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First published online 26 October 2004
doi: 10.1242/jcs.01499

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Translocation of the Dictyostelium TRAP1 homologue to mitochondria induces a novel prestarvation response

Tsuyoshi Morita^*, Aiko Amagai and Yasuo Maeda

Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan

View larger version (35K): [in a new window]   Fig. 1. Translocation of Dd-TRAP1 from the cell cortex to mitochondria is induced by prestarvation factor(s). (A) Change in subcellular localization of Dd-TRAP1 due to increased cell density. Ax-2 cells grown at the indicated cell density were fixed and immunostained with the Dd-TRAP1 antibody. Dd-TRAP1 is translocated from the cell cortex to cytoplasmic granules as the density of growth-phase cells increases. Bar, 5 µm. (B) Ax-2 cells grown at 4x106 cells/ml were incubated with 1 µM MitoTracker for 30 minutes and fixed for immunostaining with the Dd-TRAP1 antibody. The merged image indicates that Dd-TRAP1 is localized in mitochondria at a high cell density. Bar, 5 µm. (C) Effect of CGM (conditioned growth medium) on the Dd-TRAP1 translocation. CGM was prepared from Ax-2 cells that had been grown to a high cell density (1.5x107 cells/ml). Cells grown at a low cell density (3-5x105 cells/ml) were allowed to adhere to coverslips, and incubated in the CGM. After the indicated time of incubation, the cells were fixed and immunostained with the Dd-TRAP1 antibody. Bar, 5 µm. (D) Cells grown at high cell density (8x106 cells/ml) were harvested and transferred to fresh growth medium at low cell density (5x105 cells/ml). After 30 minutes of incubation, cells were fixed and immunostained with the Dd-TRAP1 antibody. Compared with cells before transfer to CGM (a), it is evident that Dd-TRAP1 goes back to the cell cortex after 30 minutes of incubation in fresh growth medium (b). Bar, 5 µm.

View larger version (24K): [in a new window]   Fig. 2. RNAi-mediated conditional knockdown of Dd-TRAP1. (A) Two PCR products (756 bp and 1198 bp) were ligated tail to tail, and inserted into the MB38 vector. The 756 bp inverted repeat (dark bar) and the 442 bp linker sequence (white bar) form a stem-loop RNA (dsRNA). (B) Growth kinetics of TRAP1-RNAi cells and parental MB35 cells. TRAP1-RNAi cells were cultured with 20 µg/ml of tetracycline () or without (), and cell-numbers were counted every 24 hours. As a control, the parental MB35 cells were also cultured without tetracycline (). At the exponential growth phase, when cell density reached over 5x106 cells/ml, cells were diluted with fresh growth medium to a density of 1x105 cells/ml. Total proteins were extracted every 24 hours for 7 days from these cultures, and western analyses were performed using the anti-Dd-TRAP1, anti-Dd-GRP94 and anti-Hsc90 antibodies.

View larger version (78K): [in a new window]   Fig. 3. TRAP1-RNAi cells showing a significant defect in prestarvation response. (A) TRAP1-RNAi cells cultured for 2 days in the absence of tetracycline, and parental MB35 cells were separately harvested, starved and plated in 24-well plates at 5x105 cells/cm2. This was followed by incubation for the indicated times at 22°C. Bars, 200 µm. (B) Expression patterns of prestarvation genes discoidin I, car1, dia1 and dia3 in TRAP1-RNAi cells and MB35 cells. TRAP1-RNAi cells cultured for 2 days in the absence of tetracycline, and MB35 cells were separately harvested at the indicated cell density or stationary growth phase (1.5-2.0x107 cells/ml, S), followed by extraction of total RNAs. The RNAs (40 µg for each) were size-fractionated and analyzed by northern blots using the cDNA probe of each gene. (C) TRAP1-RNAi cells cultured axenically in the absence of tetracycline for 2 days, and parental MB35 cells were spotted on the bacterial lawn, and incubated for 2 days at 22°C to allow them to develop. Bar, 1 mm (top panels), 500 µm (lower panels).

View larger version (84K): [in a new window]   Fig. 4. Early prestarvation response observed in TRAP1OE cells. (A) The localization of Dd-TRAP1 in growing TRAP1OE cells. Vegetatively growing TRAP1OE cells and parental Ax-2 cells were harvested at a density of 0.5-1.0x106 cells/ml and fixed for immunostaining with the Dd-TRAP1 antibody. Bar, 5 µm. (B) Expression patterns of early developmental genes discoidin I, car1, dia1 and dia3 in TRAP1OE cells and parental Ax-2 cells. TRAP1OE cells and parental Ax-2 (clone MS) cells were separately harvested at the indicated cell density of exponential growth phase (0.25-3x106 cells/ml), followed by extraction of total RNAs to detect the expression of discoidin I (upper two panels). In another experiment, cells were harvested at the exponential growth phase (1-5x106 cells/ml) or stationary growth phase (1.5-2.0x107 cells/ml, S), followed by extraction of total RNAs. The RNAs (40 µg for each) were size-fractionated and analyzed by northern blots using the cDNA probe of each gene. (C) TRAP1OE cells assemble into 3D aggregates, even in the presence of nutrients. Bar, 100 µm. (D) Early acquisition of EDTA-resistant adhesion in TRAP1OE cells. Ax-2 and TRAP1OE cells were separately starved and shaken gently in 16 mM phosphate buffer (pH 6.4) at a density of 1x107 cells/ml. After 3 hours of shaking, 10 mM EDTA was added to prevent the EDTA-sensitive cell-cell adhesion. Under this condition, only TRAP1OE cells form many aggregates. Bar, 100 µm.

View larger version (21K): [in a new window]   Fig. 5. A novel prestarvation response. (A) Gß-null and PKAcat-null cells grown at 3-5x106 cells/ml were fixed and immunostained with the Dd-TRAP1 antibody. In both transformants, Dd-TRAP1 is translocated to mitochondria, thus indicating that neither heterotrimeric G proteins nor PKA are required for the Dd-TRAP1 translocation. Bar, 5 µm (B) CGM was prepared from Ax-2 cells that had been grown up to a high cell density (8x106 cells/ml). As control cells, growth-phase Ax-2 cells at a low cell density (5x105 cells/ml) were harvested and resuspended in fresh growth medium (FGM) or CGM at 5x105 cells/ml. After 3 hours of incubation, cells were fixed and immunostained with the Dd-TRAP1 antibody. In another experiment, cells were incubated in the CGM, either pretreated with protease (30 µg/ml of pronase E) (CGM/protease) or boiled for 15 minutes (+CGM/heat). Bar, 5 µm.

View larger version (84K): [in a new window]   Fig. 6. Delayed differentiation of TRAP1OE cells and TRAP1OE/dia1– cells. (A) TRAP1OE and parental Ax-2 cells grown at 1-2x106 cells/ml were separately harvested, starved and plated either in a 24-well plate at 5x105 cells/cm2 (a-d) or on non-nutrient agar at 2.5x105 cells/cm2 (e-h). This was followed by incubation for the indicated times at 22°C. Bars, 200 µm (a-d); 500 µm (e-h). (B) Expression patterns of car1, dia3, lagC, dia1 and csA in starved TRAP1OE cells. At the indicated times of starvation, total RNAs were extracted from TRAP1OE and parental Ax-2 cells. The RNAs (20 µg for each) were size-fractionated and analyzed by northern blots using the cDNA probe of each gene. In starved TRAP1OE cells, dia1 and csA genes are expressed early, while the expression patterns of car1, dia3 and lagC genes are rather delayed. (C) dia1– and TRAP1OE/dia1– cells grown at 1-2x106 cells/ml were separately harvested, starved and plated either in a 24-well plate at 5x105 cells/cm2 (a-d) or on non-nutrient agar at 2.5x105 cells/cm2 (e-h). This was followed by incubation for the indicated times at 22°C. Bars, 200 µm (a-d); 500 µm (e-h).

View larger version (45K): [in a new window]   Fig. 7. Partial recovery of the delayed differentiation observed in TRAP1OE cells. (A) TRAP1OE cells grown at 1-3x106 cells/ml were harvested, and shaken in BSS for 6 hours with or without 50 nM cAMP pulses externally applied at 6-minute intervals, followed by incubation in 24-well plates. The incubation times after starvation are shown. Bar, 150 µm. (B) Effect of exogenously applied cAMP pulses on the induction of car1 expression in TRAP1OE cells. Total RNAs were extracted from TRAP1OE and Ax-2 cells shaken with (+) or without (–) cAMP pulses for the indicated times. Northern blots were performed using the cDNA probe of car1 gene. (C) TRAP1OE cells grown up to a high cell density (>1x107 cells/ml) exhibit no delay of differentiation. TRAP1OE and Ax-2 cells grown at the indicated cell densities were harvested, starved and developed under submerged conditions. These cells acquired aggregation competence after the indicated times of starvation, assuming elongated cell shape. The results of two independent experiments are presented. (D) The localization of Dd-TRAP1 in TRAP1OE and Ax-2 cells. Ax-2 cells (a) and TRAP1OE cells (b) grown at 1-2x106 cells/ml were separately harvested and shaken in BSS for 6 hours, followed by immunostaining with the Dd-TRAP1 antibody. A significant number of Dd-TRAP1 molecules were found to be retained in the cell cortex of TRAP1OE cells (arrowhead). By contrast, when TRAP1OE cells were harvested at the early stationary phase (>1x107 cells/ml) and starved for 6 hours, most of Dd-TRAP1 were translocated to mitochondria (c). Bar, 5 µm.

View larger version (85K): [in a new window]   Fig. 8. Loss of Dd-TRAP1 functions by introduction of the Dd-TRAP1 antibody into cells. (A) To introduce the Dd-TRAP1 antibody into Ax-2 cells, cells grown at 1x106 cells/ml were harvested and electroporated with the antiserum raised against Dd-TRAP1 (anti-TRAP1 serum). As controls, cells were also electroporated with non-immune serum (non-immune serum) or without any serum (control). After electroporation, cells were incubated in BSS for the indicated times to allow differentiation. Bar, 250 µm. (B) Ax-2 cells grown at a low density (1x106 cells/ml) were harvested and electroporated with or without the anti-TRAP1 serum. This was followed by incubation in BSS for 6 hours at 22°C and subsequent immunostaining with the Dd-TRAP1 antibody. Arrowheads indicate the Dd-TRAP1 remained in the cell cortex. Bar, 5 µm. (C) Ax-2 cells grown at a high density (1-1.5x107 cells/ml) were harvested and electroporated with or without the anti-TRAP1 serum. This was followed by incubation in BSS for the indicated times under submerged conditions. Bar, 250 µm.