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Jason M. Kinchen - One of the best experts on this subject based on the ideXlab platform.
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Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein
Nature, 2011Co-Authors: Daeho Park, Jason M. Kinchen, Michael R. Elliott, Jeffrey J. Lysiak, Claudia Z. Han, Paul C. Trampont, Soumita Das, Sheila Collins, Kyle L. Hoehn, Kodi S. RavichandranAbstract:When a phagocyte engulfs a dying cell, it essentially doubles its cellular contents, yet phagocytes are capable of ingesting several apoptotic cells one after the other. The factors regulating this impressive Engulfment capacity are not well understood. Kodi Ravichandran and colleagues show here that the mitochondrial membrane protein Ucp2, which is known to be linked to metabolic diseases and atherosclerosis, is critically important to Engulfment capacity. Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses1,2,3,4,5. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material6. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls Engulfment capacity, with lower potential enhancing Engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential7,8,9, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced Engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice10,11 were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis11,12, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.
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Unexpected requirement for ELMO1 in clearance of apoptotic germ cells in vivo
Nature, 2010Co-Authors: Michael R. Elliott, Jason M. Kinchen, Daeho Park, Shuqiu Zheng, Robin I. Woodson, Michael A. Reardon, Ignacio J. Juncadella, Jun Zhang, Jeffrey J. Lysiak, Kodi S. RavichandranAbstract:Apoptotic cell death is crucial for tissue development and function and occurs throughout mammalian life. Removal of dying cells by phagocytes is important to prevent tissue dysfunction, but the mechanisms that regulate clearance of dying cells in vivo remain unclear. In this study, Elliott et al . reveal a crucial role for a conserved Engulfment protein, ELMO1, in the phagocytic clearance of apoptotic germ cells by Sertoli cells and the maintenance of normal testicular homeostasis. Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases^ 1 , 2 , 3 . ELMO1 is an evolutionarily conserved cytoplasmic Engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells^ 4 , 5 , 6 , 7 . Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The Engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated Engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between Engulfment and tissue homeostasis in vivo . Cell death by apoptosis is crucial for tissue development and function, and occurs throughout life. Apoptotic cells must be cleared by phagocytic cells, but the mechanisms that regulate cell clearance in vivo remain unclear. Here, a conserved Engulfment protein, ELMO1, is shown to be required for the phagocytic clearance of apoptotic germ cells by Sertoli cells in mouse testes. The findings make a compelling case for the relationship between Engulfment and tissue homeostasis in vivo .
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Phagocytosis of Apoptotic Cells Is Regulated by a UNC-73/TRIO-MIG-2/RhoG Signaling Module and Armadillo Repeats of CED-12/ELMO
Current biology : CB, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Bin Kuan Chou, Li-chun Cheng, Anne BlangyAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
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phagocytosis of apoptotic cells is regulated by a unc 73 trio mig 2 rhog signaling module and armadillo repeats of ced 12 elmo
Current Biology, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Li-chun Cheng, Anne Blangy, Bin Kuan Chou, John SondekAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
Anne Blangy - One of the best experts on this subject based on the ideXlab platform.
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Phagocytosis of Apoptotic Cells Is Regulated by a UNC-73/TRIO-MIG-2/RhoG Signaling Module and Armadillo Repeats of CED-12/ELMO
Current biology : CB, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Bin Kuan Chou, Li-chun Cheng, Anne BlangyAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
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phagocytosis of apoptotic cells is regulated by a unc 73 trio mig 2 rhog signaling module and armadillo repeats of ced 12 elmo
Current Biology, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Li-chun Cheng, Anne Blangy, Bin Kuan Chou, John SondekAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
Kit Pogliano - One of the best experts on this subject based on the ideXlab platform.
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the molecular architecture of Engulfment during bacillus subtilis sporulation
bioRxiv, 2018Co-Authors: Kanika Khanna, Kit Pogliano, Javier Lopezgarrido, Ziyi Zhao, Reika Watanabe, Yuan Yuan, Elizabeth VillaAbstract:The study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation in Bacillus subtilis using cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called Engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, Engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of Engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of Engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for Engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.
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Bistable forespore Engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.
PLoS computational biology, 2014Co-Authors: Nikola Ojkic, Kit Pogliano, Javier López-garrido, Robert G. EndresAbstract:To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast Engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like Engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable Engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with Engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful Engulfment. Indeed, decreasing the medium osmolarity in experiments prevents Engulfment in line with our predictions. Forespore Engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to Engulfment in other cell types, e.g. during phagocytosis in eukaryotes.
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peptidoglycan transformations during bacillus subtilis sporulation
Molecular Microbiology, 2013Co-Authors: Elitza I Tocheva, Kit Pogliano, Javier Lopezgarrido, Velocity H Hughes, Jennifer Fredlund, Erkin Kuru, Michael S Vannieuwenhze, Yves V Brun, Grant J JensenAbstract:While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands crosslinked by peptide bridges), it has remained unclear whether PG surrounds prespores during Engulfment. To clarify this issue, we generated a slender DponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout Engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout Engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive Engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negativelike PG layers as well as its thick, archetypal Grampositive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.
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cell wall synthesis is necessary for membrane dynamics during sporulation of bacillus subtilis
Molecular Microbiology, 2010Co-Authors: Pablo Meyer, Jennifer Gutierrez, Kit Pogliano, Jonathan DworkinAbstract:Summary During Bacillus subtilis sporulation, an endocytic-like process called Engulfment results in one cell being entirely encased in the cytoplasm of another cell. The driving force underlying this process of membrane movement has remained unclear, although compo- nents of the machinery have been characterized. Here we provide evidence that synthesis of peptidoglycan, the rigid, strength bearing extracellular polymer of bacteria, is a key part of the missing force-generating mechanism for Engulfment. We observed that sites of peptidoglycan synthesis initially coincide with the engulfing membrane and later with the site of engulf- ment membrane fission. Furthermore, compounds that block muropeptide synthesis or polymerization prevented membrane migration in cells lacking a component of the Engulfment machinery (SpoIIQ), and blocked the membrane fission event at the completion of Engulfment in all cells. In addition, these compounds inhibited bulge and vesicle forma- tion that occur in spoIID mutant cells unable to initiate Engulfment, as did genetic ablation of a protein that polymerizes muropeptides. This is the first report to our knowledge that peptidoglycan synthesis is nec- essary for membrane movements in bacterial cells and has implications for the mechanism of force gen- eration during cytokinesis.
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Dual localization pathways for the Engulfment proteins during Bacillus subtilis sporulation.
Molecular microbiology, 2007Co-Authors: Stefan Aung, Shinobu Chiba, Daniel H. Broder, Jonathan Shum, Angelica Abanes-de Mello, Jennifer Fredlund-gutierrez, Kit PoglianoAbstract:Engulfment in Bacillus subtilis is mediated by two complementary systems, SpoIID, SpoIIM and SpoIIP (DMP), which are essential for Engulfment, and the SpoIIQ-SpoIIIAGH (Q-AH) zipper, which provides a secondary Engulfment mechanism and recruits other proteins to the septum. We here identify two mechanisms by which DMP localizes to the septum. The first depends on SpoIIB, which is recruited to the septum during division and provides a septal landmark for efficient DMP localization. However, sporangia lacking SpoIIB ultimately localize DMP and complete Engulfment, suggesting a second mechanism for DMP localization. This secondary targeting pathway depends on SpoIVFA and SpoIVFB, which are recruited to the septum by the Q-AH zipper. The absence of a detectable localization phenotype in mutants lacking only SpoIVFAB (or Q-AH) suggests that SpoIIB provides the primary DMP localization pathway while SpoIVFAB provides a secondary pathway. In keeping with this hypothesis, the spoIIB spoIVFAB mutant strain has a synergistic Engulfment defect at septal thinning (which requires DMP) and is completely defective in DMP localization. Thus, the Q-AH zipper both provides a compensatory mechanism for Engulfment when DMP activity is reduced, and indirectly provides a compensatory mechanism for septal localization of DMP when its primary targeting pathway is disrupted.
Colin D. Debakker - One of the best experts on this subject based on the ideXlab platform.
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Phagocytosis of Apoptotic Cells Is Regulated by a UNC-73/TRIO-MIG-2/RhoG Signaling Module and Armadillo Repeats of CED-12/ELMO
Current biology : CB, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Bin Kuan Chou, Li-chun Cheng, Anne BlangyAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
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phagocytosis of apoptotic cells is regulated by a unc 73 trio mig 2 rhog signaling module and armadillo repeats of ced 12 elmo
Current Biology, 2004Co-Authors: Colin D. Debakker, Lisa B. Haney, Cynthia Grimsley, Jason M. Kinchen, Doris Klingele, Pei Ken Hsu, Li-chun Cheng, Anne Blangy, Bin Kuan Chou, John SondekAbstract:Abstract Background: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in Engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during Engulfment and cell migration. However, the components upstream of ELMO and Dock180 during Engulfment remain elusive. Results: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during Engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. Conclusions: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in Engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO → RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during Engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during Engulfment.
Shigekazu Nagata - One of the best experts on this subject based on the ideXlab platform.
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Tim4- and MerTK- mediated Engulfment of apoptotic cells by mouse resident peritoneal macrophages
Molecular and cellular biology, 2014Co-Authors: Chihiro Nishi, Satoshi Toda, Katsumori Segawa, Shigekazu NagataAbstract:Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage Engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and Engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or Engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong Engulfment activity. Taken together, these results indicate that the Engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell Engulfment.
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Two-Step Engulfment of Apoptotic Cells
Molecular and cellular biology, 2011Co-Authors: Satoshi Toda, Rikinari Hanayama, Shigekazu NagataAbstract:Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers Engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the Engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the Engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates Engulfment in coordination with MFG-E8. A similar two-step Engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated Engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the Engulfment of apoptotic cells by macrophages.
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imaging of rab5 activity identifies essential regulators for phagosome maturation
Nature, 2008Co-Authors: Masahiro Kitano, Michio Nakaya, Shigekazu Nagata, Takeshi Nakamura, Michiyuki MatsudaAbstract:Apoptotic cells are engulfed by phagocytes in a process that involves the GTPase Rab5. This paper shows that Rab5 promotes phagosome maturation, and Rab5 activation is induced by the guanine nucleotide exchange factor Gapex-5, which is recruited to phagosomes via microtubules. Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response1,2. Rab5 is known as a key regulator of the early endocytic pathway3 and we have recently shown that Rab5 is also implicated in apoptotic cell Engulfment4; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the Engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the Engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the Engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.
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opposite effects of rho family gtpases on Engulfment of apoptotic cells by macrophages
Journal of Biological Chemistry, 2006Co-Authors: Michio Nakaya, Masato Tanaka, Yasutaka Okabe, Rikinari Hanayama, Shigekazu NagataAbstract:The efficient Engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the Engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the Engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the Engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the Engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient Engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated Engulfment.
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Opposite Effects of Rho Family GTPases on Engulfment of Apoptotic Cells by Macrophages
The Journal of biological chemistry, 2006Co-Authors: Michio Nakaya, Masato Tanaka, Yasutaka Okabe, Rikinari Hanayama, Shigekazu NagataAbstract:The efficient Engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the Engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the Engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the Engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the Engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient Engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the αv β3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated Engulfment.