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Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans
Abstract
The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation.
... GDP-GTP exchange and induction of GTPase activity are, respectively, controlled by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs), with many GEFs and GAPs regulated by efferocytic and phagocytic receptor signaling. Genomic and structural analysis of Rac1 revealed the potential homology with CED-10, which, in both mammals and C. elegans, coordinates the actin cytoskeleton [162,163]. Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [157,[162][163][164][165]. One of the pathways involves the CED-2/CED-5/CED-12 complex, for which the mammalian equivalent is the CrkII/ELMO/Dock180 complex [165]. Another pathway in Rac1 activation involves the CED-1/CED-6/CED-7 complex, which, in mammals, is equivalent to the paralogs LRP-1/GULP/ABCA1 [162,166]. ...
... Genomic and structural analysis of Rac1 revealed the potential homology with CED-10, which, in both mammals and C. elegans, coordinates the actin cytoskeleton [162,163]. Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [157,[162][163][164][165]. One of the pathways involves the CED-2/CED-5/CED-12 complex, for which the mammalian equivalent is the CrkII/ELMO/Dock180 complex [165]. ...
... Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [157,[162][163][164][165]. One of the pathways involves the CED-2/CED-5/CED-12 complex, for which the mammalian equivalent is the CrkII/ELMO/Dock180 complex [165]. Another pathway in Rac1 activation involves the CED-1/CED-6/CED-7 complex, which, in mammals, is equivalent to the paralogs LRP-1/GULP/ABCA1 [162,166]. The ELMO/DOCK180 complex and GULP protein are GEFs that activate Rac1 [157,164,167,168]. ...
Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended, they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis—the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cells and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, we review the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes for efferocytosis, with a focus on the receptors and signaling driving this process.
... GDP-GTP exchange and induction of GTPase activity are respectively controlled by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs), with many GEFs and GAPs regulated by efferocytic and phagocytic receptor signaling. Genomic and structural analysis of Rac1 reveals potential homology with CED-10, which in both mammals and C. elegans coordinates the actin cytoskeleton [139,140]. Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [134,[139][140][141][142]. One of the pathways involves the CED-2/CED-5/CED-12 complex, in which the mammalian equivalent is CrkII/ELMO/Dock180 complex [142]. Another pathway in Rac1 activation involves the CED-1/CED-6/CED-7, which in mammals are equivalent to the paralogs LRP-1/GULP/ABCA1 [139,143]. ...
... Genomic and structural analysis of Rac1 reveals potential homology with CED-10, which in both mammals and C. elegans coordinates the actin cytoskeleton [139,140]. Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [134,[139][140][141][142]. One of the pathways involves the CED-2/CED-5/CED-12 complex, in which the mammalian equivalent is CrkII/ELMO/Dock180 complex [142]. ...
... Analysis in C. elegans identified two evolutionarily conserved pathways that activate Rac1 during efferocytosis [134,[139][140][141][142]. One of the pathways involves the CED-2/CED-5/CED-12 complex, in which the mammalian equivalent is CrkII/ELMO/Dock180 complex [142]. Another pathway in Rac1 activation involves the CED-1/CED-6/CED-7, which in mammals are equivalent to the paralogs LRP-1/GULP/ABCA1 [139,143]. The ELMO/DOCK180 complex and GULP protein are GEFs that activate Rac1 [134,141,144,145]. ...
Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis—the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cell and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes to identify, remove, and process the apoptotic cells, are reviewed.
... These genes were later organized into two partially parallel phagocytic pathways: ced-1, ced-6, ced-7 and ced-2, ced-5, ced-12. Each of these pathways relays the "eat me" signal to ced-10, a GTPase responsible for regulating the cytoskeletal rearrangements that form the phagocytic cup (Kinchen et al. 2005). Orthologs for each of these pathways were later identified in Drosophila melanogaster and mammals-ced-1/ Drpr/Megf10, ced-6/Ced-6/GULP, ced-7/Eato/ABCA1/7 and ced-2/Crk/ CrkII, ced-5/Mbc/Dock180, ced-12/Ced-12/ELMO1/2, and ced-10/Rac1/ Rac1 (Ellis et al. 1991;Franc 2002;Freeman et al. 2003;Kinchen et al. 2005;Mangahas and Zhou 2005;Santoso et al. 2018). ...
... Each of these pathways relays the "eat me" signal to ced-10, a GTPase responsible for regulating the cytoskeletal rearrangements that form the phagocytic cup (Kinchen et al. 2005). Orthologs for each of these pathways were later identified in Drosophila melanogaster and mammals-ced-1/ Drpr/Megf10, ced-6/Ced-6/GULP, ced-7/Eato/ABCA1/7 and ced-2/Crk/ CrkII, ced-5/Mbc/Dock180, ced-12/Ced-12/ELMO1/2, and ced-10/Rac1/ Rac1 (Ellis et al. 1991;Franc 2002;Freeman et al. 2003;Kinchen et al. 2005;Mangahas and Zhou 2005;Santoso et al. 2018). These two pathways, however, are just a small sample of all the genes regulating phagocytosis. ...
... Future analysis will determine if the kinases act in one of the previously identified pathways (e.g., Drpr, Ced-12, JNK or cell polarity) (Ellis et al. 1991;Franc 2002;Kinchen et al. 2005;Mangahas and Zhou 2005;Meehan et al. 2015a;Hursh et al. 2016;Zheng et al. 2017) or if there is a heretofore unknown pathway waiting to be discovered. Additional tests in other phagocytic cells, such as glia and hemocytes, can determine whether the clearance effects of these kinases are ubiquitous or tissue-specific, as well as panphagocytic or specific to nonprofessional phagocytes. ...
Programmed cell death and cell corpse clearance are an essential part of organismal health and development. Cell corpses are often cleared away by professional phagocytes such as macrophages. However, in certain tissues, neighboring cells known as nonprofessional phagocytes can also carry out clearance functions. Here, we use the Drosophila melanogaster ovary to identify novel genes required for clearance by nonprofessional phagocytes. In the Drosophila ovary, germline cells can die at multiple time points. As death proceeds, the epithelial follicle cells act as phagocytes to facilitate the clearance of these cells. We performed an unbiased kinase screen to identify novel proteins and pathways involved in cell clearance during two death events. Of 224 genes examined, 18 demonstrated severe phenotypes during developmental death and clearance while 12 demonstrated severe phenotypes during starvation-induced cell death and clearance, representing a number of pathways not previously implicated in phagocytosis. Interestingly, it was found that several genes not only affected the clearance process in the phagocytes, but also non-autonomously affected the process by which germline cells died. This kinase screen has revealed new avenues for further exploration and investigation.
... GULP1 promotes phagocytosis in human macrophages and might function as a signaling adaptor downstream of CED1. CED1, CED6, and CED7 are required for actin reorganization around the apoptotic cell corpse, and CED1 and CED6 colocalize with each other and with actin around the dead cell (7). Furthermore, the guanosine triphosphatase CED10 acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED1, CED6, and CED7 signaling module as upstream regulators of Rac activation (7). ...
... CED1, CED6, and CED7 are required for actin reorganization around the apoptotic cell corpse, and CED1 and CED6 colocalize with each other and with actin around the dead cell (7). Furthermore, the guanosine triphosphatase CED10 acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED1, CED6, and CED7 signaling module as upstream regulators of Rac activation (7). Our group and others have reported that GULP1 alterations are associated with different pathophysiological conditions including cancer (8)(9)(10)(11)(12)(13). ...
... Among these interacting proteins, we have previously reported that loss of KEAP1 function leads to constitutive activation of NRF2-mediated gene expression in non-small cell lung cancer (26). Moreover, because CED6 (GULP1 homolog in C. elegans) and KEAP1 are involved in actin fiber rearrangements (7,17,26,27), we hypothesized that GULP1 may interact with KEAP1 to regulate NRF2. Consistent with our mass spectrometry analysis, we found by coimmunoprecipitation analysis that Flag-GULP1 bound to hemagglutinin (HA)-KEAP1 in human embryonic kidney (HEK) 293, T24, and BFTC905 cells (Fig. 4, A and B). ...
Disruption of the KEAP1-NRF2 pathway results in the transactivation of NRF2 target genes, consequently inducing cell proliferation and other phenotypic changes in cancer cells. Here, we demonstrated that GULP1 was a KEAP1-binding protein that maintained actin cytoskeleton architecture and helped KEAP1 to sequester NRF2 in the cytoplasm. In urothelial carcinoma of the bladder (UCB), silencing of GULP1 facilitated the nuclear accumulation of NRF2, led to constitutive activation of NRF2 signaling, and conferred resistance to the platinum drug cisplatin. Knockdown of GULP1 in UCB cells promoted tumor cell proliferation in vitro and enhanced tumor growth in vivo. In primary UCB, GULP1 silencing was more prevalent in muscle-invasive UCB compared to nonmuscle-invasive UCB. GULP1 knockdown cells showed resistance to cisplatin treatment. In parallel with decreased GULP1 expression, we observed increased expression of NRF2 , HMOX1 , and other candidate antioxidant genes in cisplatin-resistant cells. Furthermore, low or no expression of GULP1 was observed in most cisplatin nonresponder cases. Silencing of GULP1 was associated with GULP1 promoter hypermethylation in cell lines and primary tumors, and a high frequency of GULP1 promoter methylation was observed in multiple sets of primary clinical UCB samples. Together, our findings demonstrate that GULP1 is a KEAP1-binding protein that regulates KEAP1-NRF2 signaling in UCB and that promoter hypermethylation of GULP1 is a potential mechanism of GULP1 silencing.
... elegans and Drosophila) as well as mammals have been used to study how phagocytes integrate signals from apoptotic cells to drive cytoskeletal rearrangement. Upstream signals in C. elegans were discovered to converge in two parallel and independent signaling pathways:the CED-2, CED-5, and CED-12 pathway, and the CED-1, CED-6, and CED-7 pathway (Kinchen et al., 2005). Both pathways then activate CED-10, an evolutionarily highly conserved GTPase, which in turn stimulates Frontiers in Cell and Developmental Biology frontiersin.org ...
... skeletal rearrangement to form phagocytic vesicles. When it comes to regulating the clearance of apoptotic cells, the CED-2/CED-5/ CED-12 homologous signaling routes in Drosophila and mice were CG1587/myoblast city/Dmel, RKII/Dock180/ELMO1 (Kinchen et al., 2005), and the CED-1/CED-6 homologous signaling pathways were Drpr/dCed-6 (Nakano et al., 2019), MEGF10/ GULP1 (Morizawa et al., 2017). Under the influence of two pathways, phagocytes extend pseudopods for the envelopment of apoptotic cells to form phagosomes. Following maturation by a succession of Rab GTPases, phagosomes fuse with lysosomes to form phagolysosomes. ...
The clearance of apoptotic cells known as efferocytosis is the final stage of apoptosis, and includes the recognition, phagocytosis, and degradation of apoptotic cells. The maintenance of tissue homeostasis requires the daily elimination of billions of apoptotic cells from the human body via the process of efferocytosis. Accordingly, aberrations in efferocytosis underlie a growing list of diseases, including atherosclerosis, cancer, and infections. During the initial phase of apoptosis, “Eat-Me” signals are exposed and recognized by phagocytes either directly through phagocyte receptors or indirectly through secreted proteins that function as bridge molecules that cross-link dying cells to phagocytes. Here, we set out to provide a comprehensive review of the molecular mechanisms and biological significance of secreted proteins in apoptotic cell clearance. Specifically, it focuses on how these secreted proteins act as bridging molecules to facilitate the clearance process.
... The second path entails the CED-2 (CrkII), CED-5 (Dock180), CED-10 (Rac1), CED-12 (ELMO) proteins, the latter being the counterpart of the human Rac signalling. These two pathways partly converge at CED-10 involved in actin polymerisation, regulating the required cytoskeleton rearrangement for engulfment [234,235]. ...
... CED-7 has a dual role to mediate the eat-me signal, it is expressed in both the engulfed and the engulfing cell: CED-7 assists in the exposure of PtdSer and also helps CED-1 to capture the PtdSer signal [237]. Then the activated CED-1 signal is transmitted by CED-6, partly activating CED-10, but mostly triggers through DYN-1 that regulates the extension of the engulfing cell membrane [234]. ...
Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis—the engulfment and elimination of dying cells and cell debris—are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.
... Much of the machinery that regulates engulfment was first identified in C. elegans [76][77][78]. There, two partially parallel pathways were identified, Cell Death Abnormality (CED)-1/-6/-7 and CED-2/-5/-12 which both signal to the GTPase CED-10 [79]. Each of these pathways has been evolutionarily conserved. ...
... Each of these pathways has been evolutionarily conserved. In Drosophila, the pathways are Draper (Drpr)/Ced-6/Eato and Crk/Myoblast city/Ced-12, which both signal to the GTPases Rac1/2 [77][78][79][80][81][82][83]. During starvation-induced death in mid-oogenesis, both of these pathways are required in the FCs for proper clearance. ...
Throughout oogenesis, Drosophila egg chambers traverse the fine line between survival and death. After surviving the ten early and middle stages of oogenesis, egg chambers drastically change their size and structure to produce fully developed oocytes. The development of an oocyte comes at a cost, the price is the lives of the oocyte’s 15 siblings, the nurse cells. These nurse cells do not die of their own accord. Their death is dependent upon their neighbors—the stretch follicle cells. Stretch follicle cells are nonprofessional phagocytes that spend the final stages of oogenesis surrounding the nurse cells and subsequently forcing the nurse cells to give up everything for the sake of the oocyte. In this review, we provide an overview of cell death in the ovary, with a focus on recent findings concerning this phagocyte-dependent non-autonomous cell death.
... Les astrocytes expriment également un grand nombre de gènes impliqués dans le recouvrement et la phagocytose. En particulier la RhoGTPase Rac1, qui contrôle le réarrangement de l'actine nécessaire à la dynamique membranaire lors de la phagocytose, à savoir le recouvrement des débris cellulaires (Kinchen et al., 2005). De façon intéressante, la phagocytose astrocytaire a été observée in vitro et in vivo (al-Ali & al-Hussain, 1996;Kinchen et al., 2005;Park et al., 2007). ...
... En particulier la RhoGTPase Rac1, qui contrôle le réarrangement de l'actine nécessaire à la dynamique membranaire lors de la phagocytose, à savoir le recouvrement des débris cellulaires (Kinchen et al., 2005). De façon intéressante, la phagocytose astrocytaire a été observée in vitro et in vivo (al-Ali & al-Hussain, 1996;Kinchen et al., 2005;Park et al., 2007). ...
La synapse est le lieu de communication entre les neurones à l'origine de nos capacités cognitives. Les mutations des gènes codant pour des protéines synaptiques sont responsables des maladies neurodéveloppementales appelées synaptopathies, recouvrant un large spectre de pathologies, de la déficience intellectuelle aux troubles du spectre autistique. Cependant, il est actuellement établi que les neurones ne sont pas les seuls acteurs au niveau de la synapse. Les astrocytes jouent également un rôle essentiel dans la mise en place du réseau neuronal et le fonctionnement de la synapse. Ils assurent aussi l'homéostasie ionique synaptique et sont capables de sécréter des glio-transmetteurs qui modulent l'activité synaptique. Oligophrénine-1 (OPHN1) est un gène associé à la déficience intellectuelle liée à l'X chez l'Homme. OPHN1 est une protéine synaptique dont les fonctions neuronales sont bien connues. La protéine peut directement interagir avec le cytosquelette d'actine et joue un rôle dans la formation et la maturation des épines dendritiques. Cette protéine est aussi exprimée dans les astrocytes mais sa fonction astrocytaire n'est pas connue. A l'aide d'un modèle KO de souris pour Ophn1, nous avons mis en évidence les conséquences de l'absence d'Ophn1 dans les astrocytes. Nous avons démontré que la délétion d'OPHN1 altère la migration et la morphologie des astrocytes in vitro. Sachant qu'Ophn1 est capable d'inactiver la GTPase RhoA, nous avons utilisé un inhibiteur de la voie RhoA/ROCK pour retrouver un phénotype de migration normal. In vivo nous avons choisi un modèle de cicatrisation gliale cortical afin de pouvoir observer la migration et la morphologie des astrocytes au niveau de la cicatrice. Nous avons observé que la délétion d'Ophn1 altérait la cicatrisation gliale et que les astrocytes à proximité de la cicatrice était moins ramifiés. L'ensemble de ces résultats nous permet de constater que les astrocytes sont altérés dans notre modèle murin de déficience intellectuelle liée à l'X. De plus, le KO conditionel astrocytaire mis en place nous permettra à l'avenir d'étudier les conséquences de la perte d'OPHN1 uniquement dans les astrocytes, et de comprendre la contribution astrocytaire dans la physiopathologie de cette maladie neuro-développementale.
... Interestingly, the CD36-a v b 3 integrin receptor complex was reported to recognize and bind externalized PS during necrotic cell uptake as well [16]. The phagocytic receptors participating in the uptake of apoptotic cells activate two evolutionarily conserved signaling pathways, both of which trigger the activity of the small G protein Rac that regulates actin reorganization and lamellipodia formation during phagocytosis [22]. Despite the shared integrin receptors considerable differences were found in the uptake of the two cell types. ...
... Inhibition of Rac1 by the NSC23766 small GTP binding protein Rac1 inhibitor inhibited the uptake of necrotic cells as well (Fig. 5E) indicating that not only the phagocytic receptors, but the signaling pathways activated by them must be the same in the uptake of apoptotic and necrotic cells. Altogether our data indicate that both heat-and H 2 O 2 -treated necrotic thymocytes express PS and are taken up by PS-dependent mechanisms, as was indicated previously [22]. We identified several PS-dependent phagocytic receptors, such as MerTK, integrins, TG2, and Tim-4, which are known to participate in the uptake of apoptotic cells, as participating in necrotic cell uptake as well. ...
One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together, and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat‐ or H2O2‐killed necrotic and apoptotic thymocytes by mouse bone marrow‐derived macrophages (BMDMs) in vitro, and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (i) blocking phosphatidylserine on the surface of dying cells; (ii) inhibition of Mer tyrosine kinase, Tim‐4, integrin β3 receptor signaling, or Rac1 activity; or (iii) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.
... These pathways include the phagocytic receptor CED-1, homologous to Draper and MEGF11, as well as intracellular signaling molecules, such as the Crk homolog CED-2. Signaling through CED-1 or CED-2 leads to actin polymerization by activating the Rac1 ortholog CED-10 (Kinchen et al., 2005;Ou et al., 2014). Undifferentiated cells also use these engulfment pathways to phagocytose cell debris, including midbody remnants derived from mitosis (Fazeli et al., 2016;Ou et al., 2014). ...
... To test whether engulfment occurs via receptor-mediated phagocytosis, we depleted CED-1 and CED-2, which both activate the Rac1 ortholog CED-10 ( Kinchen et al., 2005). While polar bodies are robustly internalized in control embryos ( Figures 2D and 2J), few are internalized in ced-1, ced-2, or (D-I) By the six-cell stage, the second polar body (GFP::H2B, yellow) is internalized in control embryos (D), but not after knockdown of engulfment genes ced-1/ MEGF11 (E), ced-2/Crk (F), or ced-10/Rac1 (G). ...
To understand how undifferentiated pluripotent cells cope with cell corpses, we examined the clearance of polar bodies born during female meiosis. We found that polar bodies lose membrane integrity and expose phosphatidylserine in Caenorhabditis elegans. Polar body signaling recruits engulfment receptors to the plasma membrane of embryonic blastomeres using the PI3K VPS-34, RAB-5 GTPase and the sorting nexin SNX-6. The second polar body is then phagocytosed using receptor-mediated engulfment pathways dependent on the Rac1 ortholog CED-10 but undergoes non-apoptotic programmed cell death independent of engulfment. RAB-7 GTPase is required for lysosome recruitment to the polar body phagosome, while LC3 lipidation is required for degradation of the corpse membrane after lysosome fusion. The polar body phagolysosome vesiculates in an mTOR- and ARL-8-dependent manner, which assists its timely degradation. Thus, we established a genetic model to study clearance by LC3-associated phagocytosis and reveal insights into the mechanisms of phagosome maturation and degradation.
... To confirm and further examine this finding, we used established programmed cell death reporters that mark distinct time points in the process of programmed cell death [34]. The ACT-5:: YFP marker is expressed in somatic sheath cells and marks pre-disc corpses; this is used as a reporter for the early stages of apoptosis [35]. The CED-1::GFP marker is expressed in cells that have initiated the process of engulfment [36]. ...
... Using DIC microscopy, the number of cell corpses quantified include early stage apoptotic cells (corpses begin to cellularize) and the middle to late stage of apoptosis (corpses appear as "buttons" after cellularization out of the germline syncytium) [34]. The cell corpse reporter strains used include the CU1546 strain [ced-1p::ced-1::GFP + rol-6 (su1006)] and KX89 strain [ced-4(n1162); lim-7::ced-1:: GFP + lin-15] to quantify the number of cells going through the process of engulfment [36], and the WS2170 strain [lim-1p::YFP::act-5 + unc-119(ed3)], to quantify the early stages of apoptosis [35]. The acridine orange (Sigma) staining protocol was conducted as previously described [24] with the following modification; the adult hermaphrodites were incubated on NGM plates, seeded with OP50 and 500 µl of acridine orange dissolved in M9 for a final concentration of 0.02 mg/ml; animals were placed in the dark for 2 h prior to visualization. ...
Programmed cell death, which occurs through a conserved core molecular pathway, is important for fundamental developmental and homeostatic processes. The human iron–sulfur binding protein NAF-1/CISD2 binds to Bcl-2 and its disruption in cells leads to an increase in apoptosis. Other members of the CDGSH iron sulfur domain (CISD) family include mitoNEET/CISD1 and Miner2/CISD3. In humans, mutations in CISD2 result in Wolfram syndrome 2, a disease in which the patients display juvenile diabetes, neuropsychiatric disorders and defective platelet aggregation. The C. elegans genome contains three previously uncharacterized cisd genes that code for CISD-1, which has homology to mitoNEET/CISD1 and NAF-1/CISD2, and CISD-3.1 and CISD-3.2, both of which have homology to Miner2/CISD3. Disrupting the function of the cisd genes resulted in various germline abnormalities including distal tip cell migration defects and a significant increase in the number of cell corpses within the adult germline. This increased germ cell death is blocked by a gain-of-function mutation of the Bcl-2 homolog CED-9 and requires functional caspase CED-3 and the APAF-1 homolog CED-4. Furthermore, the increased germ cell death is facilitated by the pro-apoptotic, CED-9-binding protein CED-13, but not the related EGL-1 protein. This work is significant because it places the CISD family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery.
... The online version of this article includes the following figure supplement(s) for figure 2: GTPase (Wu et al., 2017;Kinchen et al., 2005;Hochreiter-hufford and Ravichandran, 2013;Wang and Yang, 2016;Figure 2-figure supplement 2). Although this engulfment process is necessary primarily for removal of the resultant corpses, it also appears to play an active role in cell killing: inhibition of the engulfment pathway diminishes occurrence of PCD, likely through a complex feedback mechanism (Reddien and Horvitz, 2004;Hoeppner et al., 2001). ...
The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). ‘Purifying selection’ mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5 , which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNA uaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNA uaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.
... The extent to which the constitution of the phagocytic synapse differs in different tissue, contexts including TMEs, is unclear; the array of deployable receptors however, provides ample scope for tuning of the recognition phase of efferocytosis for different phagocytes in preparation for various downstream responses, and possibly also for discriminatory responses to different temporal phases of the apoptosis program or to different products of the apoptotic cell including ApoBDs and other ApoEVs, in addition to the corpse itself.ResponseKnowledge of the post-recognition responses of macrophages is, unsurprisingly, dominated by the corpse engulfment signaling which is ultimately mediated by the Rho GTPase, Rac1 and its downstream effectors that regulate cytoskeletal rearrangement and phagocytic cup formation.154 During apoptosis in the nematode worm, C. elegans, two pathways converge on the Rac1 orthologue, CED-10 (cell death defective gene-10) and molecular players are conserved in mammals254 : the first pathway involves CED-1,-6, and -7 while the second sequence upstream of CED-10 comprises CED-2, -5, and -12. In the first pathway, the mammalian orthologues of CED-1 are the receptors CD91/LRP1 or MEGF10; of CED-2, the adaptor GULP1 which operates downstream of CD91/ LRP1; and of CED-7, the ATP-binding cassette transporters ABCA1 and ABCA7. ...
Cancers are genetically driven, rogue tissues which generate dysfunctional, obdurate organs by hijacking normal, homeostatic programs. Apoptosis is an evolutionarily conserved regulated cell death program and a profoundly important homeostatic mechanism that is common (alongside tumor cell proliferation) in actively growing cancers, as well as in tumors responding to cytotoxic anti‐cancer therapies. Although well known for its cell‐autonomous tumor‐suppressive qualities, apoptosis harbors pro‐oncogenic properties which are deployed through non‐cell‐autonomous mechanisms and which generally remain poorly defined. Here, the roles of apoptosis in tumor biology are reviewed, with particular focus on the secreted and fragmentation products of apoptotic tumor cells and their effects on tumor‐associated macrophages, key supportive cells in the aberrant homeostasis of the tumor microenvironment. Historical aspects of cell loss in tumor growth kinetics are considered and the impact (and potential impact) on tumor growth of apoptotic‐cell clearance (efferocytosis) as well as released soluble and extracellular vesicle‐associated factors are discussed from the perspectives of inflammation, tissue repair, and regeneration programs. An “apoptosis‐centric” view is proposed in which dying tumor cells provide an important platform for intricate intercellular communication networks in growing cancers. The perspective has implications for future research and for improving cancer diagnosis and therapy.
... The MD701 strain allows the visualization of apoptotic corpses of germline cells by marking the ced-1 gene, a caspase involved in cell apoptosis (Kinchen et al. 2005). It was possible to quantify a significant increase in the number of apoptotic cells in the germline cells of worms treated with 10 μg mL − 1 and 50 μg mL − 1 of Fe 3 O 4 @Ag-NPs ( Fig. 4A and B, p < 0.05). ...
Iron oxide nanoparticles present superparamagnetic properties that enable their application in various areas, including drug delivery at specific locations in the organism. Silver nanoparticles have potent antimicrobial effects. Although the combination of Fe3O4-NPs and Ag-NPs in one hybrid nanostructure (Fe3O4@Ag-NPs) demonstrated promising targeted biomedical applications, their toxicological effects are unknown and need to be assessed. Caenorhabditis elegans is a promising model for nanotoxicological analysis, as it allows an initial screening of new substances. After exposure to Fe3O4-NPs, Ag-NPs and Fe3O4@Ag-NPs, we observed that hybrid NPs reduced the C. elegans survival and reproduction. Higher concentrations of Fe3O4@Ag-NPs caused an increase in cell apoptosis in the germline and a decrease in egg laying, which was associated with a decrease in worm swimming movements and abnormalities in the cholinergic neurons. Fe3O4@Ag-NPs caused an increase in reactive oxygen species, along with activation of DAF-16 transcription factor. A higher expression of the target genes GST-4::GFP and SOD-3::GFP were evidenced, which suggests the activation of the antioxidant system. Our results indicate the reprotoxicity caused by high levels of Fe3O4@Ag-NPs, as well as cholinergic neurotoxicity and activation of the oxidant system in C. elegans, suggesting that high concentrations of these nanomaterials can be harmful to living organisms.
... A second classical pathway leads to Rho GTPase activation through Gulp (Ced-6 in C. elegans) and was also originally identified in C. elegans. [65,68,69]. Gulp has been implicated downstream of indirect PS receptors Megf10 and Lrp and direct PS receptor Stab2 [69][70][71], yet how Gulp regulates Rac1 activation remains to be clarified ( Figure 2c). ...
Phagocytosis triggered by the phospholipid phosphatidylserine (PS) is key for the removal of apoptotic cells in development, tissue homeostasis and infection. Modulation of PS-mediated phagocytosis is an attractive target for therapeutic intervention in the context of atherosclerosis, neurodegenerative disease, and cancer. Whereas the mechanisms of target recognition, lipid and protein signalling, and cytoskeletal remodelling in opsonin-driven modes of phagocytosis are increasingly well understood, PS-mediated phagocytosis has remained more elusive. This is partially due to the involvement of a multitude of receptors with at least some redundancy in functioning, which complicates dissecting their contributions and results in complex downstream signalling networks. This review focusses on the receptors involved in PS-recognition, the signalling cascades that connect receptors to cytoskeletal remodelling required for phagocytosis, and recent progress in our understanding of how phagocytic cup formation is coordinated during PS-mediated phagocytosis.
... Some efferocytic receptors, such as Tim-4 [10] or CD14 [11], facilitate tethering. In contrast, other receptors, such as CD36 [12], Mer tyrosine kinase (Mertk) [13], stabilin-2 [4], BAI1 [3], or integrin β3 with its coreceptor transglutaminase 2 (TG2) [14] trigger two evolutionally conserved parallel signaling pathways that initiate cytoskeletal reorganization via activating the small G protein Rac1 [15]. In addition, several scavenger receptors, such as the macrophage receptor with collagenous structure (MARCO) [16], have also been shown to contribute to efficient efferocytosis. ...
Clearance of apoptotic cells by bone marrow-derived macrophages differentiated from monocytes plays a central role in the resolution of inflammation, as the conversion of pro-inflammatory M1 macrophages to M2 macrophages that mediate the resolution process occurs during efferocytosis. Thus, proper efferocytosis is a prerequisite for proper resolution of inflammation, and failure in efferocytosis is associated with the development of chronic inflammatory diseases. Previous studies from our laboratory have shown that (13R)-all-trans-13,14-dihydroretinol (DHR), the product of retinol saturase, acting from day 4 of monocyte differentiation enhances the efferocytosis capacity of the resulted macrophages. Loss of retinol saturase in mice leads to impaired efferocytosis, and to development of autoimmunity. In the present paper, we report that in differentiating monocytes DHR, retinol, and all-trans retinoic acid all act directly on retinoic acid receptors and enhance the clearance of apoptotic cells by upregulating the expression of several efferocytosis-related genes. The effect of retinoids seems to be mediated by bone morphogenetic protein (BMP)-2, and the Smad3 transcription factor. In addition, retinoids also upregulate the expression of the vitamin D receptor and that of vascular endothelial growth factor A, indicating that altogether retinoids promote the generation of a pro-reparative M2 macrophage population during monocyte differentiation.
... The latter result is opposite to the effect of let-99 mutations on furrow ingression in centralspindlin mutants (Price and Rose, 2017). We therefore tested whether loss of CED-10 function could rescue the delay in onset of furrowing exhibited by let-99(RNAi) embryos, using a maternal effect lethal allele that is a predicted null, ced-10(t1875) (Kinchen et al., 2005). In embryos from ced-10(t1875) mothers (hereafter referred to as ced-10 embryos or mutants), there were no overt cytokinesis defects and the time from NEB to furrowing onset was comparable to controls (Fig. 1G), as reported previously for hypomorphic alleles or depletion of maternal CED-10 by RNAi. ...
During cytokinesis, signals from the central spindle stimulate the accumulation of active RhoA-GTPase and thus contractile ring components at the equator, while the astral microtubules inhibit such components at the polar cortex. The DEPDC1 family protein LET-99 is required for furrow ingression in the absence of the central spindle signal, and for timely onset of furrowing even in the presence of the central spindle signal. Here we show that LET-99 works downstream or independently of RhoA-GTP and antagonizes branched F-actin and the Rac protein CED-10 to promote furrow initiation. This interaction with CED-10 is separable from LET-99s function in spindle positioning. We also characterize a new role for LET-99 in regulating cortical stability, where LET-99 acts in parallel with the actomyosin scaffolding protein anillin, but LET-99 does not antagonize CED-10 in this case. We propose that LET-99 acts in a pathway that inhibits the Rac CED-10 to promote the proper balance of branched versus linear F-actin for cytokinesis, and that LET-99 also regulates another factor that contributes to cortical stability.
... We found that in the empty vector control RNAi-treated 210 worms (EV) there was an occurrence of several ACT-5::YFP "halos" signifying the 211 expected UV damage-induced apoptosis (Fig. 3A). As expected, knockdown of 212 either CEP-1/p53 or CED-4/Apaf-1, two key regulators of apoptosis (Lant and 213 Derry 2013; Kinchen et al. 2005), inhibited the production of the UV-induced 214 apoptotic "halos". Interestingly, upon knockdown of CCAR-1, we see an increase 215 in UV-induced ACT-5::YFP "halo"' as compared to control, signifying that CCAR-1 216 functions in protecting against UV damage-induced apoptosis (Fig. 3A). ...
The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members are putative transcription regulators that have been characterized for modulating the cell cycle, apoptosis, metabolism, and the heat shock response. Mammals have two CCAR family members, CCAR1 and CCAR2/DBC1, that evolved from the founding family member CCAR-1 that is expressed in Caenorhabditis elegans . Mammalian CCAR2, the most well-studied family member, has been shown to regulate genes involved in metabolism in cultured cells. However, the regulation of gene expression by CCAR family members at an organismal level is unknown. Here, we use whole transcriptome RNA sequencing to examine the effects of CCAR-1 on gene expression in Caenorhabditis elegans . We show that CCAR-1 regulates germline transcription, reproduction, lifespan, and DNA-damage induced apoptosis. This study shows the role of CCAR-1 in vital physiological functions in the C. elegans germline that have not been investigated before.
... One pathway includes ced-2, ced-5, and ced-12 while the other pathway includes ced-1, ced-6, and ced-7 (34). These pathways converge on actin to facilitate rearrangements that enable engulfment (35). ...
Transmembrane protein engulfment receptors expressed on the surface of phagocytes engage ligands on apoptotic cells and debris to initiate a sequence of events culminating in material internalization and immunologically beneficial outcomes. Engulfment receptors are modular, comprised of functionally independent extracellular ligation domains and cytosolic signaling motifs. Cognate kinases, adaptors, and phosphatases regulate engulfment by controlling the degree of receptor activation in phagocyte plasma membranes, thus acting as receptor-proximal signaling modules. Here, we review recent efforts to reprogram phagocytes using modular synthetic receptors composed of antibody-based extracellular domains fused to engulfment receptor signaling domains. To aid the development of new phagocyte reprogramming methods, we then define the kinases, adaptors, and phosphatases that regulate a conserved family of engulfment receptors. Finally, we discuss current challenges and opportunities for the field.
... CED-10 activation leads to actin rearrangement to form a phagocytic cup and engulf the apoptotic cell (seeFig. 3(Kinchen et al. 2005). ...
This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10. The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation. This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation.
... Signaling pathways initiated by the phagocytic receptors were reported to regulate the GTP loading of several small G proteins to achieve efficient efferocytosis. Thus, the activation of Rac1 and Cdc42 was shown to be required for efferocytosis [55,56], while the activation of RhoA was reported to be inhibitory [57]. Later, using Forster resonance energy transfer biosensors, it was discovered that these small G proteins work in a temporally regulated fashion in which Rac1 and Cdc42 are activated early to work together in facilitating phagocytic cup formation through actin polymerization followed by RhoA activation, which drives mechanical retraction and phagosome internalization [58]. ...
Heme oxygenase-1 (HO-1) plays a vital role in the catabolism of heme and yields equimolar amounts of biliverdin, carbon monoxide, and free iron. We report that macrophages engulfing either the low amount of heme-containing apoptotic thymocytes or the high amount of heme-containing eryptotic red blood cells (eRBCs) strongly upregulate HO-1. The induction by apoptotic thymocytes is dependent on soluble signals, which do not include adenylate cyclase activators but induce the p38 mitogen-activated protein (MAP) kinase pathway, while in the case of eRBCs, it is cell uptake-dependent. Both pathways might involve the regulation of BTB and CNC homology 1 (BACH1), which is the repressor transcription regulator factor of the HO-1 gene. Long-term continuous efferocytosis of apoptotic thymocytes is not affected by the loss of HO-1, but that of eRBCs is inhibited. This latter is related to an internal signaling pathway that prevents the efferocytosis-induced increase in Rac1 activity. While the uptake of apoptotic cells suppressed the basal pro-inflammatory cytokine production in wild-type macrophages, in the absence of HO-1, engulfing macrophages produced enhanced amounts of pro-inflammatory cytokines. Our data demonstrate that HO-1 is required for both the engulfment and the anti-inflammatory response parts of the efferocytosis program.
... During the removal of cell corpses, CED-10 linked different engulfment pathways by functioning downstream of CED-7, CED-6 and CED-1. 56 During the phagolysosome formation, RAB-7 was a downstream effector of CED-1. 57 VPS-34 and RAB-14 functioned in an ordered manner downstream of CED-1 to control phagosome maturation. ...
The deposition of certain amount of nanopolystyrene (NPS) could be observed in gonad of Caenorhabditis elegans. However, we still know little about the response of germline to NPS exposure. In the germline of C. elegans, NPS (1-1000 μg/L) increased expression levels of two G protein-coupled receptors (GPCRs), PAQR-2 and CED-1. Moreover, susceptibility to NPS toxicity was observed in ced-1(RNAi) worms, which suggested the protective response of germline mediated by GPCR CED-1. In the germline, five proteins (CED-10, VPS-34, SNX-1, RAB-7, and RAB-14) functioned as downstream targets of GPCR CED-1 in controlling NPS toxicity. Furthermore, these five targets in the germline regulated NPS toxicity by affecting the activities of p38 MAPK and insulin signaling pathways in intestinal cells. Therefore, we raised a GPCR CED-1-mediated signaling cascade in germline in response to NPS exposure, which is helpful for understanding the molecular basis of germline in response to NPS exposure.
... Previous research has demonstrated that CED-1, the C. elegans ortholog of Drosophila Draper and mammalian MEGF10, clears apoptotic cell bodies along with CED-6, CED-7, and CED-10 14 . Astrocytes in Drosophila also use Draper to eliminate apoptotic cell bodies and neuronal debris during developmental neuronal circuit remodeling and axonal degeneration. ...
In the central nervous system, microglia are regarded as the main cells responsible for phagocytosis, contributing to neural circuit refinement and homeostasis through synapse elimination. However, recent findings have shown that astrocytes also actively participate in synapse homeostasis through phagocytosing synapses, neuronal debris, axonal mitochondria, and pathological protein aggregates. In addition, it has been also suggested that astrocytes may regulate microglial phagocytosis by secreting molecules such as IL-33 and C3. Here, we have introduced key findings regarding direct and indirect astrocyte-mediated phagocytosis in CNS development, the sleep/wake cycle, and aging. We have also discussed current information about reactive astrocytes and their phagocytic function in the diseased brain, focusing on ischemia and Alzheimer’s disease. Through this review, we aim to provide an overview of the current status as well as future perspectives regarding the important role of astrocytic control of phagocytosis.
... Common variants within the ABCA7 locus are also associated with AD risk [26]. ABCA7 may play an important role in phagocytosis of apoptotic cells because its ortholog in Caenorhabditis elegans, ced-7, is indispensable for the efficient detection and removal of apoptotic cells [27]. In mammals, many genes harboring rare variants are highly expressed in microglial cells and modulate the functions of microglia, underscoring the possible involvement of this cell type in the etiology and progression of AD ( Figure 1) [19,28]. ...
Alzheimer's disease (AD) is a debilitating, chronic neurodegenerative disease. Genetic studies involving genome-wide association studies (GWAS) and meta-analysis have discovered numerous genomic loci associated with AD; however, the causal genes and variants remain unidentified in most loci. Integration of GWAS signals with epigenomic annotations has demonstrated that AD risk variants are enriched in myeloid-specific enhancers, implicating myeloid cells in AD etiology. AD risk variants in these regulatory elements modify disease susceptibility by regulating the expression of genes that play crucial roles in microglial phagocytosis. Several of these AD risk genes are specifically expressed in myeloid cells, whereas others are ubiquitously expressed but are regulated by AD risk variants within myeloid enhancers in a cell type-specific manner. We discuss the impact of established AD risk variants on microglial phagocytosis and debris processing via the endolysosomal system.
... This process requires C1q binding to newly exposed phosphatidylserine (PS) on the apoptotic cell surface (24). Other proteins containing multiple EGF-like repeats in the extracellular domain, such as CED-1 (a transmembrane receptor with 16 EGF-like repeats from Caenorhabditis elegans) and mammalian MEGF10 (multiple EGF-like domains-10), now called SR-F2 (1), have also been reported to play a role in host defense and in the clearance of apoptotic cells (25)(26)(27) through interaction with C1q. ...
The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs via its collagen-like region. We identified two different binding regions on SR-F1: the N-terminal moiety interacts with C1q and CRT whereas the C-terminal moiety binds AcLDL. The role of SR-F1 N-linked glycans was also tested by mutating each of the three glycosylated asparagines. The three mutants retained binding activities for both AcLDL and C1q. A stable THP-1 cell line overexpressing SR-F1 was generated and C1q was shown to bind more strongly to the surface of SR-F1 overexpressing macrophages, with C1q/SR-F1 colocalization observed in some membrane areas. We also observed a higher level of CRT internalization for THP-1 SR-F1 cells. Increasing SR-F1 negatively modulated the uptake of apoptotic cells. Indeed, THP-1 cells overexpressing SR-F1 displayed a lower phagocytic capacity as compared with mock-transfected cells, which could be partially restored by addition of C1q in the extracellular milieu. Our data shed some light on the role of SR-F1 in efferocytosis, through its capacity to bind C1q and CRT, two proteins involved in this process.
... Macrophages can both express phagocytic receptors and release bridging molecules for recognition and engulfment of apoptotic cells. Some of these receptors, such as Tim-4 [10] or CD14 [11], mediate tethering, while other receptors, such as CD36 [12], Mer tyrosine kinase (Mertk) [13], stabilin-2 [4], BAI1 [3], or integrin β 3 with is coreceptor transglutaminase 2 (TG2) [14], trigger two evolutionally conserved parallel signaling pathways that initiate cytoskeletal reorganization via activating the low molecular weight GTPase Rac1 [15]. Different macrophage populations were shown to use different phagocytic receptors or bridging molecules for efferocytosis; thus, peritoneal macrophages express high levels of Tim-4, while tingible body macrophages secrete MFG-E8 [5,16]. ...
Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvβ3/β5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.
... Conversely, overexpression of Gulp1 results in enhanced phagocytosis of phosphatidylserine-exposed RBCs by stabilin-2. The signaling pathway using ced-6 has been shown to converge on ced-10 in the nematode C. elegans [68]. Gulp1 was found to increase phosphorylation of MAPK p38 and activation of Rac1 in the signaling pathway from the scavenger receptor, SR-BI [67]. ...
Phosphatidylserine is a membrane phospholipid that is localized to the inner leaflet of the plasma membrane. Phosphatidylserine externalization to the outer leaflet of the plasma membrane is an important signal for various physiological processes, including apoptosis, platelet activation, cell fusion, lymphocyte activation, and regenerative axonal fusion. Stabilin-1 and stabilin-2 are membrane receptors that recognize phosphatidylserine on the cell surface. Here, we discuss the functions of Stabilin-1 and stabilin-2 as phosphatidylserine receptors in apoptotic cell clearance (efferocytosis) and cell fusion, and their ligand-recognition and signaling pathways.
... Genetic studies in C. elegans have highlighted two independent and partially redundant phagocytic pathways for apoptotic cell clearance. One pathway uses CrkII (ced-2), Dock180 (ced-5), and Elmo1 (ced-12) to activate Rac1 (ced-10), while the second route signals through the transmembrane receptor MEGF10 (ced-1), which activates dynamin or actin polymerization via the engulfment adaptor Gulp1 (ced-6; Liu and Hengartner, 1998;Kinchen et al., 2005). Both pathways lead to reorganization of the cytoskeleton to initiate engulfment of the target cell. ...
Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1’s presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
... The Rho-family GTPases play key roles to regulate the phagocytic process and specific GEF and GAP regulatory proteins modulate the spatio-temporal activity of Rho-family GTPases [30,[43][44][45]. ...
The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.
... The archetypical defense weapon against parasitic invasion by other organisms is the phagocytic cell (Metchnikoff 1905). Even a rather primitive organism, the nematode C. elegans that is lacking blood circulation and immune cells, has phagocytotic, although not specialized cells (Kinchen, Cabello et al. 2005). The fruit fly Drosophila melanogaster harbors so-called specialized plasmatocytes, which have high phagocytic activity (Franc 2002;Meister 2004). ...
... Entre las principales funciones de esta proteína-receptor tenemos (99)(100)(101)(102)(103)(104)(105)(106) : ...
Alzheimer's, a neurodegenerative disease in which a cognitive decline occurs, is produced by the overaccumulation of β-amyloid peptide (Aβ) in the cortex, either due to an imbalance between the rate of elimination and production or by genetic factors.
It is known that the degradation of Aβ at brain level is given by enzymes produced in neurons, neuroglias, among others, and that peripherally the Aβ has a hepatic degradation. Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic protein-receptor that is expressed in hepatocytes, neurons, astrocytes, and others. This receptor at the brain level can mediate the exit of Aβ through the blood-brain barrier and it is sLRP1 that transports the Aβ up to the hepatocytes for its degradation, being internalized by LRP1 expressed in hepatocytes. The objective was to determine the main degradation pathway of LRP1 and quantify its half-life in HepG2 cells. The methodology used consisted of first cultivating this type of cells and
then treating them with an inhibitor of the lysosomal-autophagic degradation pathway (Bafilomycin A1) and an inhibitor of the ubiquitin-proteosome degradation pathway (MG132). Then, different concentrations of Cycloheximide were tested in order to find
the optimal concentration to inhibit the synthesis of proteins in HepG2 cells, to use this concentration in the experiments for the determination of the half-life of LRP1.
Finally, the determination of the half-life of LRP1 was carried out, also in cells treated with Bafilomycin A1 and MG132. The results showed that the main degradation pathway for LRP1 is lysosomal-autophagic degradation, in addition that the half-life of LRP1 is 7.67 ± 0.05 hours and this is significantly increased in the presence of Bafilomycin A1 (22.03 ± 0.05 hours) and is significantly decreased in the presence of
MG132 (7.1 ± 0.1 hours). In conclusion, this research shows that the time of action of LRP1 can be increased by inhibiting its main degradation pathway and therefore this action would improve the role of LRP1 in the organism, especially the internalization of harmful proteins and their subsequent degradation.
... binding proteins TTR-52/transthyretin (Wang et al., 2010) and lipid transfer/LPS-binding family protein NRF-5 (Zhang et al., 2012), the membrane-bound ATP-binding cassette (ABC) transporter CED-7/ ABCA1 (Wu and Horvitz, 1998b), the transmembrane receptor CED-1/ LRP1 (Zhou et al., 2001), the intracellular adaptor CED-6/GULP (Liu and Hengartner, 1998), and DYN-1/Dynamin (Yu et al., 2006). This pathway also converges upon CED-10 for cell engulfment (Kinchen et al., 2005). In a remarkable example of biological repurposing, many of these proteins also mediate the proximal-distal recognition required for axonal fusion (Neumann et al., 2015) (Fig. 3). ...
Injuries to the nervous system can cause lifelong morbidity due to the disconnect that occurs between nerve cells and their cellular targets. Re-establishing these lost connections is the ultimate goal of endogenous regenerative mechanisms, as well as those induced by exogenous manipulations in a laboratory or clinical setting. Reconnection between severed neuronal fibers occurs spontaneously in some invertebrate species and can be induced in mammalian systems. This process, known as axonal fusion, represents a highly efficient means of repair after injury. Recent progress has greatly enhanced our understanding of the molecular control of axonal fusion, demonstrating that the machinery required for the engulfment of apoptotic cells is repurposed to mediate the reconnection between severed axon fragments, which are subsequently merged by fusogen proteins. Here, we review our current understanding of naturally occurring axonal fusion events, as well as those being ectopically produced with the aim of achieving better clinical outcomes.
... CED-10 is also important for coordinating how cells change shape and move during embryogenesis [40,41]. As a result, reduction of CED-10 function normally results in embryonic lethality and reduced brood size [42]. We therefore counted the broods of rp100, n3246 and n1993 mutant animals ( Fig 3E). ...
Rac GTPases act as master switches to coordinate multiple interweaved signaling pathways. A major function for Rac GTPases is to control neurite development by influencing downstream effector molecules and pathways. In Caenorhabditis elegans, the Rac proteins CED-10, RAC-2 and MIG-2 act in parallel to control axon outgrowth and guidance. Here, we have identified a single glycine residue in the CED-10/Rac1 Switch 1 region that confers a non-redundant function in axon outgrowth but not guidance. Mutation of this glycine to glutamic acid (G30E) reduces GTP binding and inhibits axon outgrowth but does not affect other canonical CED-10 functions. This demonstrates previously unappreciated domain-specific functions within the CED-10 protein. Further, we reveal that when CED-10 function is diminished, the adaptor protein NAB-1 (Neurabin) and its interacting partner SYD-1 (Rho-GAP-like protein) can act as inhibitors of axon outgrowth. Together, we reveal that specific domains and residues within Rac GTPases can confer context-dependent functions during animal development.
Phagocytosis, a vital defense mechanism, involves the recognition and elimination of foreign substances by cells. Phagocytes, such as neutrophils and macrophages, rapidly respond to invaders; macrophages are especially important in later stages of the immune response. They detect “find me” signals to locate apoptotic cells and migrate toward them. Apoptotic cells then send “eat me” signals that are recognized by phagocytes via specific receptors. “Find me” and “eat me” signals can be strategically harnessed to modulate antitumor immunity in support of cancer therapy. These signals, such as calreticulin and phosphatidylserine, mediate potent pro‐phagocytic effects, thereby promoting the engulfment of dying cells or their remnants by macrophages, neutrophils, and dendritic cells and inducing tumor cell death. This review summarizes the phagocytic “find me” and “eat me” signals, including their concepts, signaling mechanisms, involved ligands, and functions. Furthermore, we delineate the relationships between “find me” and “eat me” signaling molecules and tumors, especially the roles of these molecules in tumor initiation, progression, diagnosis, and patient prognosis. The interplay of these signals with tumor biology is elucidated, and specific approaches to modulate “find me” and “eat me” signals and enhance antitumor immunity are explored. Additionally, novel therapeutic strategies that combine “find me” and “eat me” signals to better bridge innate and adaptive immunity in the treatment of cancer patients are discussed.
Background
Stroke is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow to the brain or the rupture of blood vessels in the brain, which can prompt ischemic or hemorrhagic stroke. After stroke onset, ischemia, hypoxia, infiltration of blood components into the brain parenchyma, and lysed cell fragments, among other factors, invariably increase blood–brain barrier (BBB) permeability, the inflammatory response, and brain edema. These changes lead to neuronal cell death and synaptic dysfunction, the latter of which poses a significant challenge to stroke treatment.
Results
Synaptic dysfunction occurs in various ways after stroke and includes the following: damage to neuronal structures, accumulation of pathologic proteins in the cell body, decreased fluidity and release of synaptic vesicles, disruption of mitochondrial transport in synapses, activation of synaptic phagocytosis by microglia/macrophages and astrocytes, and a reduction in synapse formation.
Conclusions
This review summarizes the cellular and molecular mechanisms related to synapses and the protective effects of drugs or compounds and rehabilitation therapy on synapses in stroke according to recent research. Such an exploration will help to elucidate the relationship between stroke and synaptic damage and provide new insights into protecting synapses and restoring neurologic function.
Introduction
The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination.
Methods
Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 10⁵ CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 10³ CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment.
Results
Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%.
Discussion
These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.
Background and Objective
Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti‐inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation.
Methods
The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL‐6 secretion. Six ingredients in oral care products used as anti‐inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis‐related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G‐LISA kit.
Results
Dead cells elicited IL‐6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells.
Conclusion
Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism‐related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.
Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. A candidate GEF region on CED-5 faces towards Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies showed the GEF and GAP functions act on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.
Author Summary
GTPases in their active state promote actin nucleation that drives cellular events, from cell migrations, to cell shape changes, to cell-cell interactions. To function correctly, GTPases need to cycle from the active, GTP-bound state, to the inactive, GDP-bound state. This cycle is supported by Guanine-nucleotide Exchange Factors, or GEFs, that support activation as GDP is switched for GTP, and GTPase-Activating Proteins, or GAPs that support hydrolysis back to the GDP bound state. The Rac1/CED-10 GTPase has a well-studied GEF, CED-5/CED-12, that promotes Rac1 activation during cell engulfment of dying cells. Here we tested if CED-5/CED-12 also functioned as the activator, or GEF for Rac1 during embryonic cell migrations. Surprisingly, CED-5/CED-12 behaved completely opposite to what was expected during this cell migration. Therefore, we investigated if CED-5/CED-12 could harbor a GAP function. Comparing models of human and C. elegans protein structures suggested a putative GAP region, which we mutated to show that CED-12 likely functions as a GAP. Genetic and gene expression tests identify other GTPases, CDC-42 and RHO-1, regulated by this newly uncovered CED-12 GAP function. This places CED-5/CED-12 at a central position, where it can support the cycling of multiple GTPases, and both promote and inhibit F-actin nucleation.
Simple Summary
Macrophages are the “big eaters” of the immune system who are in charge of engulfing undesirable substances. Macrophages are vital for the human body as they are instrumental in developing organisms, regulating immune responses, and maintaining a relatively stable internal environment. When the phagocytic capacity of macrophages is impaired, the body is prone to disease. In the context of tumors, tumor cells have their ways to escape from macrophage-mediated phagocytosis. They masquerade as healthy cells by expressing “don’t eat me” signals to fool macrophages and turn the initially anti-tumoral macrophages against the human body, which results in reduced macrophage-mediated phagocytosis. Hence, promoting the phagocytosis of macrophages is an important approach to improving the efficacy of anti-tumor treatment. In this review, we introduced the underlying mechanisms of macrophage-mediated phagocytosis and reviewed the recent progress in the area of application strategies on the basis of the phagocytosis mechanism.
Abstract
Macrophages are essential for the human body in both physiological and pathological conditions, engulfing undesirable substances and participating in several processes, such as organism growth, immune regulation, and maintenance of homeostasis. Macrophages play an important role in anti-bacterial and anti-tumoral responses. Aberrance in the phagocytosis of macrophages may lead to the development of several diseases, including tumors. Tumor cells can evade the phagocytosis of macrophages, and “educate” macrophages to become pro-tumoral, resulting in the reduced phagocytosis of macrophages. Hence, harnessing the phagocytosis of macrophages is an important approach to bolster the efficacy of anti-tumor treatment. In this review, we elucidated the underlying phagocytosis mechanisms, such as the equilibrium among phagocytic signals, receptors and their respective signaling pathways, macrophage activation, as well as mitochondrial fission. We also reviewed the recent progress in the area of application strategies on the basis of the phagocytosis mechanism, including strategies targeting the phagocytic signals, antibody-dependent cellular phagocytosis (ADCP), and macrophage activators. We also covered recent studies of Chimeric Antigen Receptor Macrophage (CAR-M)-based anti-tumor therapy. Furthermore, we summarized the shortcomings and future applications of each strategy and look into their prospects with the hope of providing future research directions for developing the application of macrophage phagocytosis-promoting therapy.
Cardiovascular disease is responsible for the largest number of deaths worldwide, and atherosclerosis is the primary cause. Apoptotic cell accumulation in atherosclerotic plaques leads to necrotic core formation and plaque rupture. Emerging findings show that the progression of atherosclerosis appears to suppress the elimination of apoptotic cells. Mechanistically, the reduced edibility of apoptotic cells, insufficient phagocytic capacity of phagocytes, downregulation of bridging molecules, and dysfunction in the polarization of macrophages lead to impaired efferocytosis in atherosclerotic plaques. This review focuses on the characteristics of efferocytosis in plaques and the therapeutic strategies aimed at promoting efferocytosis in atherosclerosis, which would provide novel insights for the development of antiatherosclerotic drugs based on efferocytosis.
Pancreatic β cell apoptosis is important in the pathogenesis of type 2 diabetes mellitus (T2DM). Generally, apoptotic β cells are phagocytosed by macrophages in a process known as "efferocytosis." Efferocytosis is critical to the resolution of inflammation and is impaired in T2DM. Advanced glycation end products (AGEs), which are increased in T2DM, are known to suppress phagocytosis function in macrophages. In this study, we found that AGEs inhibited efferocytosis of apoptotic β cells by primary peritoneal macrophages in C57BL/6J mice or mouse macrophage cell line Raw264.7. Mechanistically, AGEs inhibit efferocytosis by blocking Ras-related C3 botulinum toxin substrate 1 activity and cytoskeletal rearrangement through receptor for advanced glycation end products/ras homolog family member A/Rho kinase signaling in macrophages. Furthermore, it was observed that AGEs decreased the secretion of anti-inflammatory factors and promoted the proinflammatory ones to modulate the inflammation function of efferocytosis. Taken together, our results indicate that AGEs inhibit efferocytosis through binding to receptor for advanced glycation end products and activating ras homolog family member A/Rho kinase signaling, thereby inhibiting the anti-inflammatory function of efferocytosis.
Atherosclerosis, a disease process characterized by lipid accumulation and inflammation, is the main cause of coronary heart disease (CHD) and myocardial infarction (MI). Efferocytosis involves the clearance of apoptotic cells by phagocytes. Successful engulfment triggers the release of anti-inflammatory cytokines to suppress atherosclerosis. ABCA1 is a key mediator of cholesterol efflux to apoA-I for the generation of HDL-C in reverse cholesterol transport (RCT). Intriguingly, ABCA1 promotes not only cholesterol efflux but also efferocytosis. ABCA1 promotes efferocytosis by regulating the release of “find-me” ligands, including LPC, and the exposure, release, and expression of “eat-me” ligands, including PtdSer, ANXA1, ANXA5, MEGF10, and GULP1. ABCA1 has a pathway similar to TG2, which is an “eat-me” ligand. ABCA1 has the highest known homology to ABCA7, which controls efferocytosis as the engulfment and processing ligand. In addition, ABCA1 can form several regulatory feedback axes with ANXA1, MEGF10, GULP1, TNFα, and IL-6. Therefore, ABCA1 is the central factor that links cholesterol efflux and apoptotic cell clearance. Several drugs have been studied or approved for apoptotic cell clearance, such as CD47 antibody and PD1-/PD-L1 antibody. In this article, we review the role and mechanism of action of ABCA1 in efferocytosis and focus on new insights into the ABCA1-efferocytosis axis and its potential as a novel therapeutic target in atherosclerosis.
Cilia are sensory organelles protruding from cell surface. A tight regulation of membrane receptors traffic in and out of cilia is achieved by the action of Intraflagellar Transport (IFT). Here, we show that ectosomes bud from a subset of C. elegans sensory cilia. Packing and disposal of ciliary receptors in ectosomes complement their retrieval by IFT. Mutations in ciliary retrieval genes increase export of the salt sensor GCY-22 from ASER neurons by ectosomes, preventing its accumulation in ASER cilia. Ectosomes are produced from two ciliary locations: cilia tip and/or cilia base. Ectosomes budding from cilia tip are released in the environment. Ectosomes produced from the cilia base are concomitantly phagocytosed by the associated glial cells. Although ectocytosis does not require glia to occur, ectosome phagocytosis by the contacting glia contributes to maintain cilia shape and sensory function. We suggest this coordinated neuron-glia interaction is required for proper cilia function.
Biology, microscopy, and computer vision are part of a positive feedback ecosystem where progress in one provides opportunities and promotes advancement in the others. Unfortunately, development is normally responsive rather than coordinated.
Axon regeneration is a conserved process across the animal kingdom. Recent studies using the soil worm Caenorhabditis elegans as a model system revealed that machineries regulating engulfment of dying cells also control axon regeneration and axon debris removal. In this review, the relationships between the engulfment machinery and the biological processes triggered by axon injury and subsequent axon regeneration drawn from divergent views are examined. In one study, it is found that engulfing cells directly promote axon regeneration. In this context, CED‐1 (Drosophila Draper/mouse MEGF10), an engulfment protein expressed on the surface of engulfing cells, functions as a receptor for axon debris removal and as an adhesion molecule for axon regeneration. In other studies, it is shown that those engulfment genes, previously known to function within the engulfing cells for cell corpse removal, can have a cell‐autonomous “non‐engulfing cell” role in axon regeneration. Together, these findings suggest that engulfment genes are repurposed for neuronal regeneration by acting in both engulfing cells and regenerating neurons.
Multicellular organisms are not created through cell proliferation alone. It is through cell death that an indefinite cellular mass is pared back to reveal its true form. Cells are also lost throughout life as part of homeostasis and through injury. This detritus represents a significant burden to the living organism and must be cleared, most notably through the use of specialized phagocytic cells. Our understanding of these phagocytes and how they engulf cell corpses has been greatly aided by studying the fruit fly, Drosophila melanogaster Here we review the contribution of Drosophila research to our understanding of how phagocytes respond to cell death. We focus on the best studied phagocytes in the fly: the glia of the central nervous system, the ovarian follicle cells, and the macrophage-like hemocytes. Each is explored in the context of the tissue they maintain as well as how they function during development and in response to injury.
We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
Several intracellular motility events in the Caenorhabditis elegans zygote (pseudocleavage, the asymmetric meeting of the pronuclei, the segregation of germ line-specific granules, and the generation of an asymmetric spindle) appear to depend on microfilaments (MFs). To investigate how MFs participate in these manifestations of zygotic asymmetry, the distribution of MFs in oocytes and early embryos was examined, using both antibodies to actin and the F-actin-specific probe rhodamine-phalloidin. In early-stage zygotes, MFs are found in a uniform cortical meshwork of fine fibers and dots or foci. In later zygotes, concomitant with the intracellular movements that are thought to be MF mediated, MFs also become asymmetrically rearranged; as the zygote undergoes pseudocleavage and as the germ line granules become localized in the posterior half of the cell, the foci of actin become progressively more concentrated in the anterior hemisphere. The foci remain anterior as the spindle becomes asymmetric and the zygote undergoes its first mitosis, at which time fibers align circumferentially around the zygote where the cleavage furrow will form. A model for how the anterior foci of actin may participate in zygotic motility events is discussed. Phalloidin and anti-actin antibodies have also been used to visualize MFs in the somatic tissues of the adult gonad. The myoepithelial cells that surround maturing oocytes are visibly contractile and contain an unusual array of MF bundles; the MFs run roughly longitudinally from the loop of the gonad to the spermatheca. Myosin thick filaments are distributed along the MFs in a periodic manner suggestive of a sarcomere-like configuration. It is proposed that these actin and myosin filaments interact to cause sheath cell contraction and the movement of oocytes through the gonad.
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.
Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. Germ cell death requires ras/MAPK pathway activation and is used to maintain germline homeostasis. We suggest that apoptosis eliminates excess germ cells that acted as nurse cells to provide cytoplasmic components to maturing oocytes.
The rapid engulfment of apoptotic cells is a specialized innate immune response used by organisms to remove apoptotic cells. In mammals, several receptors that recognize apoptotic cells have been identified; molecules that transduce signals from these receptors to downstream cytoskeleton molecules have not been found, however [1] [2] [3]. Our previous analysis of the engulfment gene ced-6 in Caenorhabditis elegans has suggested that CED-6 is an adaptor protein that participates in a signal transduction pathway that mediates the specific recognition and engulfment of apoptotic cells [1]. Here, we describe our isolation and characterization of a human cDNA encoding a protein, hCED-6, with strong sequence similarity to C. elegans CED-6. As is the case with the worm protein, hCED-6 contains a phosphotyrosine-binding (PTB) domain and potential Src-homology domain 3 (SH3) binding sites. Both CED-6 and hCED-6 contain a predicted coiled-coil domain in the middle region. The hCED-6 protein lacks the extended carboxyl terminus found in worm CED-6; this carboxy-terminal extension appears not to be essential for CED-6 function in C. elegans, however. Overexpression of hCED-6 rescues the engulfment defect of ced-6 mutants in C. elegans significantly, suggesting that hCED-6 is a functional homologue of C. elegans CED-6. Human ced-6 is expressed widely in most human tissues. Thus, CED-6, and the CED-6 signal transduction pathway, might be conserved from C. elegans to humans and are present in most, if not all, human tissues.
One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and discovered that TUNEL labels apoptotic cells only during a transient intermediate stage. Mutations in nuc-1 allowed the generation of TUNEL-reactive DNA but blocked the conversion of TUNEL-reactive DNA to a subsequent TUNEL-unreactive state. Completion of DNA degradation did not occur in the absence of cell-corpse engulfment. Our data suggest that the process of degradation of the DNA of a cell corpse occurs in at least three distinct steps and requires activities provided by both the dying and the engulfing cell.
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.
Similar to mammalian excitotoxic cell death, necrotic-like cell death (NCD) in Caenorhabditis elegans can be initiated by hyperactive ion channels. Here we investigate the requirements for genes that execute and regulate programmed cell death (PCD) in necrotic-like neuronal death caused by a toxic MEC-4 channel. Neither the kinetics of necrosis onset nor the total number of necrotic corpses generated is altered by any C. elegans mutation known to block PCD, which provides genetic evidence that the activating mechanisms for NCD and apoptotic cell death are distinct. In contrast, all previously reported ced genes required for phagocytotic removal of apoptotic corpses, as well as ced-12, a new engulfment gene we have identified, are required for efficient elimination of corpses generated by distinct necrosis-inducing stimuli. Our results show that a common set of genes acts to eliminate cell corpses irrespective of the mode of cell death, and provide the first identification of the C. elegans genes that are required for orderly removal of necrotic cells. As phagocytotic mechanisms seem to be conserved from nematodes to humans, our findings indicate that injured necrotic cells in higher organisms might also be eliminated before lysis through a controlled process of corpse removal, a hypothesis that has significant therapeutic implications.
In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.
Introduction The LDL receptor–related protein (LRP) is larger than but structurally similar to other members of the LDL receptor gene family, an ancient family of endocytic receptors (1–3). Whereas the LDL receptor, the founding member of this family, appears to act solely in lipoprotein metabolism, the LRP and other members of this family appear to have other distinct functions. In this article, we will focus on the diverse biological roles of the LRP, which include functions in lipid metabolism, and also in the homeostasis of proteinas-es and proteinase inhibitors, cellular entry of viruses and toxins, activation of lysosomal enzymes, cellular signal transduction, and neurotransmission.
We have identified and characterized a novel C. elegans gene, ced-12, that functions in the conserved GTPase signaling pathway mediated by CED-2/Crkll, CED-5/DOCK180, and CED-10/Rac to control cell migration and phagocytosis of apoptotic cells. We provide evidence that ced-12 likely acts upstream of ced-10 during cell migration and phagocytosis and that CED-12 physically interacts with CED-5 and forms a ternary complex with CED-2 in vitro. We propose that the formation and localization of a CED-2-CED-5-CED-12 ternary complex to the plasma membrane activates CED-10, leading to the cytoskeletal reorganization that occurs in the polarized extension of cell surfaces in engulfing cells and migrating cells. We suggest that CED-12 counterparts in higher organisms regulate cytoskeleton dynamics, as CED-12 does in C. elegans.
The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.
During body morphogenesis precisely coordinated cell movements and cell shape changes organize the newly differentiated cells of an embryo into functional tissues. Here we describe two genes, gex-2 and gex-3, whose activities are necessary for initial steps of body morphogenesis in Caenorhabditis elegans. In the absence of gex-2 and gex-3 activities, cells differentiate properly but fail to become organized. The external hypodermal cells fail to spread over and enclose the embryo and instead cluster on the dorsal side. Postembryonically gex-3 activity is required for egg laying and for proper morphogenesis of the gonad. GEX-2 and GEX-3 proteins colocalize to cell boundaries and appear to directly interact. GEX-2 and GEX-3 are highly conserved, with vertebrate homologs implicated in binding the small GTPase Rac and a GEX-3 Drosophila homolog, HEM2/NAP1/KETTE, that interacts genetically with Rac pathway mutants. Our findings suggest that GEX-2 and GEX-3 may function at cell boundaries to regulate cell migrations and cell shape changes required for proper morphogenesis and development.
WormBase (http://www.wormbase.org/) is a web-accessible central data repository for information about Caenorhabditis elegans and related nematodes. The past two years have seen a significant expansion in the biological scope of WormBase, including
the integration of large-scale, genome-wide data sets, the inclusion of genome sequence and gene predictions from related
species and active literature curation. This expansion of data has also driven the development and refinement of user interfaces
and operability, including a new Genome Browser, new searches and facilities for data access and the inclusion of extensive
documentation. These advances have expanded WormBase beyond the obvious target audience of C. elegans researchers, to include researchers wishing to explore problems in functional and comparative genomics within the context
of a powerful genetic system.
Genotoxic stress is a threat to our cells' genome integrity. Failure to repair DNA lesions properly after the induction of cell proliferation arrest can lead to mutations or large-scale genomic instability. Because such changes may have tumorigenic potential, damaged cells are often eliminated via apoptosis. Loss of this apoptotic response is actually one of the hallmarks of cancer. Towards the effort to elucidate the DNA damage-induced signaling steps leading to these biological events, an easily accessible model system is required, where the acquired knowledge can reveal the mechanisms underlying more complex organisms. Accumulating evidence coming from studies in Caenorhabditis elegans point to its usefulness as such. In the worm's germline, DNA damage can induce both cell cycle arrest and apoptosis, two responses that are spatially separated. The latter is a tightly controlled process that is genetically indistinguishable from developmental programmed cell death. Upstream of the central death machinery, components of the DNA damage signaling cascade lie and act either as sensors of the lesion or as transducers of the initial signal detected. This review summarizes the findings of several studies that specify the elements of the DNA damage-induced responses, as components of the cell cycle control machinery, the repairing process or the apoptotic outcome. The validity of C. elegans as a tool to further dissect the complex signaling network of these responses and the high potential for it to reveal important links to cancer and other genetic abnormalities are addressed.
Programmed cell death is one of the major devices controlling cellular homeostasis. However, the generation of cell debris that follows the execution phase of apoptosis has to be backed up by their efficient removal by phagocyte. This highly dynamic process requires the concerted action of a number of surface molecules able to recognize early signals of membrane modifications on the apoptotic prey. Among those, the loss of phospholipid asymmetry and exposure of phosphatidylserine on the prey to be is determinant to engage phagocyte receptors and trigger the removal of corpses. A loss of membrane lipid asymmetry occurs also on the phagocyte determining its efficiency as an undertaker. Here we will discuss how, in our mind, the ATP binding cassette transporter, ABCA1, by its action on the arrangement of lipids at the phagocyte membrane, may actively promote their competence to engulf.
Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. Germ cell death requires ras/MAPK pathway activation and is used to maintain germline homeostasis. We suggest that apoptosis eliminates excess germ cells that acted as nurse cells to provide cytoplasmic components to maturing oocytes.
The Caenorhabditis elegans genome contains three rac-like genes, ced-10, mig-2, and rac-2. We report that ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene had little effect, but inactivating two or more genes perturbed both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affected all Rac pathways in axon pathfinding and cell migration but did not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acted with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/ Tyro3 receptor tyrosine kinase family, merkd mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from merkd mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in merkd mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.
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.
After programmed cell death, a cell corpse is engulfed and quickly degraded by a neighboring cell. For degradation to occur, engulfing cells must recognize, phagocytose and digest the corpses of dying cells. Previously, three genes were known to be involved in eliminating cell corpses in the nematode Caenorhabditis elegans: ced-1, ced-2 and nuc-1. We have identified five new genes that play a role in this process: ced-5, ced-6, ced-7, ced-8 and ced-10. Electron microscopic studies reveal that mutations in each of these genes prevent engulfment, indicating that these genes are needed either for the recognition of corpses by other cells or for the initiation of phagocytosis. Based upon our study of double mutants, these genes can be divided into two sets. Animals with mutations in only one of these sets of genes have relatively few unengulfed cell corpses. By contrast, animals with mutations in both sets of genes have many unengulfed corpses. These observations suggest that these two sets of genes are involved in distinct and partially redundant processes that act in the engulfment of cell corpses.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
Genetic studies of the nematode Caenorhabditis elegans have defined a variety of single-gene mutations that have specific effects on programmed cell death. Analyses of the genes defined by these mutations have revealed that cell death is an active process that requires gene function in cells that die. Specific genes are required not only to cause cell death but also to protect cells from dying. Gene interaction studies have defined a genetic pathway for the execution phase of programmed cell death in C. elegans. Molecular and biochemical findings are consistent with the pathway proposed from these genetic studies and have also revealed that the protein products of certain cell-death genes interact directly. This pathway appears to be conserved among organisms as diverse as nematodes and humans. Important questions remain to be answered about programmed cell death in C. elegans. For example, how does a cell decide to die? How is cell death initiated? What are the mechanisms of action of the cell-death protector and killer genes? What genes lie downstream of the cell-death execution pathway? The conservation of the central cell-death pathway suggests that additional genetic analyses of programmed cell death in C. elegans will help answer these questions, not only for this nematode but also for other organisms, including ourselves.
Engulfment of apoptotic cells in Caenorhabditis elegans is controlled by two partially redundant pathways. Mutations in genes in one of these pathways, defined by the genes ced-2, ced-5 and ced-10, result in defects both in the engulfment of dying cells and in the migrations of the two distal tip cells of the developing gonad. Here we find that ced-2 and ced-10 encode proteins similar to the human adaptor protein CrkII and the human GTPase Rac, respectively. Together with the previous observation that ced-5 encodes a protein similar to human DOCK180, our findings define a signalling pathway that controls phagocytosis and cell migration. We provide evidence that CED-2 and CED-10 function in engulfing rather than dying cells to control the phagocytosis of cell corpses, that CED-2 and CED-5 physically interact, and that ced-10 probably functions downstream of ced-2 and ced-5. We propose that CED-2/CrkII and CED-5/DOCK180 function to activate CED-10/Rac in a GTPase signalling pathway that controls the polarized extension of cell surfaces.
We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.
Programmed cell death (PCD) in mammals has been implicated in several disease states including cancer, autoimmune disease, and neurodegenerative disease. In Caenorhabditis elegans, PCD is a normal component of development. We find that Salmonella typhimurium colonization of the C. elegans intestine leads to an increased level of cell death in the worm gonad. S. typhimurium-mediated germ-line cell death is not observed in C. elegans ced-3 and ced-4 mutants in which developmentally regulated cell death is blocked, and ced-3 and ced-4 mutants are hypersensitive to S. typhimurium-mediated killing. These results suggest that PCD may be involved in the C. elegans defense response to pathogen attack.
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.
The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.
Three genes, ced-2, ced-5, and ced-10, are required for both cell corpse engulfment and distal tip cell migration in C. elegans. Recently, a fourth gene, ced-12, has been identified that is required for these two processes. ced-12 encodes a novel, conserved adaptor molecule involved in the activation of Rho/Rac/CDC42-like GTPases.
The Caenorhabditis elegans genome contains three rac-like genes, ced-10, mig-2, and rac-2. We report that ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene had little effect, but inactivating two or more genes perturbed both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affected all Rac pathways in axon pathfinding and cell migration but did not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acted with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes.
Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.
Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Milk fat globule epidermal growth factor
(EGF) factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the
engulfment of apoptotic cells by macrophages. We report that tingible body macrophages in the germinal centers of the spleen
and lymph nodes strongly express MFG-E8. Many apoptotic lymphocytes were found on the MFG-E8–/– tingible body macrophages, but they were not efficiently engulfed. The MFG-E8–/– mice developed splenomegaly, with the formation of numerous germinal centers, and suffered from glomerulonephritis as a result
of autoantibody production. These data demonstrate that MFG-E8 has a critical role in removing apoptotic B cells in the germinal
centers and that its failure can lead to autoimmune diseases.
AutoimmunediseaseandimpaireduptakeofapoptoticcellsinMFG-E8-deficient mice
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Acknowledgements We would like to thank Stergiou for comments on this manuscript, S. Milstein for the use of the gla-1(op234) allele, O. Hobert for the gift of the P lim-7 plasmid
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Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We would like to thank K. S. Ravichandran, M. Spector, T. Gumienny and L. Stergiou for comments on this manuscript, S. Milstein for the use of the gla-1(op234) allele, O. Hobert for the gift of the P lim-7 plasmid, J. Austin for pDPMM0016 (unc-119(þ)), and P. Gisler letters to nature NATURE | VOL 434 | 3 MARCH 2005 | www.nature.com/nature
LRP: a multifunctional scavenger and signaling receptor
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A common set of genes mediate removal of both apoptotic and necrotic cell corpses in C. elegans
- S Chung
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Chung, S., Gumienny, T. L., Hengartner, M. O. & Driscoll, M. A common set of genes mediate removal
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