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First published online 17 August 2004
doi: 10.1242/jcs.01356

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Regulation of cytoplasmic stress granules by apoptosis-inducing factor

¹ CNRS-UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif, France
² CNRS-UMR5535, Université de Montpellier II, 34293 Montpellier CEDEX 5, France
³ INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France

View larger version (61K): [in a new window]   Fig. 1. AIF deficiency enhances the formation of cytoplasmic SGs in response to HAsO4. (A) Failure of AIF to modulate the expression of inducible HSP70 or HSP27. HeLa cells were subjected to siRNA specific for human AIF (siRNA AIF) or mouse AIF (siRNA Co) for 48 hours, followed by culture in the absence (Co) or presence of HAsO4 (3 hours, 1 mM), extraction of total cellular proteins and immunoblot detection of AIF, HSP70, HSP27 and GAPDH. (B) Abundance of cytoplasmic SGs. Cells treated as in A were stained for the detection of AIF (green fluorescence), the SG protein TIA-1 (red fluorescence) or chromatin (blue fluorescence). Note the accumulation of TIA-1+ cytoplasmic granules in HAsO4-treated, AIF-negative cells. The histogram illustrates the percentage (±s.d., n=5) of cells accumulating SGs in the cytoplasm. (C) Failure of AIF to stimulate the generation of nuclear SGs. Cells treated as in A were stained with antibodies specific for constituents of nuclear SGs (HSF1, red) and cytoplasmic SGs (TIA-1, green). As a positive control for the generation of nuclear SGs, cells were treated for 18 (instead of 3) hours with HAsO4. Note that cytoplasmic SGs form in cells that lack nuclear SGs. Results are representative of five independent experiments yielding similar results.

View larger version (55K): [in a new window]   Fig. 2. Accumulation of cytoplasmic SGs in a variety of experimental conditions. (A) Effect of genetic invalidation of the AIF gene. Male control ES cells or cells in which the AIF gene (on the X chromosome) has been invalidated (AIF–/y) were subjected to HAsO4 treatment (1 mM, 3 hours), followed by fixation, permeabilization and immunostaining for the detection of TIA-1 (red) and AIF (green, shown in the insets). (B) Redistribution of TIA-1-GFP into SGs in AIF-negative cells. HeLa cells treated with siRNA for the downmodulation of AIF (or controls) for 48 hours and then transfected with TIA-1-GFP were treated with HAsO4 during the last 3 hours of the experiment and then subjected to fixation and staining for AIF (red). (C) Redistribution of PABP-GFP into SGs in AIF-negative cells. The protocol was similar to Fig. 2B, with the difference that cells were transfected with PABP-GFP instead of TIA-1-GFP. Representative cells are depicted in the microphotographs and the percentage (±s.d., n=5) of SG+ cells was plotted in the right panels.

View larger version (48K): [in a new window]   Fig. 3. Formation of cytoplasmic SGs occurs independently from apoptosis. (A,B) SGs form before the m is lost and phoshatidylserine residues are exposed on the cell surface. Cells treated to obtain maximal SG formation (1-3 hours HAsO4, Figs 1, 2) do not manifest a loss of the m, as detectable with the m-sensitive dye DiOC(6)3 (A), nor do they stain with Annexin V (B). (C) SGs form before cytochrome c (Cyt c) is released from mitochondria. After the indicated treatment, HeLa cells were stained for the simultaneous detection of Cyt c (green) and TIA-1 (red). Note that an 18 hour incubation period with HAsO4 is required to detect diffuse or absent Cyt c staining in a fraction of cells. (D,E) Effect of the broad spectrum caspase inhibitor Z-VAD.fmk on SG formation. Cells were treated for the indicated period with HAsO4, in the presence or absence of Z-VAD.fmk and the frequency (±s.d., n=3), of SG+ cells (D) or cells exhibiting nuclear apoptosis (E) was determined by staining with anti-TIA-1 or Hoechst 33342, respectively.

View larger version (31K): [in a new window]   Fig. 4. Increased formation of G3BP-elicited SGs in the absence of AIF. Cells were treated for knock-down of AIF and then transfected with G3BP-GFP fusion constructs for 24 hours. The micrographs exemplify representative staining patterns obtained after transfection with the non-mutated G3BP-GFP construct. The histograms illustrate the quantitative difference in the formation of SG+ cells as a function of AIF expression and as a result of transfections with the mutant G3BP-GFP construct. Results are expressed as the percentage of SG+ cells exhibiting a GFP-dependent fluorescence (±s.d., n=3).

View larger version (40K): [in a new window]   Fig. 5. Functional mapping of the AIF domains regulating SG formation. (A) Repression of SG formation by retransfection with mouse AIF. HeLa cells manipulated to downregulate endogenous (human) AIF (siRNA-AIF) were re-transfected with V5-tagged mouse AIF, either full length (AIF WT) or deleted in its N-terminus (AIF1-100) which contains the mitochondrial localization sequence, together with the SG inducer G3BP-GFP. Twenty-four hours after transfection, the cells were stained with an antibody recognizing the V5 epitope. Note that no SGs are formed upon transfection with wild-type AIF, which accumulates in mitochondria (middle panel), whereas AIF1-100 (which distributes in a diffuse fashion) does not suppress SG formation. (B) Deletion mutations that affect different regions of the AIF molecule. (C,D) Effect of different mouse AIF deletion constructs on SG formation elicited with G3BP (C, done as in A) or HAsO4 (D, incubation for 3 hours with 1 mM HAsO4, 24 hours after re-transfection of AIF into cells). Note that the control for siRNA-AIF (targeting human AIF) used in C and D was a construct designed to downmodulate emerin.

View larger version (51K): [in a new window]   Fig. 6. Effect of AIF on redox metabolism. ES cells positive or negative for AIF (A,B,D,F) or HeLa subjected to AIF knock-down (C,E) were treated with HAsO4 (A,B) or a panel of different pro-oxidants (and as an internal control for GSH depletion with BSO) for 1-3 hours (A,B) or 3 hours (C-F). Then, the level of NAD(P)H was determined by cytofluorometry, based on its autofluorescence (A,C-F). Alternatively, the cells were stained with the GSH-reactive fluorochrome monochlorobimane (MCB, B,C-F). Results are expressed as the specific variation obtained in triplicate determinations (+s.d.) in one experiment representative of three. PQT, paraquat.

View larger version (25K): [in a new window]   Fig. 7. GSH represses SG formation. HeLa cells expressing AIF or not were pretreated with glutathione ethyl ester (GSH) or N-acetyl-L-cysteine (NAC) for 1 hour, before addition of HAsO4, followed by detection of NAD(P)H (by autofluorescence) (A), intracellular GSH levels (with monochlorobimane, B), or the abundance of SGs (with anti-TIA-1, C). Alternatively, cells were treated with GSH or NAC immediately after liposome-mediated tranfection of the SG inducer G3BP-GFP. After 24 hours, SG formation was quantified by assessing the cytoplasmic nucleation of G3BP-GFP.