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First published online 6 March 2007
doi: 10.1242/jcs.03417

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Interactions of DNR1 with the apoptotic machinery of Drosophila melanogaster

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB, T6G 2S2, Canada

View larger version (33K): [in this window] [in a new window]   Fig. 1. Dronc and Drice are essential for apoptosis in S2 cells. (A,B) DIC images of control S2 cells (A) and S2 cells treated with actinomycin D for 6 hours (B). Apoptotic cells undergoing membrane blebbing, a characteristic of apoptosis, are circled in (B). (C) Quantification of the apoptotic index of control S2 cells (column 1) and S2 cells incubated with actinomycin D for 6 hours (column 2). Results are the mean of three independent experiments and error bars indicate standard errors. (D) Time course of caspase activity towards peptide substrates in lysates from S2 cells treated with actinomycin D, which induces a robust increase in DEVDase and YEVDase activity. (E) Real-time PCR quantification of dcp-1 (columns 1 and 2) and decay (columns 3 and 4) transcript levels in control S2 cells (columns 1 and 3) compared with cells incubated for 72 hours with the respective dsRNA (columns 2 and 4). Dcp-1 dsRNA has no effect on dcp-1 transcript levels whereas Decay dsRNA reduces decay transcript levels by 72%. Results are the mean of three independent measurements and error bars indicate standard errors. (F) DEVDase activity of lysates from actinomycin-D-treated (6 hours) control S2 cells and S2 cells incubated with dsRNA targeting the indicated caspases. DEVDase activity is expressed as a fold-change compared with lysates from the corresponding cell population not treated with actinomycin D. Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.01, values that differ significantly from untreated cells. (G) YEVDase activity of lysates from actinomycin-D-treated control S2 cells and S2 cells incubated with dsRNA targeting the indicated caspases. YEVDase activity is expressed as a fold-change compared with lysates from the corresponding cell population not treated with actinomycin D. *P<0.01, values that differ significantly from untreated cells. Results are the mean of three independent experiments and error bars indicate standard errors. RNAi of Dronc or Drice significantly reduces DEVDase and YEVDase activity compared with control S2 cells. (H) Apoptotic indices of actinomycin-D-treated control S2 cells and S2 cells incubated with dsRNA targeting the indicated caspases. Dronc and Drice dsRNA block the onset of actinomycin-D-mediated apoptosis. Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.01, values that differ significantly from untreated cells.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Dnr1 affects Dronc protein levels. (A) Real-time PCR measurement of dnr1 transcript levels in control S2 cells (column 1) and S2 cells incubated with Dnr1 dsRNA for 72 hours (column 2). dnr1 transcript levels were normalized to actin transcript levels in both samples. Dnr1 dsRNA decreases dnr1 transcript levels by greater than 95% within three days. (B) Western blot analysis of Dronc protein in lysates from control S2 cells (lanes 2-5) and S2 cells incubated with Dnr1 dsRNA (lanes 7-10). The cells were treated with actinomycin D and lysates harvested at the indicated periods. Molecular mass markers are shown in lanes 1 and 6, actin levels are shown as loading controls (lower panel) and the full-length (FL), Pr2 and L Dronc isoforms are indicated. (C-E) Quantification of the (C) full-length (FL), (D) Pr2 and (E) large (L) forms of Dronc in control S2 cells or S2 cells treated with Dnr1 dsRNA. Dronc protein levels are normalized relative to actin protein levels. The Dronc:actin levels in control S2 cells at 0 hours actinomycin D treatment were assigned a value of 1 in (C) and the remaining Dronc:actin values are reported relative to this value. We detected a significant increase in full-length Dronc protein levels in S2 cells incubated with Dnr1 dsRNA compared with control cells in the absence of induction. Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.03, values that differ significantly from untreated cells. We also detected elevated levels of the Pr2 and large isoforms of Dronc in cells treated with Dnr1 dsRNA (D and E, respectively). Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.01, values that differ significantly from untreated cells. (F) Time course of induction of HADnr1 or HADnr1C563Y. HADnr1 and HADnr1C563Y levels are normalized to actin levels. (G) Levels of full-lenght Dronc at various times after induction of HADnr1 or HADnr1C563Y. Full-lenght Dronc levels are normalized to actin levels in both samples.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Dnr1 RNAi enhances caspase activation and activity by actinomycin D in S2 cells. (A) Western blot analysis of lysates from control S2 cells and S2 cells incubated with actinomycin D using an antibody that detects active caspase-3 (upper panel). Both treatments were performed in duplicate. Actin levels are shown as a loading control (lower panel). Actinomycin D treatment results in the appearance of an active caspase-3 signal by 6 hours. (B) Western blot analysis of lysates from control S2 cells (lanes 1-2) decay dsRNA-treated cells (lanes 3-4), Dredd dsRNA-treated cells (lanes 5-6), Drice dsRNA-treated cells (lanes 7-8) and Dronc dsRNA-treated cells (lanes 9-10). Where indicated, cells had been incubated with actinomycin D for 6 hours. Lysates were probed with an active-caspase 3 antibody. An active caspase-3 signal is detected in S2 cells, as well as Dredd and Decay dsRNA-treated cells, whereas depletion of Drice or Dronc abrogates the signal. A crossreacting band is indicated by an asterisk. (C) Time course of caspase activation by actinomycin D in lysates from S2 cells (first panel) and lysates from S2 cells treated with Dnr1 dsRNA (third panel). Actin levels are shown as a loading control for both samples (second and fourth panel, respectively). Dnr1 RNAi results in an earlier and stronger appearance of the active caspase isoform at all times measured. (D,E) Time courses of DEVDase (D) and YEVDase (E) activity from actinomycin-D-treated lysates from control S2 cells and Dnr1 RNAi treated S2 cells. Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.01, values that differ significantly from untreated cells. Dnr1 depletion results in a significant increase of caspase activity in response to actinomycin D.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Dnr1 depletion sensitizes cells to induction of apoptosis. Control S2 cells, GFP dsRNA-treated S2 cells or Dnr1 dsRNA-treated S2 cells were incubated with 0.01, 0.1 or 1 µM actinomycin D (A-C, respectively) and the apoptotic index of the different cell populations were determined as indicated. Results are the mean of three independent experiments and error bars indicate standard errors. **P<0.01, values that differ significantly from Dnr1 dsRNA-treated cells; *P<0.05, values that differ significantly from Dnr1 dsRNA-treated cells. Dnr1 RNAi results in an elevated apoptotic index of actinomycin-D-treated S2 cells compared with control S2 cells or GFP dsRNA-treated S2 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Overexpression of Dnr1 blocks caspase activity and apoptosis in S2 cells. (A) Anti-HA western blot analysis of lysates from control S2 cells, S2 cells expressing HADnr1 and S2 cells expressing HADnr1C563Y treated with actinomycin D as indicated. Equal amount of lysate was loaded in each lane. The levels of HADnr1C563Y are considerably higher than HADnr1, consistent with a role for the RING domain in regulating Dnr1 stability. (B) Caspase activity assays in lysates from S2 cell, S2 cells treated with actinomycin D, HADnr1-expressing cells treated with actinomycin D and HADnr1C563Y-expressing cells treated with actinomycin D. HADnr1 expression blocks actinomycin-D-induced caspase activity, whereas the RING-domain-inactive version fails to impact on caspase activity. (C) Quantification of the apoptotic index of S2 cells, HADnr1-expressing S2 cells and HADnr1C563Y expressing S2 cells before (columns 1-3) or after exposure to actinomycin D (columns 4-6). Results are the mean of three independent experiments and error bars indicate standard errors. (D) Quantification of the apoptotic index of S2 cells, HADnr1-expressing S2 cells and HADnr1C563Y-expressing S2 cells before (columns 1-3) or after exposure to ultra-violet irradiation (columns 4-6) or cycloheximide (columns 7-9). Results are the mean of three independent experiments and error bars indicate standard errors.

View larger version (128K): [in this window] [in a new window]   Fig. 6. (A-I) Time-lapse imaging of apoptotic progression in control S2 cells (A-C), HADnr1-expressing S2 cells (D-F) and HADnr1C563Y-expressing S2 cells (G-I). All cell lines were treated with dIAP1 dsRNA and imaged at the indicated time. Whereas DIAP1 depletion induces rapid apoptosis in control S2 cells, overexpression of Dnr1 or Dnr1C563Y suppresses the loss of DIAP1 phenotype.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Dnr1-dependent destruction of Dronc is pro-domain independent. (A) Western blot analysis of lysates from control S2 cells (lanes 1 and 2), and S2 cells constitutively expressing HADnr1 or HADnr1C563Y (lanes 3 or 4, respectively). The individual lines were transfected with a plasmid that drives expression of 6xmycDronc where indicated. Lysates were probed with anti-myc (red) and anti-actin antibodies (green). Three distinct Dronc isoforms were detected corresponding to the full-length (FL), D135 and D113 isoforms. Each isoform is indicated by an arrow. (B) Serial dilutions of lysates from S2 cells, HADnr1-expressing cells and HADnr1C563Y-expressing cells transfected with a 6xmycDronc expressing plasmid were analyzed by western blot analysis. The levels of the individual Dronc isoforms were quantified and normalized to the level of a control protein (actin). Results are the mean of three independent experiments and error bars indicate standard errors. The level of each Dronc isoform is significantly reduced in HADnr1-expressing cells compared with S2 cells, whereas HADnr1C563Y expression fails to impact on Dronc protein levels. (C) Equal amounts of a plasmid that drives expression of 6xmycDroncPro was transfected into S2 cells, HADnr1-expressing cells and HADnr1C563Y-expressing cells as indicated. The myc tag was visualized by western blot analysis (upper panel) and quantified (lower panel) relative to the levels of a control protein (actin, middle panel). Overexpression of Dnr1 decreases the levels of DroncPro, whereas the C563Y variant of Dnr1 has no significant impact on DroncPro levels. Results are the mean of three independent experiments and error bars indicate standard errors.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Identifying functional domains within Dnr1. (A) Schematic representation of Dnr1, showing the FERM domain (purple), FERM_C motif (hatched) and RING domain (yellow). The indicated series of GFP-tagged expression constructs were prepared. (B) Anti-GFP western blot analysis of lysates from control S2 cells and a series of stable S2 cell lines expressing GFP-tagged versions of the indicated Dnr1 expression constructs. Molecular mass markers are indicated. A cross-reacting signal is indicated with an asterisk. (C) The individual stable lines were transfected with equal amounts of a plasmid that drives constitutive expression of 6xmycDronc and the ratio of full-length Dronc (C) to actin were quantified for the respective lines. Dnr1324-677 and Dnr1397-677-expressing cells diminished Dronc levels to a similar extent as wild-type Dnr1. Results are the mean of three independent measurements and error bars indicate standard errors. (D) Anti-GFP western blot of lysates from control S2 cells and S2 cells transfected with equal amounts of plasmids that drive expression of GFP-tagged versions of Dnr1, Dnr1324-677 and Dnr1397-677 as indicated. Molecular mass markers are indicated and actin levels are shown as loading controls. (E-O) Confocal images of S2 cells transfected with GFPDnr1142-677 (E-L) and GFPDnr1324-677 (M-O). In the merged images of panels H and O GFP is shown in green, DNA is visualized in blue and filamentous actin is labeled in red. In the merged image of panel L, GFP is shown in green, DNA is visualized in blue and the nuclear envelope is visualized in red with Alexa Fluor-568-labeled wheat germ agglutinin. Whereas GFPDnr1324-677 is cytoplasmic GFPDnr1142-677 is a nuclear protein, indicating the existence of an NLS between residues 142 and 324. Panels E-H and M-O are shown at 100x magnification and panels I-L are shown at 200x magnification. (P) Primary sequence of residues 150-193 in Dnr1. Three potential NLS are indicated, two are boxed blue or red boxes, a putative bipartite NLS is boxed green.