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First published online September 15, 2004
doi: 10.1242/10.1242/jcs.01339

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ß-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death

¹ The Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China
² Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
³ Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (50K): [in a new window]   Fig. 1. ß-actin mutation leads to a resistance of TNF-induced cell death in L929 cells. (A) The fused mRNA of neo and an endogenous gene in a TNF-resistant L929 clonal cell line was amplified by 3'-RACE. The junction sequence of the fused cDNA is shown, which reveals that the viral insertion occurred 5' to the coding region of the ß-actin gene. The amino acid sequence at the C terminus of neo is shown beneath the cDNA sequence. The sequence introduced by viral vectors is shown in lowercase. The number in parentheses indicates the position relative to the start codon of ß-actin. (B) ß-actin protein is reduced in ß-actin mutant cells (Actinmut). Western blotting was performed using anti-ß-actin antibody. Equal loading of total cell lysates was determined by staining an identical SDS-PAGE with Coomassie Blue and a portion of the picture is shown. (C) Wild-type and Actinmut cells were treated with TNF (100 ng/ml) for different periods of time and cell viability was assessed by propidium iodide (PI) exclusion. Results represent the means±s.e. (n=3). (D) Stable cell lines were generated from Actinmut cells by transfection of ß-actin expression vector (Actinmut+actin) or empty vector (Actinmut+vector). The expression level of ß-actin was determined as in (B). (E) The sensitivity to TNF-induced cell death in Actinmut, Actinmut+actin and Actinmut+vector was measured as in (C). (F) Parental wild-type L929 cells were treated with siRNA for ß-actin as described in Materials and Methods for different periods of time as indicated. The level of ß-actin protein was determined as in (B). (G) The viability of the RNAi-treated L929 cells was measured as in (C) after 48 hours TNF treatment.

View larger version (39K): [in a new window]   Fig. 2. Actinmut cells are selectively resistant to TNF-induced killing. (A) Wild-type and Actinmut cells were treated with indicated doses of TNF for 48 hours, H2O2 for 9 hours, UV, mytomycin for 96 hours, 5-fluorouracil for 65 hours and vincristine for 96 hours. Cell viability was measured. (B) Wild-type and Actinmut cells were treated with TNF (100 ng/ml) and/or zVAD (1 µM or 10 µM) in different combinations for 18 hours. Cell viability was measured. Results represent the means±s.e. (n=3).

View larger version (39K): [in a new window]   Fig. 3. Actin mutation interferes with the TNF-induced death signal but not with TNF-induced NF-B and p38 activation. (A) Wild-type and Actinmut cells were transiently transfected with the GFP expression vector and an expression vector of TNFR1(CD), TRADD, FADD, RIP or TRAF2. The death of the transfected cells (GFP positive) was calculated by counting sixteen random areas, and the differences in cell death were statistically significant (P<0.05, Student's t test). Three separate experiments were carried out with comparable results. (B) Wild-type and Actinmut cells were treated with TNF (100 ng/ml) for different time periods as indicated. IB- and phospho (P)-p38 levels were determined by Western blotting. Equal loading of total cell lyates is shown by p38 protein levels.

View larger version (52K): [in a new window]   Fig. 4. Actin cleavage is not involved in TNF-induced L929 cell death. (A) L929 or HeLa cells were treated with TNF (100 ng/ml) or etoposide (Eto 40 µg/ml) for times as indicated. Actin and the 31 kDa fragment of actin were detected with the anti-actin polyclonal antibody against the C-terminal 11 residues. (B) Stable cell lines were generated from wild-type and Actinmut cells by transfection of an expression vector of flag-tagged 15 kDa ß-actin fragment (Wt+15K and Actinmut+15K). The expression of the 15 kDa fragment was detected with anti-flag antibody M2. (C) The sensitivity to TNF-induced cell death in Wt, Actinmut, Wt+15K and Actinmut+15K was measured with propidium iodide (PI) exclusion. Results represent the means±s.e. (n=3).

View larger version (47K): [in a new window]   Fig. 5. Actin deficiency blocks TNF-induced perinuclear redistribution of mitochondria and ROS production. (A) Mitochondria of wild-type and Actinmut cells were stained with MitoTracker, and the distribution of mitochondria was analyzed under fluorescence microscopy (x100). (B) Wild-type and Actinmut cells were stained with hydroethidine (HE) or dichlorofluorescein-diacetate (DCFH-DA) together with PI at different times of TNF treatment. Levels of ethidium, the oxidation product of HE, or dichlorofluorescein (DCF), the fluorescent product of DCFH-DA, in PI-negative cells (live cells) were determined and shown. Results represent the means±s.e. (n=3).

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