The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis.

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation.

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death.

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Multilamellar bodies as potential scattering particles in human age-related nuclear cataracts.

PURPOSE: To characterize within human age-related nuclear cataracts rare spherical objects covered by multiple membranes, termed multilamellar bodies (MLBs). METHODS: Adult human normal, transparent lenses were obtained from eye bank donors and age-related nuclear cataracts were obtained immediately after extracapsular extraction. Each sample was Vibratome sectioned fresh into 200 microm thick sections that were fixed and embedded for light or electron microscopy. Confocal images were recorded from sections stained with the lipid soluble dye, DiI. RESULTS: Light micrograph montages of the equatorial plane containing the fetal and embryonic nuclei were examined. Rare, but distinct, circular 1-3 microm diameter objects were observed consistently in the cataracts. These objects did not appear to be components of the complex intercellular interfaces. Serial sections indicated that the objects were spherical, or contained a spherical component. For about 20,000 fiber cell cross-sections in each lens, the frequency of MLBs was 10 times higher in cataracts than in the normal lens nuclei. Although extensive searching with the electron microscope was necessary, the size, circular profile and multiple layers of thin (5 nm) membranes easily identified the MLBs. Interiors of the MLBs displayed variable textures. Confocal images indicated that the coverings were enriched in lipid compared to the adjacent plasma membranes. The calculated density of the MLBs in the cataractous nuclei was about 3800/mm3, which represents a volume fraction of 0.00003. CONCLUSIONS: Because the MLBs are large compared to the wavelength of light, display interiors with variable staining textures and have lipid-rich coverings, they appear to be ideal candidates for large scattering particles that may contribute to the forward light scattering in nuclear cataracts.

Structural evidence of human nuclear fiber compaction as a function of ageing and cataractogenesis.

This study was conducted to quantify structural change associated with human nuclear fiber compaction as a function of ageing and nuclear cataract formation. Normal donor lenses in three age ranges, young (15--25 years), middle-aged (36--46 years) and aged (59--81 years) were compared to each other and to age-related nuclear cataracts (55--81 years) surgically removed by extracapsular extraction. Several structural modifications which occurred as a manifestation of fiber compaction were noted. In the fetal nucleus (FN), the average anterior and posterior fiber angles decreased approximately 20% with age. Additionally, there was a reduction in the thickness of both the anterior and posterior segments of fetal fibers with age. On average, the anterior--posterior (A--P) axis in the embryonic nucleus (EN) decreased 33% with age. The average length of EN fibers decreased significantly (37%) as a function of age. This change in EN fiber length was accomplished by effecting compaction folds along fiber length. By comparison, in nuclear cataracts the anterior and posterior angles of FN fibers were about 12% smaller than comparably aged normal lenses. Similarly, the A--P axis and the length of EN fibers were 13% smaller than age-matched normals. Nuclear fiber compaction in early adulthood was significant and may contribute to the lens hardening and loss of accommodative ability symptomatic of presbyopia. 3D-CAD reconstructions of fiber compaction show how the reduction in the spacing of lateral interdigitations along fiber length causes an increase in the fiber membrane complexity along the A--P axis in relation to fiber cytoplasm as light passes through lenses. These results may explain, at least in part, how an increase in large particle scatter occurs as light is transmitted through fiber membranes, resulting in reduced lens optical quality as a function of age. By extrapolation, the significantly increased compaction of nuclear fibers in age-related nuclear cataracts may be a contributing factor for excessive scatter in nuclear opacification.