Role of SERCA1 truncated isoform in the proapoptotic calcium transfer from ER to mitochondria during ER stress - PubMed (original) (raw)

Figure 1

Figure 1. S1T Is an ER Stress Protein

(A) Detection of ER stress proteins (phosphorylated alpha-subunit of eukaryotic Initiation Factor 2 [P-eIF2α], GRP78, SERCA2b, Calreticulin [CRT], CHOP, GRP94, and ATF6 p50) in pcDNA3.1 (Control) or S1T-GFP-transfected HeLa cells for 40 hr. (B and C) Expression of endogenous S1T and of ER stress proteins in HeLa cells treated for 20 hr with 5 μg/ml BFA, 10 μg/ml TUN, 0.5 μM TG, 10 μM TBU, or 2 mM EGTA (B), and in hepatitis C virus “replicon” (HCV) and in control cell lines. (C) HCV core antibody was used as control. (D and E) Detection of S1T and of ER stress proteins in HeLa cells treated or not treated with 0.5 μg/ml BFA for 20 hr (D) and in HCV “replicon” cells (E) transfected or not transfected with S1TRNAi or ControlRNAi. S1T, S1T-GFP, and SERCA2b were detected on microsomal fraction in (A) and (C) and in total extracts in (B), (D), and (E). All the other proteins were detected on total extracts. GFP antibody was used to detect S1T-GFP protein.

Figure 2

Figure 2. S1T Is Induced through the PERK-eIF2α-ATF4 Pathway

All the experiments were performed in HeLa cells treated or not treated with 0.5 μg/ml BFA. (A) Induction of S1T and of ER stress proteins in cells treated for the indicated time points. Graphs: time course induction. Where indicated (asterisk), traces are referring to right scale. (B) (Upper panel) Alignment of ATF/Cre-responsive element found in SERCA1, GRP78, and Growth Arrest and DNA Damage-inducible protein 34 (GADD34) genes. (Lower panel) Alignment of CHOP-responsive element found in SERCA1, carbonic anyhdrase VI (CA VI), tribbles-related protein 3 (TRB3), caseinolytic serine protease (ClpP), mitochondrial heat shock protein DnaJ (mtDnaJ), and chaperonins 60/10 (Cpn 60/10) genes. The base positions of the consensus are shaded and indicated 5′→3′. Positions are relative to transcription start site or where indicated (asterisk) to initiation codon. (C) Electrophoresis mobility shift assay with ATF/Cre and CHOP probes derived from the SERCA1, the GADD34, and the Cpn60/10 promoters performed on nuclear extracts from cells left nontreated (NT) or treated with BFA for 40 min (for ATF/Cre probes) or 20 hr (for CHOP probes). The shifted ATF4 and CHOP complexes are indicated by arrows a, a′, and a″ and b, b′, b″, respectively. (D) Oligonucleotide pull-down assay with biotin-labeled probes used in (C), and western blot analyses of DNA-protein complexes in nuclear extracts (input) and on pull-down complexes (PD). (E) Detection of PERK, P-eIF2α, and S1T in HeLa cells treated or not treated with 0.5 μg/ml BFA for 1 hr transfected or not transfected with ControlRNAi or PERKRNAi. In (A) and (E), total extracts were used for western blot analyses.

Figure 3

Figure 3. S1T Mediates Ca2+ Deregulation Related to ER Stress

In (A), (C), and (D), [Ca2+]er was measured using erAEQ probe 40 hr posttransfection. (A and C) Steady-state [Ca2+]er (presented as a percentage of control considered as 100%) in nontreated HeLa cells (Control), 0.5 μg/ml BFA, 1 μg/ml TUN, 2.5 nM TG-treated cells for 20 hr, in S1T-pcDNA3.1-transfected cells for 40 hr (S1T) (A) (Student's t test, *p < 0.05, **p < 0.005 versus Control) and in nontreated HeLa cells (Control), in BFA-treated cells for 20 min or 2 hr (Student's t test, ns = nonsignificant, *p < 0.005 versus Control) (C). (B) Expression of S1T in HeLa cells treated with 0.5 μg/ml BFA for the indicated time points. (D) Representative traces of [Ca2+]er in nontreated HeLa cells (Control) and in 0.5 μg/ml BFA, 10 μM TBU, or 2.5 nM TG-treated cells for 20 hr transfected or not transfected with S1TRNAi. The effect of S1TRNAi was recorded in 40% of aequorintransfected cells and thus underestimated (see the Experimental Procedures). Data in (A), (C), and (D) are represented as mean ± SEM. Numbers of experiments are indicated in Table S1.

Figure 4

Figure 4. S1T Is Localized to Mitochondria-Associated Membranes and Determines Mitochondrial Immobilization and Docking at the ER Surface

(A) Subcellular fractionation. Tot, total; mito, crude mitochondria; Mic, microsomes; MAM, mitochondrial-associated membranes. All endogenous proteins except S1T-GFP were detected in nontransfected HeLa cells. S1T and GFP antibodies were used to detect endogenous S1T and S1T-GFP 40 hr posttransfection, respectively. SERCA2b, VDAC, and cytochrome c oxydase (COX) were used as microsomal, MAM, and mitochondrial fractions markers, respectively. (B) Quantification of colocalization of S1T with mitochondria (labeled with MitoTracker Deep Red) in living HeLa cells transfected with S1T-GFP (S1T-GFP, n = 20) or with erGFP probe (Control, n = 10). Representative magnified image shows S1T-GFP signal in green, mitochondria in red, and colocalization in white. Student's t test, *p < 0.0005 versus Control. (C) Mitochondrial movements analyzes in HeLa cells treated with 0.5 μg/ml BFA (3 hr) transfected or not transfected with S1TRNAi-fluorescein or transfected with mtGFP or S1T-GFP. In S1TRNAi conditions, quantification was performed exclusively in S1TRNAi-fluorescein-positive cells. Mitochondrial movements was evaluated before and after 100 μM histamine stimulation (used as control of ER Ca2+ release-related mitochondrial immobilization) or upon BAPTA-AM (5 μM, 20 hr) application. Student's t test, ns‡ = nonsignificant, ‡p < 0.005 versus nontreated cells, Δp < 0.005 versus BFA-treated cells, ns* = nonsignificant, *p < 0.05, and **p < 0.0005 versus mtGFP-transfected cells, #p < 0.0005 versus S1T-transfected cells. Numbers of experiments are indicated in Table S3. (D) Graph: quantitative analysis of the colocalization of ER and mitochondria (as percentage of total mitochondrial volume) in HeLa cells cotransfected with erGFP and S1T-pcDNA3.1 (S1T 8 hr, n = 16; S1T 40 hr, n = 11) or SERCA1-pcDNA3.1 (SERCA1, n = 9) or in cells transfected with erGFP left untreated (Control, n = 30) or treated with 5 μM BAPTA-AM for 3 hr (Control + BAPTA-AM, n = 17) or with 0.5 μg/ml BFA (BFA, n = 19), or with BFA and BAPTA-AM (BFA + BAPTA-AM, n = 8). Representative images of ER (erGFP, green), mitochondria (MitoTracker Deep Red, red) and magnified overlay images showing colocalization between ER and mitochondria (white). Student's t test, ns = nonsignificant, *p < 0.005, **p < 0.0005 versus Control, and #p < 0.05 versus BFA. (E) Representative fields of electron micrographs and ER-mitochondria distance (graph) in HeLa cells transfected for 40 hr with pcDNA3.1, S1T-pcDNA3.1, or S1T-GFP (Control, n = 31; S1T, n = 46; S1T-GFP, n = 44). M, mitochondria; ER, endoplasmic reticulum. Arrows indicate proximity of ER to mitochondria. Student's t test, *p < 0.0005 versus Control. Data in (B)-(E) are represented as mean ± SEM.

Figure 5

Figure 5. S1T Overexpression Enhances Basal and Agonist-Evoked Mitochondrial Ca2+ Load, Leading to Mitochondrial Structure Alteration

(A) Mitochondrial basal Ca2+ level as evaluated with X-rhod-1, AM dye in HeLa cells transfected with S1T-pcDNA3.1 (S1T) for 8 hr and 40 hr or in cells treated with 0.5 μg/ml BFA for 20 hr and transfected or not transfected with S1TRNAi. Calibrated values of basal mitochondrial Ca2+ load are presented as percentages of controls (pcDNA3.1-transfected or nontreated cells) considered as 100%. The effect of S1TRNAi was recorded in S1TRNAi-positive cells (fluorescein-positive cells). Values of basal mitochondrial Ca2+ level are provided in μM in Table S4 and were determined as described in the Supplemental Experimental Procedures. Student's t test, *p < 0.01, **p < 0.0001 versus Control, #p < 0.005 versus BFA. (B) Mitochondrial agonist-evoked Ca2+ uptake (peak value) after 100 μM histamine application, as measured with mitAEQmut probe, in HeLa cells transfected or treated as in (A), and presented as percentage of controls considered as 100%. The effect of S1TRNAi was recorded in 40% of aequorin-transfected cells and thus underestimated. Student's t test, *p < 0.005, **p < 0.0001 versus Control, #p < 0.005 versus BFA. (C) Percentage of cells with tubular mitochondrial network and those with altered mitochondrial network in HeLa cells (see representative images) cotransfected with mitBFP and pcDNA3.1 (Control, n = 58) or S1T-GFP (S1T 8 hr, n = 83; S1T 40 hr, n = 105) or SERCA1-GFP (SERCA1, n = 50) and in Control and S1T cells (40 hr) treated with 5 μM BAPTA-AM for 20 hr posttransfection (n = 64 and n = 74, respectively). (D) Relative fluorescence intensity changes (dF/F) following 100 μM histamine stimulation in HeLa cells 40 hr after cotransfection of mitochondrial pericam probe and pcDNA3.1 (Control) or S1T-pcDNA3.1 [S1T(T) with tubular-like mitochondrial network and S1T(A) with altered mitochondrial network]. Table: representative images of the mitochondrial network of S1T (T) and (A) cells, percentage of each population, and maximal rate of mitochondrial Ca2+ uptake evaluated from the ascending phase of dF/F kinetics versus Control considered as 100%. The Control trace shows the mean ± SEM of cells with tubular versus altered mitochondrial network (Table S5). Data in (A)-(D) are represented as mean ± SEM. For (A), (B), and (D), numbers of experiments are indicated in Tables S4 and S5.

Figure 6

Figure 6. S1T Alters Mitochondrial Structure and Function

All these analyses were performed in HeLa cells 40 hr posttransfection. (A) Representative high-magnification 3D-reconstructed images of cells transfected as described in Figure 4D. Mitochondrial and ER volume were evaluated (as compared to Control cells considered as 100%) (Control, n = 18; S1T, n = 18; SERCA1, n = 10). (B) Representative fields of electron micrographs of cells transfected with pcDNA3.1 (Control, n = 109) or with S1T-pcDNA3.1 (S1T, n = 110). M, mitochondria. Graph: mean of mitochondria cross-sectional area. (C) Mitochondrial potential change was evaluated after transfection of GFP empty vector (Control, n = 326), S1T-GFP (S1T, n = 155), or SERCA1-GFP (SERCA1, n = 201). (D) Representative images and graph showing cytochrome c fluorescence intensity in cells transfected with pcDNA3.1 (Control, n = 262) or S1T-GFP (S1T, n = 189). (E) Western blot analyses of cells transfected with pcDNA3.1 or with S1T-pcDNA3.1 and treated or not treated with 100 μM ZVAD-fmk for 20 hr. As positive control, cells were treated with 1 μM staurosporine (STS) for 2 hr. (F) Cellular viability in S1T-GFP or SERCA1-GFP-overexpressing cells treated (20 hr posttransfection) or not treated with 12 μg/ml cyclosporine A (CsA), 5 μM BAPTA-AM, and 100 μM ZVAD-fmk, presented as percentages of GFP-fluorescent cells among the whole cell population (determined by Hoechst staining) versus the nontreated transfected cells considered as 100%. Student's t test, ns = nonsignificant, *p < 0.05, and **p < 0.0005 versus Control. Data are represented as mean ± SEM.

Figure 7

Figure 7. S1T Knockdown Reduces BFA-Induced Apoptosis

All the experiments were performed in HeLa cells left untreated or treated with 0.5 μg/ml BFA for 20 hr and transfected or not transfected with S1TRNAi. (A) Percentage of cells with tubular or altered mitochondrial network in Control (n = 428), BFA (n = 293), and BFA + S1TRNAi (n = 197) cells. (B) Mitochondrial volume recorded as in Figure 6A in Control (n = 19), in BFA (n = 24), and in BFA cells cotreated with 5 μM BAPTA-AM (BFA + BAPTAAM; n = 12). (C) Quantification of cytochrome c fluorescence intensity evaluated as in Figure 6D in Control (n = 262), in BFA (n = 335), and in BFA + S1TRNAi (n = 242) cells. (D) Percentage of cells with activated Bax (intense mitochondrial staining in representative images) in Control (n = 747), BFA (n = 596), and BFA + S1TRNAi cells (n = 300). (E) Western blot analyses performed as in Figure 6E. (F) Percentage of apoptotic bodies in nontreated cells (Control) or treated for 20 hr with 0.5 μg/ml BFA, 5 μg/ml TUN, 0.5 μM TG, and 10 μM TBU after being transfected or not transfected with S1TRNAi. Inset: Percentage of necrotic cells (propidium-iodide-positive cells) recorded in the same conditions. (A, E, and F) Effect of S1TRNAi is recorded in total cell population and thus underestimated. (C and D) Quantification was performed exclusively in S1TRNAi-fluorescein-positive cells. (B-D and F) Student's t test, ns = nonsignificant, *p < 0.0001 versus Control, #p < 0.0001 versus BFA, TUN, TG, or TBU. Data are represented as mean ± SEM.