The rapid engulfment (phagocytosis) of cells undergoing programmed cell death (apoptosis) is a fundamental biological process that is not well understood. Here we report the cloning and functional characterization of ced-6, a gene specifically required for the engulfment of apoptotic cells in the nematode C. elegans. The CED-6 protein contains a phosphotyrosine binding domain at its N terminus and a proline/serine-rich region in its C-terminal half. Genetic mosaic analysis demonstrates that ced-6 acts within engulfing cells. We also show that ced-6 can promote the engulfment of cells at both early and late stages of apoptosis. Our data suggest that CED-6 is an adaptor molecule acting in a signal transduction pathway that specifically mediates the recognition and engulfment of apoptotic cells.
... One pathway includes the membrane receptor CED-1/MEGF10 which is thought to recognize the PtdSer on the surface of dying cells . CED-6/GULP is an adapter protein in this pathway that may play a role in signal transduction (Liu and Hengartner 1998;Liu and Hengartner 1999). CED-7 is a membrane ATP-binding cassette (ABC) transporter that may be involved in PtdSer presentation (Wu and Horvitz 1998). ...
... These include PSR-1 (Reddien and Horvitz 2000;Wang et al., 2003), the Frizzled homolog MOM-5, and the integrin heterodimer INA-1/PAT-3 (Wang et al., 2003;Cabello et al., 2010;Hsu and Wu 2010). A partially redundant engulfment pathway to PSR-1 consists of the secreted PtdSer-binding protein TTR-52/transthyretin (Wang et al., 2010) the lipid-binding protein NRF-5 (Zhang et al., 2012), the membrane-bound CED-7/ABC transporter (Wu and Horvitz 1998), the transmembrane receptor CED-1/MEGF10 , and the intracellular adaptor CED-6/GULP (Liu and Hengartner 1998). ...
... Second, it functions in a pathway parallel to its canonical pathway that involves the transthyretin protein TTR-52/ transthyretin which binds to PtdSer to promote fusion following injury. This pathway also involves CED-7/ABC transporter, NRF-5 and CED-6/GULP (Liu and Hengartner 1998;Wu and Horvitz 1998;Wang et al., 2003;Zhang et al., 2012). In this model, PtdSer serves as a 'save-me' signal for the distal fragment to re-establish axonal integrity. ...
Here we highlight the increasingly divergent functions of the Caenorhabditis elegans cell elimination genes in the nervous system, beyond their well-documented roles in cell dismantling and removal. We describe relevant background on the C. elegans nervous system together with the apoptotic cell death and engulfment pathways, highlighting pioneering work in C. elegans . We discuss in detail the unexpected, atypical roles of cell elimination genes in various aspects of neuronal development, response and function. This includes the regulation of cell division, pruning, axon regeneration, and behavioral outputs. We share our outlook on expanding our thinking as to what cell elimination genes can do and noting their versatility. We speculate on the existence of novel genes downstream and upstream of the canonical cell death pathways relevant to neuronal biology. We also propose future directions emphasizing the exploration of the roles of cell death genes in pruning and guidance during embryonic development.
... In C. elegans, two parallel redundant regulatory pathways control the process of engulfment of ACs. In the ced-1/6/7 pathway [4][5][6], the phagocytic receptor CED-1 recognizes 'eat-me' signal phosphatidylserine (PS) [7][8][9]. In the ced-2/5/12 pathway [10][11][12][13], several receptors including the phosphatidylserine receptor PSR-1 [14], MOM-5, and integrin INA-1 [15] are proposed to recognize and bind to the PS. ...
As the final step in apoptosis, apoptotic cells (ACs) are swiftly removed by specialized phagocytes, such as macrophages, or nonprofessional phagocytes, such as epidermal cells. Genetic studies of model organisms such as Caenorhabditis elegans have helped to elucidate the mechanisms of AC clearance and the underlying causes of disorders related to dysregulation of these pathways. C. elegans possesses six class B scavenger receptor homologs, but it is unknown if they affect apoptosis. Here we show that only the loss of function of scav-3, the C. elegans homolog of human LIMP-2, resulted in a considerable accumulation of cell corpses, which was caused by a failure in degradation rather than engulfment. SCAV-3 was found to be widely distributed and localized in lysosomes to maintain the integrity of the lysosomal membrane. Further study revealed that loss of scav-3 had no effect on phagosome maturation or the recruitment of lysosomes to phagosomes carrying cell corpses. Moreover, we discovered that the hydrolytic enzymes contained in the lysosomes were reduced in phagosomes in scav-3 mutants. Thus, hydrolases may leak from the damaged lysosome during phagolysosome formation due to the loss of scav-3 function, which reduces lysosome digestion activity and thus directly contributes to the elimination of apoptotic cells.
... Macrophages integrate signals from ACs to promote cytoskeletal rearrangement, a process that has been studied in the model organisms C. elegans and Drosophila, as well as in mammals. In C. elegans, upstream signals were found to converge in two parallel and independent signaling pathways:CED-2, CED-5, CED-12 pathway and CED-1, CED-6, CED-7 pathway which both subsequently activate CED-10, an evolutionarily highly conserved GTPase (Park and Kim, 2017) and thus stimulates skeletal rearrangement to form phagocytic vesicles (Wu and Horvitz, 1998a;Wu and Horvitz, 1998b;Liu and Hengartner, 1998;Reddien and Horvitz, 2000;Gumienny et al., 2001). CED-2/CED-5/CED-12 homologous signaling pathways in Drosophila and mouse were CG1587/myoblast city/Dmel, RKII/ Dock180/ELMO1, and CED-1/CED-6 homologous signaling pathways were Drpr/dCed-6, MEGF10/GULP1 (Zheng et al., 2017), which are are highly conserved to regulate ACs clearance. ...
The human body generates 10–100 billion cells every day, and the same number of cells die to maintain homeostasis. The genetically controlled, autonomously ordered cell death mainly proceeds by apoptosis. Apoptosis is an important way of programmed cell death in multicellular organisms, timely and effective elimination of apoptotic cells plays a key role in the growth and development of organisms and the maintenance of homeostasis. During the clearance of apoptotic cells, transcription factors bind to specific target promoters and act as activators or repressors to regulate multiple genes expression, how transcription factors regulate apoptosis is an important and poorly understood aspect of normal development. This paper summarizes the regulatory mechanisms of transcription factors in the clearance of apoptotic cells to date.
... CED-7, a homolog of the mammalian ABC transporter, functions in both dying and engulfing cells, is required for the enrichment of CED-1 around cell corpses (Mapes et al., 2012;Wu and Horvitz, 1998). After the phagocytic receptor CED-1 recognizes PS, the adaptor protein CED-6 (GULP) interacts with the cytoplasmic tail of CED-1 through its phosphotyrosine-binding domain (PTB) to transduce engulfment signals to downstream effectors (Liu and Hengartner, 1998;Su et al., 2002), including the large GTPase dynamin (DYN-1) (Yu et al., 2006), clathrin, and clarithin adaptors AP2 or epsin (Chen et al., 2013;Shen et al., 2013), to promote actin rearrangement for the internalization of ACs. The ced-1/6/7 and ced-2/5/12 pathways lead to the internalization of ACs and formation of phagosomes. ...
The phagocytic receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans . Whether appropriate levels of CED-1 are maintained for executing the engulfment function remains unknown. Here, we identified the C. elegans E3 ubiquitin ligase tripartite motif containing-21 (TRIM-21) as a component of the CED-1 pathway for apoptotic cell clearance. When the NPXY motif of CED-1 was bound to the adaptor protein CED-6 or the YXXL motif of CED-1 was phosphorylated by tyrosine kinase SRC-1 and subsequently bound to the adaptor protein NCK-1 containing the SH2 domain, TRIM-21 functioned in conjunction with UBC-21 to catalyze K48-linked poly-ubiquitination on CED-1, targeting it for proteasomal degradation. In the absence of TRIM-21, CED-1 accumulated post-translationally and drove cell corpse degradation defects, as evidenced by direct binding to VHA-10. These findings reveal a unique mechanism for the maintenance of appropriate levels of CED-1 to regulate apoptotic cell clearance.
... Genetic screening in C. elegans identified two partially redundant phagocytic pathways (ced-1/6/7 and ced-2/5/10/12) that are highly evolutionarily conserved (Reddien et al., 2001). CED-1 (Draper in flies, MEGF10 in mammals) (Zhou et al., 2001) interacts with CED-6 (dCed-6 in flies, GULP in mammals) (Liu and Hengartner, 1998;Su et al., 2002) to recruit the clathrin protein CHC-1 and its junctional components AP2 and epsin (Chen et al., 2013b;Shen et al., 2013), rearranging the cytoskeleton and facilitating phagocytic pseudopod expansion. CED-2, CED-5, CED-10, CED-12, and PSR-1 (homologs of CrkⅡ, DOCK180, RacGTPase, PH-SH3 domain, and PSR, respectively, in mammals) constitute another phagocytic pathway (Gumienny et al., 2001;Hsu and Wu, 2010;Wu et al., 2017). ...
Ubiquitination, a critical post-translational modification of proteins, refers to the covalent attachment of ubiquitin to the substrate and is involved in various biological processes such as protein stability regulation, DNA damage repair, and apoptosis, among others. E3 ubiquitin ligases are essential enzymes of the ubiquitin pathway with high substrate specificity and precisely regulate specific proteins’ turnover. As one of the most well-studied forms of programmed cell death, apoptosis is substantially conserved across the evolutionary tree. The final critical stage in apoptosis is the removal of apoptotic cells by professional and non-professional phagocytes. Apoptosis and apoptotic cell clearance are crucial for the normal development, differentiation, and growth of multicellular organisms, as well as their association with a variety of inflammatory and immune diseases. In this review, we discuss the role of ubiquitination and deubiquitination in apoptosis and apoptotic cell clearance.
CED-1 is a transmembrane receptor involved in the recognition of “eat-me” signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.
Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin‐dependent kinase inhibitor p21. The activation of the TGF‐β/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF‐β/SMAD2/3 pathway.
The engulfment adaptor phosphotyrosine‐binding domain containing 1 (GULP1) is an adaptor protein involved in the engulfment of apoptotic cells via phagocytosis. Gulp1 was first found to promote the phagocytosis of apoptotic cells by macrophages, and its role in various tissues, including neurons and ovaries, has been well studied. However, the expression and function of GULP1 in bone tissue are poorly understood. Consequently, to determine whether GULP1 plays a role in the regulation of bone remodeling in vitro and in vivo, we generated Gulp1 knockout (KO) mice. Gulp1 was expressed in bone tissue, mainly in osteoblasts, while its expression is very low in osteoclasts. Microcomputed tomography and histomorphometry analysis in 8‐week‐old male Gulp1 KO mice revealed a high bone mass in comparison with male wild‐type (WT) mice. This was a result of decreased osteoclast differentiation and function in vivo and in vitro as confirmed by a reduced actin ring and microtubule formation in osteoclasts. Gas chromatography‐mass spectrometry analysis further showed that both 17β‐estradiol (E2) and 2‐hydroxyestradiol levels, and the E2/testosterone metabolic ratio, reflecting aromatase activity, were also higher in the bone marrow of male Gulp1 KO mice than in male WT mice. Consistent with mass spectrometry analysis, aromatase enzymatic activity was significantly higher in the bone marrow of male Gulp1 KO mice. Altogether, our results suggest that GULP1 deficiency decreases the differentiation and function of osteoclasts themselves and increases sex steroid hormone‐mediated inhibition of osteoclast differentiation and function, rather than affecting osteoblasts, resulting in a high bone mass in male mice. To the best of our knowledge, this is the first study to explore the direct and indirect roles of GULP1 in bone remodeling, providing new insights into its regulation.
During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.
A new method for producing genetic mosaics, which involves the spontaneous somatic loss of free chromosome fragments, is demonstrated. Four genes that affect the behavior of C. elegans were studied in mosaic animals. The analysis was greatly aided by the fact that the complete cell lineage of wild-type animals is known. Two of the mutant genes affect certain sensory responses and prevent uptake of fluorescein isothiocyanate (FITC) by certain sensory neurons. Mosaic analysis indicated that one of these mutant genes is cell autonomous with respect to its effect on FITC uptake and the other is cell nonautonomous. In the latter case, the genotype of a non-neuronal supporting cell that surrounds the processes of the neurons that normally take up FITC probably is critical. The other two mutant genes affect animal movement. Mosaic analysis indicated that the expression of one of these genes is specific to certain neurons (motor neurons of the ventral and dorsal nerve cords are prime candidates) and the expression of the other gene is specific to muscle cells.
Metamorphosing animals provide the most obvious models for the study of cell death. In insects and amphibia, large masses of relatively homogeneous tissue degenerate rapidly and predictably. In many instances, the extracellular controls are well understood and the control manipulatable at the experimenter’s convenience; the response may be evocable in vitro. In other instances of rapid development—the metamorphosis of embryonic invertebrates (molluscs, echinoderms or ascidians) or tissue metamorphosis (Müllerian and Wolffian ducts, ovulation, involution of the corpus luteum) — there are equal opportunities for experimentation, but they have not been as thoroughly studied (Glücksmann, 1951).
In the nematode C. elegans, cells undergoing programmed death in the developing ventral nerve cord were identified by Nomarski optics and prepared for ultrastructural study at various times after their birth in mitosis. The sequence of changes observed suggests that the hypodermis recognizes the dying cell before completion of telophase. The dying cell is engulfed and digestion then occurs until all that remains within the hypodermal cytoplasm is a collection of membranous whorls interspersed with condensed chromatin-like remnants. The process shares several features with apoptosis, the mode of programmed cell death observed in vertebrates and insects. The selection of cells for programmed death appears not to involve competition for peripheral targets.
As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.
Although Fc receptor-mediated phagocytosis is accompanied by a variety of transmembrane signaling events, not all signaling events are required for particle ingestion. For example, Fc receptor-mediated phagocytosis in mouse inflammatory macrophages (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106:657; Greenberg, S., J. El Khoury, F. Di Virgilio, and S. C. Silverstein. 1991. J. Cell Biol. 113:757) and neutrophils (Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. J. Immunol. 144:1411) occurs in the absence of cytosolic calcium transients. We sought to identify transmembrane signaling events that are essential for phagocytosis. Here we show that tyrosine phosphorylation is an early event after Fc receptor ligation in mouse inflammatory macrophages, and that the formation of tyrosine phosphoproteins coincides temporally with the appearance of F-actin beneath phagocytic cups. The distribution of tyrosine phosphoproteins that accumulated beneath phagocytic cups was punctate and corresponded to areas of high ligand density on the surface of the antibody-coated red blood cells, which provided the phagocytic stimulus. A tyrosine kinase inhibitor, genistein, but not several inhibitors of protein kinase C, blocked the appearance of tyrosine phosphoproteins as assessed by immunofluorescence, the focal accumulation of F-actin beneath immunoglobulin G-opsonized particles, and the ingestion of these particles as well. We suggest that tyrosine phosphorylation is a critical signaling event that underlies Fc receptor-mediated phagocytosis in mouse macrophages, and is necessary for the engulfment per se.
Phagocyte recognition and ingestion of intact cells undergoing apoptosis are key events in this generally important program of cell death. Insufficient phagocyte capacity for apoptotic cells can result in failure to clear dying cells before membrane integrity is lost, resulting in leakage of noxious cell contents and severe tissue damage. However, no means has been available to increase phagocytic clearance of apoptotic cells. We now report that transfection of the macrophage adhesion molecule CD36 into human Bowes melanoma cells specifically conferred greatly increased capacity to ingest apoptotic neutrophils, lymphocytes, and fibroblasts, comparable to that exhibited by macrophages. Furthermore, when CD36 was transfected into another cell type with limited capacity to take up apoptotic bodies, the monkey COS-7 cell, similar effects were observed. Therefore, CD36 gene transfer can confer "professional" capacity to ingest apoptotic cells upon "amateur" phagocytes.
In the nematode C. elegans, cells undergoing programmed death in the developing ventral nerve cord were identified by Nomarski optics and prepared for ultrastructural study at various times after their birth in mitosis.
The sequence of changes observed suggests that the hypodermis recognizes the dying cell before completion of telophase. The dying cell is engulfed and digestion then occurs until all that remains within the hypodermal cytoplasm is a collection of membranous whorls interspersed with condensed chromatin-like remnants. The process shares several features with apoptosis, the mode of programmed cell death observed in vertebrates and insects. The selection of cells for programmed death appears not to involve competition for peripheral targets.
![Overexpression of ced-6 Promotes the Engulfment of Both Early and Persistent Cell Corpses (A) Cell death during embryonic development. Embryonic development was divided into 50 min intervals, and the number of cells dying in each interval plotted as a function of developmental age. Embryos were heat shocked (arrows) either prior to or well after the main wave of embryonic cell death, and scored for the number of persistent cell corpses in the head region shortly after hatching (arrowhead). The experiments shown were performed with ced-6(n1813). (B) Overexpression of ced-6 promotes the engulfment of both early and persistent embryonic cell corpses. Embryos laid by transgenic mothers were heat-shocked either before (open symbols) or after (filled symbols) the wave of embryonic cell death (see [A]), and scored for number of persistent cell corpses shortly after hatching. (C) Overexpression of ced-6 rescues the germ cell engulfment defect. Transgenic animals were heat shocked 24 hr after the L4/adult molt (arrow), and germ cell corpses were scored over the next 60 hr. In (C) and (D), symbols represent means 95% confidence interval. (D) Overexpression of ced-6 DNA promotes the engulfment of persistent germ cell corpses. Adult transgenic animals were submitted to heat shock at various times after the L4/adult molt (arrows), and germ cell corpses counted 12 hr after heat shock.](https://www.researchgate.net/profile/Qiong-Liu-17/publication/13650022/figure/fig4/AS:630944746704896@1527440358454/Overexpression-of-ced-6-Promotes-the-Engulfment-of-Both-Early-and-Persistent-Cell-Corpses_Q320.jpg)
