Figure 1.
Derlin-1 modifies the neurodegeneration associated with the pathogenic TER94 mutants.
Scanning electron micrographs (SEM) of adult eyes (upper rows) and confocal sections of retina (lower rows) stained with phalloidin (red) and anti-Lamin antibody (green). (A) The compound eye of GMR>LacZ exhibits a highly ordered structure composed of approximately 750 facets known as ommatidia. The organization of underlying photoreceptors is revealed by phalloidin, which stains the light-sensing rhabdomeres. Lamin antibody marks the nuclear envelopes. (B) Eye phenotypes from GMR>TER94A229E with RNAi-mediated knockdown of sip3 and derlin-1, and with overexpression of ufd1, sip3, and derlin-1. (C) Eye phenotypes of two additional TER94 disease mutants, GMR>TER94R188Q and GMR>TER94R152H, with or without derlin-1 co-expression. (D) Eye phenotypes from GMR>TER94A229E>derlin-1 with RNAi-mediated knockdown of sip3 and ufd1. All images are collected from 1-day-old adult except GMR>TER94R152H group in (C), which are 18-day-old adult. For the SEM images, anterior is to the left and dorsal is up. Scale bars: 100 µm (SEM), 10 µm (confocal).
Figure 2.
The suppression of TER94A229E by Derlin-1 overexpression requires direct interaction.
(A) Western analysis of lysates and anti-Derlin-1 immunoprecipitates from hs>LacZ; tub-GAL80ts (control, hs-GAL4 in combination with tub-GAL80ts to drive LacZ expression; see Materials and Methods), hs>derlin-1; tub-GAL80ts (derlin-1 expression), derlin-1null, and hs>TER94A229E; tub-GAL80ts (TER94A229E expression). The lysate (input) blot was detected by anti-Derlin-1 and then stripped to re-probe with anti-β-Tubulin for loading control, whereas the IP blot was probed with anti-VCP and anti-Derlin-1 to detect co-IPed TER94 and Derlin-1, respectively. The band corresponding to Ig heavy chains is indicated by asterisk. (B) A schematic diagram of Derlin-1 domains, depicting the N-terminal cytoplasmic segment (denoted N), the six transmembrane domains (beige box), the α-domain (yellow box) and the C-terminal SHP domain (green box). A ClustalW sequence alignment of the putative C-terminal SHP domains from human and fly Derlin homologs. Identical (asterisks) and similar (dots) residues shared by the homologs are denoted. (C) GST pull-down of TER94 from GMR>TER94A229E head extract by Derlin-1 truncations or C-terminally FLAG-tagged Derlin-1. The pull-downed TER94 proteins were detected by immunoblotting with anti-VCP (upper panel) antibodies, and the blot was re-probed by anti-GST antibodies (middle panel) and anti-Derlin-1 antibodies (lower panel). GST alone is included as a control. (D) Pull-down of bacterially expressed His-tagged TER94 truncations by GST-Derlin-1 C-terminal fragment. The blot was probed with anti-Derlin-1 antibodies (input), followed by re-probing with anti-6XHis antibodies. Only full-length His-TER94 and His-TER94N-L1 interact with the Derlin-1 C-terminal fragment. (E) SEM (upper row) and confocal section of retinas (lower row) from 1-day-old adults with indicated transgenes expressed under GMR-GAL4 control. The retinas are stained with phalloidin (red) and anti-Lamin antibody (green) to visualize the rhabdomeres and the nuclear envelopes, respectively. Overexpression Derlin-1, but not Derlin-1ΔSHP, Derlin-2, or GFP-Derlin-1-CT, suppresses pathogenic TER94A229E-induced eye degeneration. Scale bars: 100 µm (SEM), 10 µm (confocal).
Figure 3.
Overexpressing Derlin-1 suppresses the ATPase activity of pathogenic TER94 mutant.
(A, C and D) In-gel ATPase activity assay of indicated transgenes driven by hs-GAL4; tub-GAL80ts. The ATPase activity presented with bands intensity (see Materials and Methods) from the clear-native (CN)-PAGE was normalized by TER94 protein levels in SDS-PAGE. A bracket marks the measured bands for this representative CN-PAGE. Quantification of ATPase activities from flies expressing LacZ control and TER94 transgenes in panel A is shown. Values represent the mean± SE from five independent experiments. **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). (B) Measurement of cellular ATP levels in flies carrying indicated transgenes driven by hs-GAL4. Values represent the means ± SE from six independent experiments (**p<0.01; one-way ANOVA with Bonferroni's multiple comparison test). (E) Measurement of ATPase activities from C and D. Values represent the mean± SE from six independent experiments. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test).
Figure 4.
ER stress increases Derlin-1 expression and promotes the recruitment of TER94 to the ER.
(A) Confocal images of control GMR>LacZ, GMR>TER94K2A, and derlin-1null larval eye discs expressing CD3δ-YFP (green). The eye discs are stained with anti-Elav antibodies (red) to label neuronal nuclei. Expression of TER94K2A serves as a positive control for CD3δ-YFP. The boxed region in the derlin-1null panel is shown at a higher magnification. (B) Quantitative RT-PCR analysis of derlin-1 and bip transcripts from eye discs with (+) and without (−) 5 mM DTT treatment. Results from three independent quantitative RT-PCR experiments, after being normalized to rp49 levels, are shown in fold change (compared to untreated). Values shown represent mean ± SE. *p<0.05; **p<0.01 (Student's t-test). (C) Confocal images of wild-type mid-pupal eyes with and without (−) 5 mM DTT treatment, stained with phalloidin (red) and anti-Derlin-1 (green). (D) Quantitative Western of endogenous Derlin-1 protein levels from flies subjected to 2 hrs cold shock at 0°C. Lysates from wild-type flies (con) and those recovered after the cold shock for the indicated time periods are probed with anti-Derlin-1 antibody. β-Tubulin levels serve as loading control. (E) Results from eight independent experiments in D are shown. Derlin-1 protein levels, normalized to loading controls, are shown in fold change as compared to untreated control. Values shown represent mean ± SE. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). (F and G) Confocal images of GMR>KDEL-eGFP mid-pupal eyes, before (“−“ in F; left panels in G) and after the cold treatment (cold shock), stained with anti-Derlin-1 (F) or anti-VCP (G) antibodies (red). KDEL-eGFP (green) labels the ER, and the co-localization with KDEL-eGFP in merged panels is shown in white. (H) Pearson's co-localization coefficient analyses of images from four independent experiments as in G (see Materials and Methods for details). Cold shock treatment shows enhanced correlation of pixel pairs that label TER94 and the ER in a Derlin-1-dependent manner. Scale bars: 10 µm. (I) Western analysis of lysates and anti-VCP immunoprecipitates from flies treated with (4 hrs) or without (con) cold shock. The IP blot was probed with anti-VCP and anti-Derlin-1 to detect TER94/Derlin-1 complexes. The lysate (input) blot was detected by anti-VCP, and then stripped and re-probed with anti-β-Tubulin for loading control.
Figure 5.
Derlin-1 involves in ER stress-induced caspase activation.
(A) Confocal images of GMR>CD8-PARP-Venus eye discs treated with 5 mM DTT for 1-, 2- and 3.5-hrs. In the lower row, Derlin-1 expression is reduced by derlin-1 RNAi. The eye discs are stained with anti-PARP (red) and anti-Elav (blue) to mark activated caspase activity and neuronal nuclei, respectively. The anti-PARP signals are shown separately from the merged images for comparison. (B) Quantification of anti-PARP puncta from DTT-treated eye discs shown in A. For each eye disc, anti-PARP signals were normalized to the disc size (numbers of PARP puncta/rows of ommatidia), and ten eye discs for each time point was plotted. (C) Confocal images of wild-type and derlin-1null eye discs stained with anti-Elav (red) and anti-cleaved caspase-3 (green) antibodies. The eye discs in the right panels were treated with 5 mM DTT for 2 hours. The caspase-3 signals are shown separately from the merged images for comparison. (D) Quantification of anti-cleaved caspase-3 puncta as shown in C. Punta numbers were normalized to the disc size as described in B. Seven eye discs were analyzed. Values shown in B and D represent mean ± SE. *p<0.05; ***p<0.001 (Student's t-test). (E) Confocal images of GMR>LacZ (upper row) and GMR>derlin-1 (lower row) eyes at the larval, pupal, and adult stages, stained with anti-Elav antibodies to label photoreceptor nuclei (blue). These eyes carry a membrane-tethered CD8-PARP-Venus (green), and are stained with anti-PARP antibodies (red) to detect caspase-mediated cleavage events. Scale bars: 10 µm. (F) Confocal images of larval, pupal, and adult GMR>LacZ (upper row) and GMR>derlin-1 (lower row) eyes stained with anti-Elav (red) and anti-cleaved caspase-3 (green) antibodies. Scale bars: 10 µm.
Figure 6.
TER94 overexpression suppresses Derlin-1-associated cytotoxicity.
(A and B) SEM (upper row) and confocal micrographs (lower row) of 1-day-old adult eyes with indicated transgenes expressed by GMR-GAL4. In all confocal panels, whole mount retinas are labeled with phalloidin (red) and anti-Lamin (green). (A) Eye-specific overexpression of Derlin-1 or SHP box deleted Derlin-1 causes a rough eye and photoreceptor disorganization (compared to the LacZ control). (B) Overexpression of either TER94 or human VCP suppresses Derlin-1-induced eye phenotypes (GMR>derlin-1), whereas reduction of TER94 (heterozygous for loss-of-function mutation; TER94−/+) in GMR>derlin-1 results in lethality. A reciprocal genetic experiment shows mild phenotype in wild-type TER94 expressing eyes (GMR>TER94WT) is enhanced by reducing a copy of derlin-1 (heterozygous for derlin-1null; derlin-1−/+). Insets in SEM panels show enlarged views of the areas outlined in yellow. Fused ommatidia are evident in GMR>TER94WT, derlin-1−/+. Scale bars: 100 µm (SEM), 10 µm (confocal). (C) TER94 overexpression does not reduce Derlin-1 protein level. Western analysis of head lysates from GMR>derlin-1 adults carrying indicated transgenes was probed with anti-Derlin-1 antibodies. The β-Tubulin bands serve as loading control.
Figure 7.
Prolonged ER stress increases Derlin-1 proteins that are not bound to TER94.
(A) Confocal micrographs of retinas from GMR>xbp1-eGFP flies fed with different concentrations of Tunicamycin (Tm). The ER stress response to Tm treatment is dose-dependent, with 24 µM Tm eliciting robust Xbp1-eGFP signals. (B) Western analyses of lysates from flies fed with 24 µM Tm for 2, 4, and 6 days. The levels of endogenous Derlin-1 and TER94 (revealed by anti-Derlin-1 and anti-VCP antibodies) in response to continuous Tm treatment are compared to those from untreated (con). The β-Tubulin level is included as loading control. In the bar graph, endogenous Derlin-1 and TER94 levels (as shown in the Western in B) are normalized to loading controls and presented in fold change as compared to untreated control. Values shown represent mean ± SE from five independent experiments. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). (C) Sequential IP (1st IP by anti-VCP and 2nd IP by anti-Derlin-1) of lysates from flies fed with 24 µM Tm for 2, 4, and 6 days. The immunoprecipitates from control and Tm-treated flies are probed with anti-VCP and anti-Derlin-1.
Figure 8.
Derlin-1 overexpression impairs ER homeostasis and produces mitochondrial abnormality.
(A and B) Confocal images of larval eye discs expressing CD3δ-YFP (A) and Xbp1-eGFP (B) probes (green), stained with anti-Elav antibodies (red) to label neuronal nuclei. The genotypes of eye discs include GMR>LacZ (control), GMR>derlin-1 (derlin-1 overexpression), GMR>derlin-1>TER94WT (overexpression of both derlin-1 and TER94), and GMR>derlin-1>TER94A229E (overexpression of derlin-1 and TER94A229E). (C–F) TEM micrographs of 18-day-old Rh1>LacZ control (C) and Rh1>derlin-1 eyes (D–F). Unlike Rh1>LacZ (C), Derlin-1-overexpressing photoreceptors (D) contain an elevated level of ER-resembling tubular membranes. (E) Another TEM section shows that Rh1>derlin-1 outer photoreceptors contain excessive ER membrane, as well as abnormal mitochondria with intracristal swelling (white arrowheads) and discontinuous membrane (red arrowhead). Inset shows higher magnification of mitochondrion pointed by red arrowhead. (F) A representative TEM micrograph shows that Derlin-1-overexpressing photoreceptors contain smaller mitochondria (white arrowheads). As Rh1 promoter is active only in the outer photoreceptors, the mitochondria (arrows) in the inner photoreceptor (outlined in yellow) serve as an internal control. Scale bars: 10 µm (confocal). 1 µm (TEM). (G and H) Scatter dot plots of individual mitochondria in photoreceptor cells from four ultrathin sections. Mitochondrial size in outer photoreceptor cells of Rh1>LacZ and Rh1>derlin-1 (G, n = 50 mitochondria per genotype), and in both inner and outer photoreceptor cells of Rh1>derlin-1 (H, n = 21 and 37 mitochondria for inner and outer cells, respectively) were manually outlined to measure the size by ImageJ. Magenta bar and blue line represent mean ± SE in each group. ***p<0.001 (unpaired Student's t-test).
Figure 9.
Derin-1 overexpression elicits canonical mitochondrial apoptosis.
(A) Confocal images of 1-day-old GMR>derlin-1 adult eyes co-expressing RNAi constructs of various caspases or in heterozygous damm background. Whole-mount adult eyes are stained with phalloidin (red) and anti-Lamin (green) to mark photoreceptor rhabdomeres and nuclear envelopes, respectively. Scale bar: 10 µm. (B) Quantification of the percentage of ommatidia (10 eyes for each genotype) containing normal complement of photoreceptors as shown in (A). Values shown represent mean ± SE. **p<0.01; ***p<0.001 (Student's t-test).
Figure 10.
The C-terminal α-domain is required for Derlin-1 overexpression-induced cytotoxicity.
(A and B) SEM (upper row) and confocal section (lower row) of 1-day-old adult fly eyes expressing the indicated transgenes with (A) or without (B) TER94A229E using GMR-GAL4 driver. Phalloidin (red) and anti-Lamin antibodies (green) are used to label the rhabdomeres and the nuclear envelopes, respectively. Scale bars: 100 µm (SEM), 10 µm (confocal). (C–G) Structural prediction of Derlin-1 constructs by I-TASSER. (C) Full-length Derlin-1 features six major helixes (colored in blue, green, yellow, brown, red, and magenta from first to sixth helix), corresponding to the transmembrane domain. The C-terminal cytoplasmic tail contains the seventh helix (colored in purple). (D–G) The predicted sixth (magenta) transmembrane helix (shown as sticks view) and the C-terminal cytoplasmic tail (shown as cartoon view) of wild-type Derlin-1 (D), FLAG-tagged Derlin-1 (E), Myc-tagged Derlin-1 (F), and Derlin-1L204G (G). The last residue of Derlin-1 is marked in red. Epitope tags and altered residue are marked in cyan (E and F) and gold (G), respectively.