Caenorhabditis elegans gene ced-9 protects cells from programmed cell death.

The gene ced-9 of the nematode Caenorhabditis elegans acts to protect cells from programmed cell death. A mutation that abnormally activates ced-9 prevents the cell deaths that occur during normal C. elegans development. Conversely, mutations that inactivate ced-9 cause cells that normally live to undergo programmed cell death; these mutations result in embryonic lethality, indicating that ced-9 function is essential for development. The ced-9 gene functions by negatively regulating the activities of other genes that are required for the process of programmed cell death.     

Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans.

Genetic studies have identified over a dozen genes that function in programmed cell death (apoptosis) in the nematode Caenorhabditis elegans. Although the ultimate effects on cell survival or engulfment of mutations in each cell death gene have been extensively described, much less is known about how these mutations affect the kinetics of death and engulfment, or the interactions between these two processes. We have used four-dimensional-Nomarski time-lapse video microscopy to follow in detail how cell death genes regulate the extent and kinetics of apoptotic cell death and removal in the early C. elegans embryo. Here we show that blocking engulfment enhances cell survival when cells are subjected to weak pro-apoptotic signals. Thus, genes that mediate corpse removal can also function to actively kill cells.     

Programmed elimination of cells by caspase-independent cell extrusion in C. elegans

The elimination of unnecessary or defective cells from metazoans occurs during normal development and tissue homeostasis, as well as in response to infection or cellular damage. Although many cells are removed through caspase-mediated apoptosis followed by phagocytosis by engulfing cells, other mechanisms of cell elimination occur, including the extrusion of cells from epithelia through a poorly understood, possibly caspase-independent, process. Here we identify a mechanism of cell extrusion that is caspase independent and that can eliminate a subset of the Caenorhabditis elegans cells programmed to die during embryonic development. In wild-type animals, these cells die soon after their generation through caspase-mediated apoptosis. However, in mutants lacking all four C. elegans caspase genes, these cells are eliminated by being extruded from the developing embryo into the extra-embryonic space of the egg. The shed cells show apoptosis-like cytological and morphological characteristics, indicating that apoptosis can occur in the absence of caspases in C. elegans. We describe a kinase pathway required for cell extrusion involving PAR-4, STRD-1 and MOP-25.1/-25.2, the C. elegans homologues of the mammalian tumour-suppressor kinase LKB1 and its binding partners STRADα and MO25α. The AMPK-related kinase PIG-1, a possible target of the PAR-4–STRD-1–MOP-25 kinase complex, is also required for cell shedding. PIG-1 promotes shed-cell detachment by preventing the cell-surface expression of cell-adhesion molecules. Our findings reveal a mechanism for apoptotic cell elimination that is fundamentally distinct from that of canonical programmed cell death.     

DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans

Genetic analyses in Caenorhabditis elegans have been instrumental in the elucidation of the central cell-death machinery, which is conserved from C. elegans to mammals. One possible difference that has emerged is the role of mitochondria. By releasing cytochrome c, mitochondria are involved in the activation of caspases in mammals. However, there has previously been no evidence that mitochondria are involved in caspase activation in C. elegans. Here we show that mitochondria fragment in cells that normally undergo programmed cell death during C. elegans development. Mitochondrial fragmentation is induced by the BH3-only protein EGL-1 and can be blocked by mutations in the bcl-2-like gene ced-9, indicating that members of the Bcl-2 family might function in the regulation of mitochondrial fragmentation in apoptotic cells. Mitochondrial fragmentation is independent of CED-4/Apaf-1 and CED-3/caspase, indicating that it occurs before or simultaneously with their activation. Furthermore, DRP-1/dynamin-related protein, a key component of the mitochondrial fission machinery, is required and sufficient to induce mitochondrial fragmentation and programmed cell death during C. elegans development. These results assign an important role to mitochondria in the cell-death pathway in C. elegans.     

Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans

Interplay among four genes--egl-1, ced-9, ced-4 and ced-3--controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4-CED-9 complex at 2.6 A resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.     

Social controls on cell survival and cell death.

Programmed cell death occurs in most animal tissues at some stage of their development, but the molecular mechanism by which it is executed is unknown. For some mammalian cells, programmed death seems to occur by default unless suppressed by signals from other cells. Such dependence on specific survival signals provides a simple way to eliminate misplaced cells, for regulating cell numbers and, perhaps, for selecting the fittest cells. But how general is this dependence on survival signals?     

Suppression of CED-3-independent apoptosis by mitochondrial betaNAC in Caenorhabditis elegans

To ensure cell survival, it is essential that the ubiquitous pro-apoptotic machinery is kept quiescent. As death is irreversible, cells must continually integrate developmental information with regulatory inputs to control the switch between repressing and activating apoptosis. Inappropriate activation or suppression of apoptosis can lead to degenerative pathologies or tumorigenesis, respectively. Here we report that Caenorhabditis elegans inhibitor of cell death-1 (ICD-1) is necessary and sufficient to prevent apoptosis. Loss of ICD-1 leads to inappropriate apoptosis in developing and differentiated cells in various tissues. Although this apoptosis requires CED-4, it occurs independently of CED-3--the caspase essential for developmental apoptosis--showing that these core pro-apoptotic proteins have separable roles. Overexpressing ICD-1 inhibits the apoptosis of cells that are normally programmed to die. ICD-1 is the beta-subunit of the nascent polypeptide-associated complex (betaNAC) and contains a putative caspase-cleavage site and caspase recruitment domain. It localizes primarily to mitochondria, underscoring the role of mitochondria in coordinating apoptosis. Human betaNAC is a caspase substrate that is rapidly eliminated in dying cells, suggesting that ICD-1 apoptosis-suppressing activity may be inactivated by caspases.     

Corpse clearance defines the meaning of cell death

While philosophers seek the meaning of life, cell biologists are becoming ever more interested in the meaning of death. Apoptosis marks unwanted cells with 'eat me' signals that direct recognition, engulfment and degradation by phagocytes. Far from being the end of the story, these clearance events allow scavenger cells to confer meaning upon cell death. But if the phagocytic 'spin doctors' receive or transmit the wrong messages, trouble ensues.     

玉米中的细胞程序性死亡及其遗传机制

玉米是主要农作物之一 ,也是一种重要的模式研究植物。玉米生长发育过程中自始至终伴随着细胞程序性死亡 (programmedcelldeathPCD)。这些PCD大致分为三大类 ,一类是正常生长发育过程中的PCD ;一类是抗病过程中的PCD或突变模拟病斑 ;另一类是外界逆境因素诱导的PCD。在玉米中已发现许多细胞死亡突变体 ,导致这些突变的一般是一个已知的可转座元素 ,这样便可通过转座子标签法来分离这些突变体的相应基因。对这些突变体的分析将阐明玉米PCD的分子机制及遗传调控 ,从而为人工控制玉米PCD提供理论基础 ,并将为其它植物的PCD研究提供一种模型。     

Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein

Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.     

Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death.

During normal vertebrate development, about half of spinal motoneurons are lost by a process of naturally occurring or programmed cell death. Additional developing motoneurons degenerate after the removal of targets or afferents. Naturally occurring motoneuron death as well as motoneuron death after loss of targets or after axotomy can be prevented by in vivo treatment with putative target (muscle) derived or other neurotrophic agents. Motoneurons can also be prevented from dying in vitro and in vivo (Y.Q.-W., R.W., D.P., J. Johnson and L. Van Eldik, unpublished data and refs 7, 13, 14) by treatment with central nervous system extracts (brain or spinal cord) and purified central nervous system and glia-derived proteins. Here we report that in vivo treatment of chick embryos with brain-derived neurotrophic factor rescues motoneurons from naturally occurring cell death. Furthermore, in vivo treatment with brain-derived neurotrophic factor (and nerve growth factor) also prevents the induced death of motoneurons that occurs following the removal of descending afferent input (deafferentation). These data indicate that members of the neurotrophin family can promote the survival of developing avian motoneurons.     

Identification of Tim4 as a phosphatidylserine receptor

In programmed cell death, a large number of cells undergo apoptosis, and are engulfed by macrophages to avoid the release of noxious materials from the dying cells. In definitive erythropoiesis, nuclei are expelled from erythroid precursor cells and are engulfed by macrophages. Phosphatidylserine is exposed on the surface of apoptotic cells and on the nuclei expelled from erythroid precursor cells; it works as an 'eat me' signal for phagocytes. Phosphatidylserine is also expressed on the surface of exosomes involved in intercellular signalling. Here we established a library of hamster monoclonal antibodies against mouse peritoneal macrophages, and found an antibody that strongly inhibited the phosphatidylserine-dependent engulfment of apoptotic cells. The antigen recognized by the antibody was identified by expression cloning as a type I transmembrane protein called Tim4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as Timd4). Tim4 was expressed in Mac1+ cells in various mouse tissues, including spleen, lymph nodes and fetal liver. Tim4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of Tim4 in fibroblasts enhanced their ability to engulf apoptotic cells. When the anti-Tim4 monoclonal antibody was administered into mice, the engulfment of apoptotic cells by thymic macrophages was significantly blocked, and the mice developed autoantibodies. Among the other Tim family members, Tim1, but neither Tim2 nor Tim3, specifically bound phosphatidylserine. Tim1- or Tim4-expressing Ba/F3 B cells were bound by exosomes via phosphatidylserine, and exosomes stimulated the interaction between Tim1 and Tim4. These results indicate that Tim4 and Tim1 are phosphatidylserine receptors for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved.     

Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans

Necrotic cell death underlies the pathology of numerous human neurodegenerative conditions. In the nematode Caenorhabditis elegans, gain-of-function mutations in specific ion channel genes such as the degenerin genes deg-1 and mec-4, the acetylcholine receptor channel subunit gene deg-3 and the G(s) protein alpha-subunit gene gsa-1 evoke an analogous pattern of degenerative (necrotic-like) cell death in neurons that express the mutant proteins. An increase in concentrations of cytoplasmic calcium in dying cells, elicited either by extracellular calcium influx or by release of endoplasmic reticulum stores, is thought to comprise a major death-signalling event. But the biochemical mechanisms by which calcium triggers cellular demise remain largely unknown. Here we report that neuronal degeneration inflicted by various genetic lesions in C. elegans requires the activity of the calcium-regulated CLP-1 and TRA-3 calpain proteases and aspartyl proteases ASP-3 and ASP-4. Our findings show that two distinct classes of proteases are involved in necrotic cell death and suggest that perturbation of intracellular concentrations of calcium may initiate neuronal degeneration by deregulating proteolysis. Similar proteases may mediate necrotic cell death in humans.     

Pleiotropy as a mechanism to stabilize cooperation

Most genes affect many traits. This phenomenon, known as pleiotropy, is a major constraint on evolution because adaptive change in one trait may be prevented because it would compromise other traits affected by the same genes. Here we show that pleiotropy can have an unexpected effect and benefit one of the most enigmatic of adaptations--cooperation. A spectacular act of cooperation occurs in the social amoeba Dictyostelium discoideum, in which some cells die to form a stalk that holds the other cells aloft as reproductive spores. We have identified a gene, dimA, in D. discoideum that has two contrasting effects. It is required to receive the signalling molecule DIF-1 that causes differentiation into prestalk cells. Ignoring DIF-1 and not becoming prestalk should allow cells to cheat by avoiding the stalk. However, we find that in aggregations containing the wild-type cells, lack of the dimA gene results in exclusion from spores. This pleiotropic linkage of stalk and spore formation limits the potential for cheating in D. discoideum because defecting on prestalk cell production results in an even greater reduction in spores. We propose that the evolution of pleiotropic links between cheating and personal costs can stabilize cooperative adaptations.     

Structure and function of Rac genes in higher plants

As the sole ubiquitous signal GTP-binding protein in higher plants, Rac genes act as pivotal molecular switches and participate in regulations of many life activities, such as cell morphogenesis and polarity growth, programmed cell death, production of H2O2, cell differentiation, and hormone reaction. Based on our work on rice Rac genes, this paper summarizes the researches on Rac genes in higher plant of the last ten years. It will help us to understand the relation between the signal tranduction and the biological functions of plant Rac.     

Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis.

The survival, differentiation, proliferation and development of haemopoietic precursor cells and the functional activity of mature blood cells are all influenced by colony stimulating factors (CSFs). As haemopoietic cells rapidly die in the absence of appropriate CSF, the promotion of cell survival mediated by CSFs, or growth factors, is fundamental to all the other effects exerted by these factors. This enhancement of cell survival is distinct from the stimulation of proliferation. Here we show that the death of haemopoietic precursor cells on withdrawal of the relevant CSF. is due to active cell death, or apoptosis, indicating that CSFs promote cell survival by suppression of the process of apoptosis. The existence of a positive control mechanism regulating precursor cell survival has important implications both for the regulation of normal haemopoiesis and for tumorigenesis.     

Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency

Apoptosis is a form of programmed cell death that is controlled by aspartate-specific cysteine proteases called caspases. In the immune system, apoptosis counters the proliferation of lymphocytes to achieve a homeostatic balance, which allows potent responses to pathogens but avoids autoimmunity. The CD95 (Fas, Apo-1) receptor triggers lymphocyte apoptosis by recruiting Fas-associated death domain (FADD), caspase-8 and caspase-10 proteins into a death-inducing signalling complex. Heterozygous mutations in CD95, CD95 ligand or caspase-10 underlie most cases of autoimmune lymphoproliferative syndrome (ALPS), a human disorder that is characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly and autoimmunity. Mutations in caspase-8 have not been described in ALPS, and homozygous caspase-8 deficiency causes embryonic lethality in mice. Here we describe a human kindred with an inherited genetic deficiency of caspase-8. Homozygous individuals manifest defective lymphocyte apoptosis and homeostasis but, unlike individuals affected with ALPS, also have defects in their activation of T lymphocytes, B lymphocytes and natural killer cells, which leads to immunodeficiency. Thus, caspase-8 deficiency in humans is compatible with normal development and shows that caspase-8 has a postnatal role in immune activation of naive lymphocytes.     

XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome

The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.     

Effects of an Rb mutation in the mouse.

The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.