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First published online 10 December 2003
doi: 10.1242/dev.00933

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Caspases function in autophagic programmed cell death in Drosophila

¹ Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA
² Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA

View larger version (64K): [in a new window]   Fig. 1. Salivary gland cells fragment as they die. (A) Twelve hour apf salivary glands have large vacuoles (asterisks) and nuclei (arrows). (B) Vacuoles (asterisks) are smaller in salivary glands of 14 hour apf animals, and nuclei (arrows) begin to separate from the cytoplasm. (C-E) Salivary glands rapidly degenerate 14.5 hours to 15 hours apf. (C,D) Nuclei (arrows) and cytoplasm of salivary glands separate 14.5 hours apf, and fragments of cytoplasm (enclosed in circle) are present. (E) By 15 hours apf, fragments of cellular debris are scattered throughout the area that was occupied by salivary glands. All images are at the same magnification.

View larger version (60K): [in a new window]   Fig. 2. Salivary glands have several characteristics of apoptosis, and appear to degrade without the assistance of phagocytes. (A-C) TUNEL assay was used to distinguish nuclei that contain fragmented DNA from cytoplasmic fragments and possible phagocytes. (A) Salivary glands possess TUNEL-positive nuclei (arrows) 14 hours apf. (B) TUNEL-positive nuclei (arrows) are surrounded by TUNEL-negative structures that are presumably fragments of salivary gland cytoplasm 15 hours apf, as well as by a small number of unidentified cells (asterisks) that did not contain obvious cell corpses. (C) At 16 hours apf, few TUNEL-positive structures are present in the location where salivary glands reside prior to destruction, and unidentified cells (asterisks) are present, but do not contain obvious dead cell corpses. (D) Transmission electron micrograph of salivary glands 14.5 hours apf, showing the separation of the nucleus (n) from the cytoplasm (c). (E) Membranous structures reminiscent of smooth endoplasmic reticulum are present in some of the balls of cytoplasm (c). Images in A-C are the same magnification. Scale bars: in D, 10 µm; in E, 1 µm.

View larger version (64K): [in a new window]   Fig. 3. Structural proteins exhibit dynamic changes during salivary gland autophagic cell death. Salivary glands of staged wild-type Canton S animals stained with antibodies against -Tubulin (red) and -Spectrin (green) (top panels), with rhodamine phalloidin to visualize filamentous Actin (red; middle panels), and with antibodies against nuclear Lamin DmO (red) and Croquemort (green) (lower panels). Nuclei are labeled with TOTO-3 (blue) in all images.

View larger version (57K): [in a new window]   Fig. 4. Changes in nuclear Lamins are associated with increased levels of active caspase. Salivary glands from wild-type Canton S were dissected and stained with antibodies against: nuclear Lamin DmO (red) and active Caspase-3/Drice (green) (top panels); and nuclear Lamin DmO (red) and cleaved nuclear Lamin (green) (bottom panels). Nuclear Lamin DmO is present 8 and 10 hours apf, but decreases by 12 hours apf. At this stage, active Caspase-3/Drice and cleaved nuclear Lamin are present in the cytoplasm; levels of these increase by 14 hours apf.

View larger version (62K): [in a new window]   Fig. 5. Changes in the localization of Lamins coincide with their degradation, whereas Actin changes localization prior to degradation. Western blot analysis of wild-type salivary gland protein extracts show that full-length 76 kD Lamin is cleaved to 45 kD (the expected size following caspase cleavage) starting at 10 hours apf. By contrast, full-length Actin persists through 12 hours apf, but is not detected in extracts 14 hours apf.

View larger version (69K): [in a new window]   Fig. 6. Animals with mutations in ßFTZ-F1, E93, BR-C and E74A have altered levels and localization of structural proteins. Salivary glands were dissected from mutants 24 hours apf, and analyzed for the levels and localization of: -Tubulin (red) and -Spectrin (green) (top panels); filamentous Actin (red) (middle panels); and nuclear Lamin DmO (red) and Croquemort (green) (lower panels). The -Tubulin network is present in the salivary glands of all of these mutants, but it appears to be at slightly lower levels in the cytoplasm of E93 mutants. -Tubulin appears to be absent near the cortex in BR-C and E74A mutant animals, given the lack of overlap with -Spectrin staining. -Spectrin is present along the cortex in ßFTZ-F1, E93 and BR-C, and in fragments in the cytoplasm of E74A mutant salivary glands. Filamentous Actin is aggregated in clumps in the cytosol, and is expressed along the cortical region of salivary gland cells in ßFTZ-F1, E93, BR-C and E74A mutant animals, although differences clearly exist in the localization of this protein in the different mutants. Nuclear Lamin DmO is present and is evenly expressed in association with the nuclear membrane in ßFTZ-F1 mutant salivary gland cells, is expressed at a lower level with punctate spots in E93 and BR-C mutant salivary glands, and is difficult to detect in E74A mutant salivary gland cells. Croquemort is not detectable in ßFTZ-F1 mutant salivary glands, is expressed at a lower level in E93 and BR-C salivary glands, and is highly expressed in E74A mutant salivary gland cells.

View larger version (58K): [in a new window]   Fig. 7. Mutations in ßFTZ-F1, E93, BR-C and E74A impact caspase activation during autophagic cell death. Salivary glands were dissected from mutants 24 hours apf, and either analyzed for abundance and localization of nuclear Lamin DmO (red) and active Caspase-3/Drice (green) (top panels), or nuclear Lamin DmO (red) and cleaved nuclear Lamin (green) (bottom panels). Active Caspase-3/Drice and cleaved nuclear Lamins are absent in ßFTZ-F1 salivary glands, are present at extremely low levels in E93 and BR-C salivary gland cells, and are expressed at elevated levels in E74A mutant salivary gland cells.

View larger version (105K): [in a new window]   Fig. 8. Inhibition of caspases prevents protein changes and salivary gland cell death. (A) The TUNEL assay was used to distinguish nuclei that contain fragmented DNA from nuclei with intact DNA. Animals staged 24 hours apf that express the dominant-negative form of Dronc C318A possess persistent salivary glands (SG) that lack vacuoles, but have nuclei with intact DNA (arrows), even though the DNA of the larval midgut (MG) is fragmented (dark stain, arrows). The brain (Br) is indicated to assist with orientation. (B) Expression of p35 and dominant-negative Dronc C318A prevent changes in the expression of -Tubulin (red) and -Spectrin (green), shown in the left panels, and nuclear Lamin DmO (red) (middle panels). By contrast, expression of p35 and dominant-negative Dronc C318A did not prevent the increase in abundance of Croquemort (green in the middle panels) in the cytoplasm, even though nuclear Lamin DmO (red) is expressed in the same cell, indicating that some cytoplasmic changes are not regulated by caspases. Similarly, filamentous Actin (red in the right panels) changes proceed and resemble 12-hour apf wild-type salivary gland cells, even though expression of p35 and dominant-negative Dronc C318A prevent the death of these cells.