent cultures by treatment with enzyme-free cell dissociation solution (Life Technologies, Paisley, UK). 5 × 105 cells were transferred to a 1.5-ml reaction tube in a volume of 0.1 ml of medium. mAb was added to a final concentration of 0.48 mg/ml, with an unlabeled/fluorescent labeled ratio of 15:1, followed by the addition of Pluronic F-68 (Sigma; 2% (v/v)). This solution was transferred 20 times through a 30-G injection needle. Excess Ab and pluronic F-68 were washed away, and the cells were seeded in chambered coverslips for CLSM analysis. The cells were allowed to recover for 12 h before the experiment. There was typically 10% cell death resulting from loading; dead cells were removed by washing after the recovery period. Loaded cells were identified by the presence of fluorescence in the cytosol and represented typically 50–60% of the total cell population. L929hR55wt and L929hR55ΔMPR cells were harvested from adherent, subconfluent cultures by treatment with enzyme-free cell dissociation solution. 106 cells were incubated for 1 h with the primary Ab,viz. htr-9 (200 ng; a gift of Dr. M. Brockhaus), in the case of hTNF-R55 or anti-mTNF-R55 mAb (200 ng). FITC-conjugated goat anti-mouse Ig or goat anti-hamster Ig (both from Harlan Sera-Lab) were used as secondary Ab. FITC fluorescence intensity was analyzed on an Epics 753 flow cytometer. Activation of NF-κB was measured in an electrophoretic mobility shift assay. Subconfluent monolayers of L929r2 cells were treated with TNF for 1 h. Nuclear protein and binding reactions were performed as described previously (33Patestos N.P. Haegeman G. Vandevoorde V. Fiers W. Biochimie. 1993; 75: 1007-1018Crossref PubMed Scopus (20) Google Scholar). L929 cells were lysed in lysis buffer containing 1% CHAPS and various protease and phosphatase inhibitors (10 mm Tris-HCl, pH 7.4, 25 mm NaCl, 50 mm EDTA, 10 μm Pefabloc (Pentapharm, Basel, Switzerland), 40 mm β-glycerophosphate, 10 mmNaF, aprotinin (100 times diluted), 1 mmNaVO3). 100 μg of protein was separated on a 7.5% SDS gel, transferred to a nitrocellulose membrane by electroblotting, and processed for ECL detection (Amersham Pharmacia Biotech). Primary Ab, SUK4 mAb, or irrelevant mAb (mouse anti-hamster IgG mAb; PharMingen, San Diego, CA) was used at 500 ng/ml. TNF treatment of the murine fibrosarcoma cell line L929 results in cell death by an atypical, necrosis-like process, which is characterized by cell swelling, disruption of plasma membrane integrity, and subsequently cellular collapse (8Grooten J. Goossens V. Vanhaesebroeck B. Fiers W. Cytokine. 1993; 5: 546-555Crossref PubMed Scopus (87) Google Scholar). Mitochondrial dysfunction is crucial in this TNF-induced cytotoxic process; ROS are formed in the mitochondria, and interference with the generation or scavenging of these ROS arrests cell death (9Schulze-Osthoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Abstract Full Text PDF PubMed Google Scholar, 10Schulze-Osthoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 11Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (555) Google Scholar). Using CLSM, we observed that within 1 h of treatment of L929 cells with TNF the spatial distribution of mitochondria evolved in the majority of the cells from an originally scattered, bipolar or nearly symmetric distribution to an asymmetric, clustered distribution (Fig. 1). Analysis of the kinetics of this mitochondrial translocation revealed that the response preceded mitochondrial ROS production and subsequent cell death by several hours (Fig. 2 A). For these experiments, the mitochondria-specific dye R123 was preferred because preliminary experiments had shown that this dye did not interfere with cell viability or responsiveness to TNF. However, since the specificity of R123 derives from its ΔΨm-dependent accumulation in the mitochondria, a disruption of ΔΨmin a fraction of the mitochondria could be perceived as a translocation. Yet, revealing the spatial distribution of mitochondria with MitoTracker, a mitochondria-specific dye that is retained in the mitochondria by the thiol reactivity of its chloromethyl moiety (34Macho A. Decaudin D. Castedo M. Hirsch T. Susin S.A. Zamzami N. Kroemer G. Cytometry. 1996; 25: 333-340Crossref PubMed Scopus (157) Google Scholar), yielded an altered distribution identical to the one observed after treatment with TNF (data not shown). Consequently, the modified distribution of R123 and MitoTracker fluorescence in TNF-treated L929 cells reflects the modified spatial distribution of mitochondria and not a loss of ΔΨm in a fraction of the organelles.Figure 2TNF-induced translocation of mitochondria precedes cell death. A, the increment of cells exhibiting translocated mitochondria (line), of dead cells (bar) and of ROS production (inset) was measured following the addition of TNF. Translocation was analyzed by CLSM in approximately 150–200 cells using the mitochondria-specific probe R123. Cell death was quantified simultaneously by PI uptake. The production of mitochondrial ROS was measured in a parallel flow-cytometric assay as described previously (11Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (555) Google Scholar) using the ROS-sensitive probe dihydrorhodamine 123 and is expressed as the increment in fluorescence relative to untreated cells. The data shown are representative of five experiments (n = 5). B, U937 cells. Similarly to L929 cells, TNF-induced translocation of mitochondria (line) was analyzed by CLSM (n = 5). The percentage of dead cells (bar) was obtained by flow cytometry based on the number of cells exhibiting hypoploidy (n = 5). C, cell death was induced in L929 cells by treatment with either TNF or the cytotoxic agent menadione (Men), tert-butyl hydroperoxide (t-BHP), H2O2, or staurosporine (STS). After 4 h of treatment, the occurrence of mitochondrial translocation (lower half) and cell death (upper half) was analyzed as described above (n = 5).View Large Image Figure ViewerDownload (PPT) In contrast to L929, cell death induced by TNF in the human histiocytic lymphoma cell line U937 is accompanied by plasma membrane blebbing, DNA degradation, and subsequent formation of apoptotic bodies (data not shown), features characteristic of apoptosis. To verify whether also in these cells mitochondria converge in response to treatment with TNF, TNF-treated U937 cells were stained with R123 and analyzed by CLSM. As shown in Fig. 2 B, cells exhibiting a clustered spatial distribution of mitochondria became apparent within 1 h of TNF treatment, preceding the onset of the apoptotic response by several hours. Clearly, translocation of mitochondria occurs independently of the mechanism of cell death, viz. necrosis-like or apoptotic, and is an early feature relative to the implementation of the cell death program. Cell death is accompanied by morphological changes that occur independently of the type of signal triggering the process. Since the observed translocation of mitochondria could be such a phenomenon, we induced cell death in L929 cells by administration of the cytotoxic agent menadione, tert-butyl hydroperoxide, H2O2, or staurosporine, after which the distribution of mitochondria was analyzed (Fig. 2 C). Despite clear cytotoxicity, no changes in the spatial distribution of mitochondria were observed. This negative result clearly demonstrates that induction of mitochondrial translocation is an intrinsic feature of the TNF signal transduction pathway. To establish the relationship between the observed clustering of mitochondria and the cytocidal versusnoncytocidal activities of TNF, we studied the occurrence of mitochondrial translocation, cell death, activation of NF-κB, and gene induction in the L929 variant L929r2 (29Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). L929r2 cells are resistant to the cytocidal activity of TNF but retain responsiveness to its noncytocidal activities exemplified by activation of NF-κB and subsequent expression of IL-6. However, the addition of the RNA or protein synthesis inhibitor actinomycin D or CHX, respectively, fully restores the cytocidal response to TNF. As shown in Fig. 3 A, mitochondria converged after TNF treatmen