Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

Abstract

Background: During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.

Results: We define early and late apoptotic stages and find that early in apoptosis Dalpha-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis.

Conclusion: This study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

Figure 1

DIAP1 loss of function mutation results in altered cell polarity and adhesion. (A-F) Apoptosis in th¹⁰⁹ heterozygous and homozygous embryos; (A-C) th¹⁰⁹ heterozygous embryo stained for fragmented DNA (TUNEL) (A), activation of the executioner caspase drICE (B) and expression of ftz::lacZ (B); the ftz::lacZ transgene results in a striped expression pattern of beta-galactosidase (beta-gal) and indicates a copy of the wild type allele of the DIAP1 gene. Arrowheads in (A-C) indicate apoptotic cells in the head region. (D-F) th¹⁰⁹ homozygous embryo stained for fragmented DNA (D) and expression of ftz::lacZ and active drICE (E). (C, F) Merged images show anti beta-galactosidase and anti active-drICE staining in red and TUNEL signal in green/yellow. (G-R) th¹⁰⁹ mutant embryos stained for Bazooka (Baz, in blue) (H, L, P), Neurotactin (Nrt, red) (I, M, Q) and Dα-Catenin (Dα-Cat, green) (J, N, R). (G-J) th¹⁰⁹ heterozygous embryo; (K-N) th¹⁰⁹ homozygous embryo 45 min after cephalic furrow formation. (O-R) th¹⁰⁹ homozygous embryo 60 min after cephalic furrow formation. Scale bars in (F) 40 μm valid for (A-F, G, K, O); scale bar in (R) 10 μm for (H-J, L-N, P-R).

Figure 2

Arm is degraded during apoptosis. (A-C') th¹⁰⁹ heterozygous embryo (stage 9, i.e. 60 min after the onset of the morphogenetic arrest in th¹⁰⁹ homozygotes), stained for Arm (A, A') and DE-cadherin (B, B'). (A, B) confocal cross-section; surface view (A', B'). (D-F') Same stage th¹⁰⁹homozygous embryo stained for Arm (D, D') and DE-cadherin (E, E'). (D, E) confocal cross section and surface view(D', E'). (C, C', F, F') Merged images with Arm in green and DE-cadherin in red. Scale bar in (F') for (A-F) represents 10 μm. (G) Western blot probing protein levels of DE-cadherin, Dα-Cat, Arm and Actin in protein extracts of th¹⁰⁹ heterozygous (wt) and th¹⁰⁹homozygous embryos 45 min and 60 min after cephalic furrow formation (indicated as +45 and +60, respectively). (H) Western blot using anti-Arm^Nterm antibody to probe extracts of balancer controls (TM3) and th¹⁰⁹ homozygous embryos at time points 60 min after cephalic furrow formation; note that anti-Arm^Nterm antibody recognizes full length Arm protein in controls, but no degradation products in th¹⁰⁹ homozygotes. Actin was used as a loading control, because we found that Actin is not apparently cleaved in th¹⁰⁹ homozygous embryos at the time points indicated.

Figure 3

Arm is cleaved by caspases in vitro. (A) Cleavage of Arm and Lamin Dmo by embryonic extracts in vitro. ³⁵S-labeled Arm (lane a-c) or Lamin Dmo (d-f) was incubated with protein extracts of wildtype embryos (wt extract; c, f) or extracts prepared from embryos obtained from th¹⁰⁹ heterozygous flies (1/4 of the embryos are homozygous for th¹⁰⁹ indicated as th¹⁰⁹ extract; b, e). The th¹⁰⁹ extract contains proteolytic activity that leads to quantitative degradation of Lamin Dmo (e) and Arm (b). ³⁵S-labeled Arm appears as a range of bands suggesting premature termination during in vitro translation. Arm cleavage results in proteolytic fragments with the apparent M_r between 90 kDa and 70 kDa suggesting N-terminal cleavage of all isoforms produced in vitro (bracket in b). Addition of the pan-caspase inhibitor z-VAD-FMK completely prevents degradation of Arm (a) and Lamin Dmo (d). (B) In vitro cleavage of Arm by recombinant drICE, but not DCP-1. ³⁵S-labeled Arm was incubated with 0.5 μg and 1 μg recombinant drICE (b+c) or DCP1 (d, e), respectively. drICE cleavage of Arm leads to stable proteolytic products with the M_r of 90 kDa and 70 kDa, the 70 kDa product again likely represents a cleaved isoform produced by premature termination of the substrate (lower stars in b and c). (C) drICE and DCP1 are both active in vitro. (a) In vitro translated, S³⁵-methionine labelled Arm is cleaved by drICE resulting into the 90 kDa Arm cleavage product. (b) Cleavage of S³⁵-methionine labelled Arm can be prevented by addition of z-VAD-FMK to the lysate prior to incubation with drICE. (c) S³⁵-methionine labelled Lamin Dmo is cleaved by drICE into 20 kDa and 50 kDa fragments (d) DCP1 cleaves Lamin Dmo into 20 kDa, 50 kDa and 55 kDa fragments. Another caspase3 (and -7) related caspase Decay [55] is unable to cleave Lamin Dmo (lane e).

Figure 4

The N-terminus of Arm is cleaved by drICE. (A) Schematic representation of the domain structure of Arm. Arm repeats are indicated as red boxes. Putative caspase cleavage sites are indicated in green boxes. Blue bars depict the extent of the antigens used to produce Arm^Nterm (monoclonal antibody 7A1) and Arm^Central, respectively. (B) Cleavage of in vitro translated, ³⁵S-methionine labelled mutated forms of Arm. As control full length Arm was incubated with buffer (control). Mutated variants of Arm (D⁸⁸A, D¹²³A, D¹⁷⁰A, D¹⁷²A and D⁷⁵⁵A) were incubated with recombinant drICE. Upon incubation with drICE stable products were identified of all of the Arm mutants except for D⁸⁸A. The cleavage products have a M_r of approximately 90 kDa and are marked as Arm^ΔN. Full length Arm (Arm^Full, M_r 110 kDa) is reduced except for D⁸⁸A, which remains stable. (C) Western blot of embryo extracts of th¹⁰⁹ homozygous embryos (- Arm^D88A) and th¹⁰⁹ homozygous embryos expressing Arm^D88A from a transgene (+ Arm^D88A); expression of Arm^D88A was achieved using the Gal4/UAS system with Gal4 expressed under the control of a maternal beta-tubulin promoter (mat15::Gal4). Hand selected embryos of either genotype were subjected to Western blotting using Arm^Nterm antibody; the blot was stripped and re-probed using an anti-Actin antibody for loading control; note that Actin remains stable in th¹⁰⁹ apoptotic cells.

Figure 5

A stable Arm cleavage product persists in apoptotic cells. (A) Arm^Central antibody immunoprecipitates full length Arm; Full length Arm (M_r 110 kDa; indicated as Arm^Full) is immunoprecipitated (IP) by the Arm^Nterm or the Arm^Central antibody. In Western blots of these immunoprecipitates Arm^Full is detected by the Arm^Nterm antibody. DE-cadherin or Arm antibodies were used to immunoprecipitate proteins from embryo extracts (IP) as indicated. Immunoprecipitates were then subjected to Western blotting using Arm^Central antiserum. Anti Arm^Central detects full length Arm in the input lane (in) and in immunoprecipitates by all three antibodies. (B) Detection of Arm^ΔN in protein extracts of th¹⁰⁹ homozygous embryos. Western blot of protein extracts from th¹⁰⁹ heterozygous (wt) and th¹⁰⁹ homozygous embryos. Levels of Arm^Full are decreased in th¹⁰⁹ homozygous embryos and a short form of Arm with M_r ~90 kDa is detected corresponding to Arm^ΔN. Actin was used as loading control. (C-F) Localization of Arm^ΔN in th¹⁰⁹ homozygous embryos (45 min after onset of morphogenesis arrest) stained for Arm^Nterm (C), Arm^Central (D) and Dα-Cat (E). Arrows in (C-E) point to an apoptotic cell where full length Arm is absent from the plasma membrane but Arm^Central is still present (F) Merged image with Arm^Nterm in green, Arm^Central in red and Dα-Cat in blue. (G-J) th¹⁰⁹ homozygous embryo (60 min after onset of morphogenesis arrest) (inset in J) stained for (G) TUNEL, (H) Arm^Central and (I) Dα-Cat; (J) Overlay with TUNEL in green, Arm^Central in red and Dα-Cat in blue.

Figure 6

Expression of Arm^D88A interferes with redistribution of DE-cadherin during apoptosis. (A, B) th¹⁰⁹ homozygous embryo (stage 9, 60 min post morphogenetic block) expressing Arm^D88A stained for TUNEL (A, A') and expression of the Flag-tagged Arm^D88A transgene (B, B' anti-Flag). (C, D) th¹⁰⁹ homozygous embryo (60 min post cephalic furrow formation) expressing Arm^D88A and stained for Arm (C) and DE-cadherin (D); note persistent localization of DE-cadherin at the plasma membrane. (E-H) DE-cadherin localization in th¹⁰⁹ homozygous embryos with or without expression of Arm^D88A. (E, F) th¹⁰⁹ homozygous embryo (60 min post cephalic furrow formation) expressing Arm^D88A stained for DE-cadherin (E, E'; red in F, F') and TUNEL (green in F, F'). (G, H) th¹⁰⁹ homozygous embryo (60 min post cephalic furrow formation) stained for DE-cadherin (red in H) and TUNEL (green in H). Scale bar in (B) and (F) 40 μm, scale bar in B') and F') 10 μm.

Figure 7

Rescue potential of Arm^ΔN-88. Larval cuticles were prepared from wildtype (A), arm^YD35 hemizygous (B), arm^YD35 expressing wildtype Arm (C) or arm^YD35 expressing an N-terminal deletion of Arm, Arm^ΔN-88 (D). Ectopic expression was achieved using the UAS/Gal4 system and arm::Gal4 as a driver line. Note that arm^YD35 has a strongly shortened cuticle lacking anterior and head structures. Wildtype Arm and Arm^ΔN-88 are able to partially suppress both the segmentation defect and cuticle formation.