Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.     

Mitochondrial intermembrane proteins in cell death

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.     

klumpfuss regulates cell death in the Drosophila retina

Programmed cell death (PCD) plays a central role in the sculpting and maturation of developing epithelia. In adult tissue, PCD plays a further role in the prevention of malignancy though removal of damaged cells. Here, we report that mutations in klumpfuss result in an excess of support cells during maturation of the developing Drosophila pupal retina. These ectopic cells are the result of a partial and specific failure of apoptotic death during normal cell fate selection. klumpfuss is required and differentially expressed in the cells that choose the life or death cell fate. We also provide genetic and biochemical evidence that klumpfuss regulates this process through down-regulation of the Epidermal Growth Factor Receptor/dRas1 signaling pathway. Based on its sequence Klumpfuss is an EGR-class nuclear factor, and our results suggest a mechanism by which mutations in EGR-class factors such as Wilms' Tumor Suppressor-1 may result in oncogenic events such as pediatric kidney tumors.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.     

Progression from mitotic catastrophe to germ cell death in Caenorhabditis elegans lis-1 mutants requires the spindle checkpoint

Deletion of the lissencephaly disease gene LIS-1 in humans causes an extreme disorganization of the brain associated with significant reduction in cortical neurons. Here we show that deletion or RNA interference (RNAi) of Caenorhabditis elegans lis-1 results in a reduction in germline nuclei and causes a variety of cellular, developmental, and neurological defects throughout development. Our analysis of the germline defects suggests that the reduction in nuclei number stems from dysfunctional mitotic spindles resulting in cell cycle arrest and eventually programmed cell death (apoptosis). Deletion of the spindle checkpoint gene mdf-1 blocks lis-1(lf)-induced cell cycle arrest and germline apoptosis, placing the spindle checkpoint pathway upstream of the programmed cell death pathway. These results suggest that apoptosis may contribute to the cell-sparse pathology of lissencephaly.     

Comparative transcriptional profiling and evolutionary analysis of the GRAM domain family in eukaryotes

The GRAM domain was found in glucosyltransferases, myotubularins and other membrane-associated proteins. So far, functions for majority of these proteins are yet to be uncovered. In order to address the evolutionary and functional significance of this family members, we have performed a comprehensive investigation on their genome-wide identification, phylogenetic relationship and expression divergence in five different organisms representing monocot/dicot plants, vertebrate/invertebrate animals and yeast, namely, Oryza sativa, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster and Saccharomyces cerevisiae, respectively. We have identified 65 members of GRAM domain family from these organisms. Our data revealed that this family was an ancient group and various organisms had evolved into different family sizes. Large-scale genome duplication and divergence in both expression patterns and functions were significantly contributed to the expansion and retention of this family. Mouse and Drosophila members showed higher divergences in their proteins as indicated by higher Ka/Ks ratios and possessed multiple domains in various combinations. However, in plants, their protein functions were possibly retained with a relatively low divergence as signified by lower Ka/Ks ratios and only one additional domain was combined during evolution. On the other hand, this family in all five organisms exhibited high divergence in their expression patterns both at tissue level and under various biotic and abiotic stresses. These highly divergent expression patterns unraveled the complexity of functions of GRAM domain family. Each member may play specialized roles in a specific tissue or stress condition and may function as regulators of environmental and hormonal signaling.     

Heat shock proteins,cellular chaperones that modulate mitochondrial cell death pathways

Stress or heat shock proteins (HSPs) are ubiquitous and highly conserved proteins whose expression is induced in response to a wide variety of physiological and environmental insults. They allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. These proteins play an essential role in intracellular "house-keeping" by assisting the correct folding of nascent and stress-accumulated misfolded proteins and preventing their aggregation. Several HSPs have also demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream, and downstream of the mitochondrial events. Finally, HSPs could play a role in the proteasome-mediated degradation of selected proteins under stress conditions. Altogether, these properties could make HSPs appropriate targets for modulating cell death pathways.     

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.     

X-marks the spot: X-chromosome identification during dosage compensation

Dosage compensation is the essential process that equalizes the dosage of X-linked genes between the sexes in heterogametic species. Because all of the genes along the length of a single chromosome are co-regulated, dosage compensation serves as a model system for understanding how domains of coordinate gene regulation are established. Dosage compensation has been best studied in mammals, flies and worms. Although dosage compensation systems are seemingly diverse across species, there are key shared principles of nucleation and spreading that are critical for accurate targeting of the dosage compensation complex to the X-chromosome(s). We will highlight the mechanisms by which long non-coding RNAs function together with DNA sequence elements to tether dosage compensation complexes to the X-chromosome. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

Augmentation of drug-induced cell death by ER protein BRI3BP

To determine the contribution of the endoplasmic reticulum (ER) to cell fate decision, we focused on BRI3-binding protein (BRI3BP) residing in this organelle. BRI3BP, when overexpressed, augmented the apoptosis of human embryonic kidney 293T cells challenged with drugs including the anti-cancer agent etoposide. In contrast, the knockdown of BRI3BP reduced the drug-triggered apoptosis. BRI3BP overexpression enhanced both mitochondrial cytochrome c release and caspase-3 activity in etoposide-treated cells. In response to etoposide, the ER reorganized into irregularly shaped lamellae in mock-transfected cells, whereas in BRI3BP-overexpressing cells, such reorganization was not observed. These observations suggest that BRI3BP is involved in the structural dynamics of the ER and affects mitochondrial viability. Taken together, BRI3BP, widely expressed in animal cell types, seems to possess a pro-apoptotic property and can potentiate drug-induced apoptosis.     

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. In this paper, we present data indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity had no effect on this death. Instead, our evidence indicates a role for Wingless signaling to provoke this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern.     

C. elegans PlexinA PLX-1 mediates a cell contact-dependent stop signal in vulval precursor cells

PLX-1 is a PlexinA transmembrane protein in Caenorhabditis elegans, and the transmembrane-type semaphorin, SMP-1, is a ligand for PLX-1. The SMP-1/PLX-1 system has been shown to be necessary for proper epidermal morphogenesis in the male tail and seam cells. Here, we show that the SMP-1/PLX-1 system also regulates vulval morphogenesis. In plx-1 and smp-1 mutants, hermaphrodites sometimes exhibit a protruding vulva or multiple vulva-like protrusions. Throughout the vulval development of plx-1 and smp-1 mutants, the arrangement of vulval cells is often disrupted. In the initial step of vulval morphogenesis, vulval precursor cells (VPCs) are generated normally but are subsequently arranged abnormally in mutants. Continuous observation revealed that plx-1 VPC fails to terminate longitudinal extension after making contact with neighbor VPCs. The arrangement defects of VPCs in plx-1 and smp-1 mutants are rescued by expressing the respective cDNA in VPCs. plx-1::egfp and smp-1::egfp transgenes are both expressed in all vulval cells, including VPCs, throughout vulval development. We propose that the SMP-1/PLX-1 system is responsible for a cell contact-mediated stop signal for VPC extension. Analyses using cell fate-specific markers showed that the arrangement defects of VPCs also affect cell fate specification and cell lineages, but in a relatively small fraction of plx-1 mutants.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.     

Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

Proteolytic mechanisms in necrotic cell death and neurodegeneration

Programmed neuronal cell death is required during development to achieve the accurate wiring of the nervous system. However, genetic or accidental factors can lead to the premature, non-programmed death of neurons during adult life. Inappropriate death of cells in the nervous system is the cause of multiple neurodegenerative disorders. Pathological neuronal death can occur by apoptosis, by necrosis or by a combination of both. Necrotic cell death underlies the pathology of devastating neurological diseases such as neurodegenerative disorders, stroke or trauma. However, little is known about the molecular mechanisms that bring about necrotic cell death. Proteases play crucial roles in neuron degeneration by exerting both regulatory and catabolic functions. Elevated intracellular calcium is the most ubiquitous feature of neuronal death with the concomitant activation of cysteine calcium-dependent proteases, calpains. Calpains and lysosomal, catabolic aspartyl proteases, play key roles in the necrotic death of neurons. In this review, we survey the recent literature on the role of cysteine and aspartyl proteases in necrosis and neurodegeneration, aiming to delineate common proteolytic mechanisms mediating cellular destruction.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.