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Reddien, P. W. & Horvitz, H. R. CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nature Cell Biol. 2, 131-136
Abstract
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.
... During apoptotic cell phagocytosis in C. elegans, CED-10 Rac cooperates with CED-2 CrkII, an SH2-SH3-SH3 adaptor protein, and CED-5 DOCK180 (Reddien and Horvitz, 2000;Wu and Horvitz, 1998). CED-5 DOCK180 functions upstream of CED-10 Rac for phagocytosis (Reddien and Horvitz, 2000), and mammalian DOCK180 can increase the amount of GTPbound Rac1 in cell culture (Kiyokawa et al., 1998;Nolan et al., 1998); DOCK180 probably functions as a Rac activator. ...
... During apoptotic cell phagocytosis in C. elegans, CED-10 Rac cooperates with CED-2 CrkII, an SH2-SH3-SH3 adaptor protein, and CED-5 DOCK180 (Reddien and Horvitz, 2000;Wu and Horvitz, 1998). CED-5 DOCK180 functions upstream of CED-10 Rac for phagocytosis (Reddien and Horvitz, 2000), and mammalian DOCK180 can increase the amount of GTPbound Rac1 in cell culture (Kiyokawa et al., 1998;Nolan et al., 1998); DOCK180 probably functions as a Rac activator. Myoblast city, a DOCK180-like molecule, acts in myoblast fusion in Drosophila, a process that is sensitive to gain-offunction and dominant-negative Rac activity (Luo et al., 1994;Nolan et al., 1998). ...
... The full-length mig-2 cDNA was derived from the clone yk344c5 (kindly provided by Y. Kohara). The ced-10 cDNA has been previously described (Reddien and Horvitz, 2000). gfp expression vectors were kindly provided by A. Fire. ...
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.
... Then CED-6 interacts with CED-1 to transduce engulfment signals to downstream effectors. CED-7, an ABC transporter homolog, can expose PS to the surface of apoptotic cell [5,9]. In the ced-2/5/12 pathway, CED-2, an adaptor protein containing SH2 and SH3 domains, activates the CED-5/CED-12 complex [5,9]. ...
... CED-7, an ABC transporter homolog, can expose PS to the surface of apoptotic cell [5,9]. In the ced-2/5/12 pathway, CED-2, an adaptor protein containing SH2 and SH3 domains, activates the CED-5/CED-12 complex [5,9]. The CED-5/CED-12 complex acts as a bipartite nucleotide exchange factor to activate Rac GTPase CED-10, which in turn causes rearrangement of the actin cytoskeleton for cell corpse engulfment [5,9]. ...
... In the ced-2/5/12 pathway, CED-2, an adaptor protein containing SH2 and SH3 domains, activates the CED-5/CED-12 complex [5,9]. The CED-5/CED-12 complex acts as a bipartite nucleotide exchange factor to activate Rac GTPase CED-10, which in turn causes rearrangement of the actin cytoskeleton for cell corpse engulfment [5,9]. The ced-1/6/7 and ced-2/5/12 pathways lead to the internalization of apoptotic cell and the formation of phagosomes [7]. ...
Appropriate clearance of dead cells generated by apoptosis is critical to the development of multicellular organisms and tissue homeostasis. In mammals, the removal of apoptotic cell is mediated by polarized monocyte/macrophage populations of the innate immune system. The innate immune system is essential for anti-viral and anti-microbial defense. However, our current understanding of the relationship between apoptotic cell clearance and the innate immune response has remained rather limited. Here, we study how apoptotic cell clearance programs contribute to the innate immune response in C. elegans. We find apoptotic cell clearance mutant worms are more resistant to pathogenic bacteria of Pseudomonas aeruginosa PA14 and Salmonella typhimurium SL1344 due to significant upregulation of innate immune-dependent pathogen response genes. In addition, genetic epistasis analysis indicates that defects in apoptotic cell clearance can activate the innate immune response through PMK-1 p38 MAPK and MPK-1/ERK MAPK pathways in C. elegans. Taken together, our results provide evidence that insufficient clearance of apoptotic cell can protect Caenorhabditis elegans from bacterial infection through innate immune response activation.
... Salmonella encodes two proteins (SopE and SptP) which co-ordinate their uptake by sequentially activating and then antagonising die function of host cell Cdc42 and Rac (Hardt et al., 1998;Fu and Galan, 1999). Finally, the Rho GTPases are also emerging as critical components for the engulfinent of apoptotic cells in organisms ranging fi*om C.elegans to mammals (Reddien and Horvitz, 2000;Leverrier and Ridley, 2001). ...
... elegans (Nolan et al., 1998;Wu and Horvitz, 1998;Reddien and Horvitz, 2000). However, neither is a known exchange factor and so far, no GEF has been isolated using the genetic screens. ...
... How this occurs is unknown. Two gene products acting upstream of Rac, Ced2/CrkII and Ced-5/DOCKl 80 have been identified through the genetic analysis of apoptotic cell uptake in C. elegans (Reddien and Horvitz, 2000). Crkll and DOCK180 (180kDa protein downstream of CRK) both are adapter proteins (Hasegawa et al., 1996;Kiyokawa et al., 1997). ...
Phagocytosis is the process whereby cells bind and engulf particulate materials via the localised remodelling of the actin cytoskeleton. Small GTPases of the Rho family have been widely implicated in co-ordinating actin dynamics during diverse cellular processes, including phagocytosis, yet the signalling mechanisms controlling their recruitment and activation remain unclear. This thesis investigates the signalling pathways involved in regulating small GTPase function during phagocytosis via two distinct receptors, the FcγR, which is known to require Cdc42 and Rac and the complement receptor 3 (CR3, αMβ2, CD11b/CD18), which is known to couple to Rho. Data is presented showing that in response to FcγR ligation, Cdc42 and Rac are activated. Moreover, the guanine nucleotide exchange factor, Vav translocates to nascent phagosomes and promotes GTP-loading on Rac, but not Cdc42. The Vav induced Rac activation proceeds independently of Cdc42 function suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. This suggests that Rac is recruited to Fcγ membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac. In contrast, phagocytosis via the integrin receptor CR3 (αMβ2) proceeds via a two-step mechanism involving inside-out signalling to activate the receptor and ligand directed engulfment. Evidence supporting independent roles for the cytoplasmic tails of CR3 in regulating these activities is presented, with the β2 (CD18) tail directing receptor activation and the αM (CD11b) tail promoting the activation of Rho, necessary for engulfment.
... ced-10 alleles were originally isolated in genetic screens for mutants that are unable to execute apoptotic corpse engulfment [34,35]. It was further shown that CED-10 acts in engulfing cells to coordinate cytoskeletal remodeling required for engulfment [36]. Two viable alleles isolated from these screens, n3246 and n1993, affect the Switch 2 (G60R) and membrane targeting regions (V190G), respectively ( Fig 3A). ...
... We wanted to determine whether these differential phenotypic consequences are also observed for CED-10 function outside the nervous system. Therefore, we compared the effect of the rp100, n3246 and n1993 alleles in other well-characterized ced-10 phenotypes-apoptotic corpse engulfment and fecundity (Fig 3D and 3E) [16,36]. The number of persistent apoptotic cell corpses was counted in the heads of freshly hatched larval stage 1 (L1) animals harboring mutations in three ced-10 alleles ( Fig 3D). ...
... The number of persistent apoptotic cell corpses was counted in the heads of freshly hatched larval stage 1 (L1) animals harboring mutations in three ced-10 alleles ( Fig 3D). As previously reported, multiple persistent cell corpses were present in n3246 and n1993 mutant animals ( Fig 3D) [36]. In stark contrast, we did not detect any persistent cell corpses in rp100 mutant animals, as in wild type animals ( Fig 3D). ...
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.
... ced-10 alleles were originally isolated in genetic screens for mutants that are unable to execute apoptotic corpse engulfment 34,35 . It was further shown that CED-10 acts in engulfing cells to coordinate cytoskeletal remodeling required for engulfment 36 . Two viable alleles isolated from these screens, n3246 and n1993, affect the Switch 2 (G60R) and membrane targeting regions (V190G), respectively ( Figure 3A). ...
... We wanted to determine whether these differential phenotypic consequences are also observed for CED-10 function outside the nervous system. Therefore, we compared the effect of the rp100, n3246 and n1993 alleles in other well-characterized ced-10 phenotypesapoptotic corpse engulfment and fecundity ( Figure 3D-E) 16,36 . The number of persistent apoptotic cell corpses was counted in the heads of freshly hatched larval stage 1 (L1) animals harboring mutations in three ced-10 alleles ( Figure 3D). ...
... The number of persistent apoptotic cell corpses was counted in the heads of freshly hatched larval stage 1 (L1) animals harboring mutations in three ced-10 alleles ( Figure 3D). As previously reported, multiple persistent cell corpses were present in n3246 and n1993 mutant animals ( Figure 3D) 36 . In stark contrast, we did not detect any persistent cell corpses in rp100 mutant animals, as in wild type animals ( Figure 3D). ...
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.
... Previous work showed that rho-1 does not play a major role in ExCa outgrowth (Marcus-Gueret et al., 2012); thus, we focused our analysis on the three other Rho/ Rac-encoding genes. We analyzed several ced-10 loss-of-function alleles: a genomic deletion, hereafter ced-10(0), resulting in maternally rescued homozygous-sterile animals; a mutation in the C-terminal 'CAAX' box, which is required for CED-10 membrane localization; and mutations in the 'switch 1' or 'switch 2' regions, which interact with upstream regulators and/or downstream effectors (see Fig. 4A; Reddien and Horvitz, 2000;Lundquist et al., 2001;Zhuravlev et al., 2017;Nørgaard et al., 2018). These loss-of-function mutations all resulted in significant ExCa shortening (Fig. 4B), indicating that ced-10 promotes ExCa outgrowth. ...
... Scale bars: 100 µm. Right: Quantification of ExCa outgrowth in control animals and in animals homozygous for: ced-10 loss-of-function mutations affecting the switch 1 region [ced-10(rp100); Norgaard et al., 2018], the CAAX sequence or the switch 2 regions [ced-10(n1993) or ced-10(n3246), respectively; see Reddien and Horvitz, 2000), and the ced-10(0) deletion. (C) ExCa outgrowth in control and in animals homozygous for: a mig-2 mutation affecting the second GBM, leading to reduced function (rf ), an early stop predicted to be a mig-2(0) mutant, and an activating (act) mutation [mig-2(gm38), mig-2(mu28) and mig-2(gm103), respectively; see Forrester and Garriga, 1997;Zipkin et al., 1997;Forrester et al., 1998]. ...
Chloride intracellular channels (CLICs) are conserved proteins whose cellular and molecular functions remain mysterious. An important insight into CLIC function came from the discovery that C. elegans EXC-4/CLIC regulates morphogenesis of the excretory canal (ExCa) cell, a single-cell tube. Subsequent work showed that mammalian CLICs regulate vascular development and angiogenesis, and human CLIC1 can rescue exc-4 mutants, suggesting conserved function in biological tube formation (tubulogenesis) and maintenance. However, the cell behaviors and signaling pathways regulated by EXC-4/CLICs during tubulogenesis in vivo remain largely unknown. We report a new exc-4 mutation, affecting a C-terminal residue conserved in virtually all metazoan CLICs, that revealed a specific role for EXC-4/CLIC in ExCa outgrowth. Cell culture studies suggest a function for CLICs in heterotrimeric G-protein (Ga/b/g)-Rho/Rac signaling, and Rho-family GTPases are common regulators of cell outgrowth. Using our new exc-4 mutant we describe a previously unknown function for Ga-encoding genes (gpa-12/Ga12/13, gpa-7/Gai, egl-30/Gaq, and gsa-1/Gas), ced-10/Rac, and mig-2/RhoG in EXC-4-mediated ExCa outgrowth. Our results demonstrate that EXC-4/CLICs are primordial players in Ga-Rho/Rac-signaling—a pathway critical for tubulogenesis in C. elegans and in vascular development.
... D. melanogaster Rab14 regulates Staphylococcus aureus phagocytosis and participates in the phagosome maturation (Garg & Wu, 2014). Rac regulates apoptotic cells phagocytosis in Caenorhabditis elegans (Reddien & Horvitz, 2000). Rac, Rho, and Rab can regulate the hemocytes phagocytic capacity to bacteria in the Mediterranean fruit fly Ceratitis capitata and Kuruma shrimp Marsupenaeus japonicus (Soldatos et al., 2003;Zong et al., 2008). ...
... Apart from its role in egg development regulation in females, MmRho1 facilitates the development of the eggs and larvae of the wasp in its host by suppressing host cellular immune responses. Studies revealed that small G proteins from different families participate in cellular immune responses (Reddien & Horvitz, 2000;Soldatos et al., 2003;Zettervall et al., 2004;Williams et al., 2005;Williams et al., 2006;Avet-Rochex et al., 2007;Zong et al., 2008;Shim et al., 2010;Lee et al., 2011;Ye et al., 2012;Garg & Wu, 2014;Li et al., 2015a). Rab3 in the cotton bollworm mediates cellular immune responses, including phagocytosis, nodulation, and encapsulation (Li et al., 2015a). ...
Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast two-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses. This article is protected by copyright. All rights reserved.
... The second main pathway comprises the proteins CED-2/ CrkII, CED-5/DOCK180, CED-10/Rac GTPase, and CED-12/ ELMO (Reddien and Horvitz 2000;Gumienny et al., 2001;Wu et al., 2001;, which are thought to function together as a guanine nucleotide exchange factor (GEF) signaling module to control CED-10/Rac GTPase activity. CED-10/Rac GTPase is thought to control actin cytoskeletal assembly during the extension of the engulfing cell pseudopods around the dying cell. ...
... CED-5/DOCK180 and CED-12/ELMO have been shown to act downstream of at least 3 receptors involved in cell corpse recognition. These include PSR-1 (Reddien and Horvitz 2000;Wang et al., 2003), the Frizzled homolog MOM-5, and the integrin heterodimer INA-1/PAT-3 (Wang et al., 2003;Cabello et al., 2010;Hsu and Wu 2010). A partially redundant engulfment pathway to PSR-1 consists of the secreted PtdSer-binding protein TTR-52/transthyretin (Wang et al., 2010) the lipid-binding protein NRF-5 (Zhang et al., 2012), the membrane-bound CED-7/ABC transporter (Wu and Horvitz 1998), the transmembrane receptor CED-1/MEGF10 , and the intracellular adaptor CED-6/GULP (Liu and Hengartner 1998). ...
Here we highlight the increasingly divergent functions of the Caenorhabditis elegans cell elimination genes in the nervous system, beyond their well-documented roles in cell dismantling and removal. We describe relevant background on the C. elegans nervous system together with the apoptotic cell death and engulfment pathways, highlighting pioneering work in C. elegans . We discuss in detail the unexpected, atypical roles of cell elimination genes in various aspects of neuronal development, response and function. This includes the regulation of cell division, pruning, axon regeneration, and behavioral outputs. We share our outlook on expanding our thinking as to what cell elimination genes can do and noting their versatility. We speculate on the existence of novel genes downstream and upstream of the canonical cell death pathways relevant to neuronal biology. We also propose future directions emphasizing the exploration of the roles of cell death genes in pruning and guidance during embryonic development.
... 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]. ...
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]. ...
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.
... CED-5 is a huge bridging protein (1781 amino acids) similar to mammalian DOCK180: CED-5 contains a Docker domain that specifically binds nucleotide-free Rac, and CED-5 physically interacts with CED-2 and CED-12 (Gumienny et al., 2001;Wu and Horvitz, 1998b). ced-2 encodes an adaptor protein with a SH2-domain and two SH3-binding sites, and is similar to the mammalian protein CrkII (Reddien and Horvitz, 2000). CrkII can regulate cell shape and motility, and is also known to directly bind DOCK180 (Klemke et al., 1998). ...
... CED-10 is similar to Rac1, a small GTPase that controls cell morphology by regulating the cytoskeleton (Reddien and Horvitz, 2000). Genetically, ced-10 acts downstream of ced-2, -5, and -12 because its over-expression rescues the engulfment phenotype of these different mutant strains. ...
Warum werden Zellen geboren, die später Selbstmord begehen? Welches sind die Signale, die dieses Zellschicksal bestimmen? Diese Fragen, die Biologen seit Jahrzehnten faszinieren, bleiben immer noch weitgehend unbeantwortet. Während meiner Studien für die Doktorarbeit habe ich untersucht, wie sich in der Keimbahn von C.elegans eine Zelle zwischen Leben und Tod entscheidet, um das genetische Netzwerk zu finden, welches Apoptose in Tieren reguliert. Ich verwendete sowohl forward als auch reverse genetisch Methoden und identifizierte einige neue Gene, welche entweder das Überleben von Keimzellen fördern oder Apoptose in der Keimbahn von C.elegans auslösen. Da die apoptotischen Kaskaden konserviert sind bringt die weitere Charakterisierung der Wurmgene, die in dieser Dissertation präsentiert werden, auch Erkenntnisse über die Mechanismen, welche genomische Stabilität, p53 Aktivierung und Fertilität in Säugetieren kontrollieren. Why are some cells born to eventually commit suicide? And what are the cues that determine this cell fate? These questions, which have fascinated biologists for decades, still remain largely unanswered. During my doctoral studies, I have investigated the life-versus-death decision of cells in the germ line of C. elegans to dissect the genetic network that regulates apoptosis in animals. Using both forward and reverse genetic approaches, I identified several new genes that either promote germ cell survival or trigger germline apoptosis in C. elegans. Because apoptotic pathways are conserved, further characterization of the worm genes presented in this dissertation will shed light onto the mechanisms that control genomic stability, p53 activation, and fertility in mammals.
... In addition, CED-7 may facilitate adhesion between these two cells by transporting adhesion-related molecules to the cell surface 26 . The other involves INA-1/PAT-3, PSR-1 (phosphatidylserine receptor), MOM-5 (Frizzled), CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), and CED-10 (Rac GTPase) [28][29][30][31][32][33][34][35][36][37] . INA-1/PAT-3, PSR-1, and MOM-5 receptors transduce the "eat-me" signal through CED-2 [34][35][36] . ...
... INA-1/PAT-3, PSR-1, and MOM-5 receptors transduce the "eat-me" signal through CED-2 [34][35][36] . As a canonical component, CED-2 recruits CED-5 and CED-12 proteins to the cell membrane of engulfing cells, where CED-5 and CED-12 function together as a guanine nucleotide exchange factor to facilitate the exchange of GDP for GTP on CED-10, leading to cytoskeleton rearrangement and engulfment of dying cells [28][29][30][31][32][33]37 . ...
Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury.
... Several other key components act in this engulfment pathway alongside CED-1: CED-7, an ABC transporter homolog that exposes PS on the sur- face of apoptotic cells; CED-6, a cytoplasmic adaptor for CED-1; and DYN-1, an ortholog of the large GTPase dynamin that promotes "focal exocytosis" during pseudopod extension and stabilizes the cytoskeleton underneath extending pseudopods in response to CED-1 activation [8,28,29,30]. In the other canonical engulfment pathway, CED-2 regulates the activity of the CED-5/CED-12 complex, presumably through its N-terminus that contains SH2 and SH3 domains [31][32][33][34][35]. The CED-5/CED-12 complex, in turn, functions as a bipartite nucleotide exchange factor to activate the Rac GTPase CED-10 [31,33]. ...
... Several other key components act in this engulfment pathway alongside CED-1: CED-7, an ABC transporter homolog that exposes PS on the sur- face of apoptotic cells; CED-6, a cytoplasmic adaptor for CED-1; and DYN-1, an ortholog of the large GTPase dynamin that promotes "focal exocytosis" during pseudopod extension and stabilizes the cytoskeleton underneath extending pseudopods in response to CED-1 activation [8,28,29,30]. In the other canonical engulfment pathway, CED-2 regulates the activity of the CED-5/CED-12 complex, presumably through its N-terminus that contains SH2 and SH3 domains [31][32][33][34][35]. The CED-5/CED-12 complex, in turn, functions as a bipartite nucleotide exchange factor to activate the Rac GTPase CED-10 [31,33]. CED-10 promotes the reorganiza- tion of the actin cytoskeleton and the extension of pseudopods around cell corpses [34,35]. ...
In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.
... In migrating cells, Rac proteins generally function downstream of a variety of receptors to promote actin polymerization, protrusion dynamics, and motility (1,4,47). Rac has also long been known to stimulate macropinocytosis, which is fluid engulfment (1), as well as phagocytosis (30,48,49). Proteomic studies have identified hundreds of different proteins within phagosomes from multiple species but surprisingly little overlap from one cell type to the next (32). ...
The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2 E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2 E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2 +/E62K bone-marrow-derived macrophages also cannibalize primary Rac2 +/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2 +/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2 E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2 +/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.
... In the ced-1/6/7 pathway [4][5][6], the phagocytic receptor CED-1 recognizes 'eat-me' signal phosphatidylserine (PS) [7][8][9]. In the ced-2/5/12 pathway [10][11][12][13], several receptors including the phosphatidylserine receptor PSR-1 [14], MOM-5, and integrin INA-1 [15] are proposed to recognize and bind to the PS. ...
As the final step in apoptosis, apoptotic cells (ACs) are swiftly removed by specialized phagocytes, such as macrophages, or nonprofessional phagocytes, such as epidermal cells. Genetic studies of model organisms such as Caenorhabditis elegans have helped to elucidate the mechanisms of AC clearance and the underlying causes of disorders related to dysregulation of these pathways. C. elegans possesses six class B scavenger receptor homologs, but it is unknown if they affect apoptosis. Here we show that only the loss of function of scav-3, the C. elegans homolog of human LIMP-2, resulted in a considerable accumulation of cell corpses, which was caused by a failure in degradation rather than engulfment. SCAV-3 was found to be widely distributed and localized in lysosomes to maintain the integrity of the lysosomal membrane. Further study revealed that loss of scav-3 had no effect on phagosome maturation or the recruitment of lysosomes to phagosomes carrying cell corpses. Moreover, we discovered that the hydrolytic enzymes contained in the lysosomes were reduced in phagosomes in scav-3 mutants. Thus, hydrolases may leak from the damaged lysosome during phagolysosome formation due to the loss of scav-3 function, which reduces lysosome digestion activity and thus directly contributes to the elimination of apoptotic cells.
... Macrophages integrate signals from ACs to promote cytoskeletal rearrangement, a process that has been studied in the model organisms C. elegans and Drosophila, as well as in mammals. In C. elegans, upstream signals were found to converge in two parallel and independent signaling pathways:CED-2, CED-5, CED-12 pathway and CED-1, CED-6, CED-7 pathway which both subsequently activate CED-10, an evolutionarily highly conserved GTPase (Park and Kim, 2017) and thus stimulates skeletal rearrangement to form phagocytic vesicles (Wu and Horvitz, 1998a;Wu and Horvitz, 1998b;Liu and Hengartner, 1998;Reddien and Horvitz, 2000;Gumienny et al., 2001). CED-2/CED-5/CED-12 homologous signaling pathways in Drosophila and mouse were CG1587/myoblast city/Dmel, RKII/ Dock180/ELMO1, and CED-1/CED-6 homologous signaling pathways were Drpr/dCed-6, MEGF10/GULP1 (Zheng et al., 2017), which are are highly conserved to regulate ACs clearance. ...
The human body generates 10–100 billion cells every day, and the same number of cells die to maintain homeostasis. The genetically controlled, autonomously ordered cell death mainly proceeds by apoptosis. Apoptosis is an important way of programmed cell death in multicellular organisms, timely and effective elimination of apoptotic cells plays a key role in the growth and development of organisms and the maintenance of homeostasis. During the clearance of apoptotic cells, transcription factors bind to specific target promoters and act as activators or repressors to regulate multiple genes expression, how transcription factors regulate apoptosis is an important and poorly understood aspect of normal development. This paper summarizes the regulatory mechanisms of transcription factors in the clearance of apoptotic cells to date.
... It acts via a phosphatidylserine signal present at the cell surface of apoptotic cells. An apoptotic cell-dependent attractive signal was also demonstrated in mammals (named the "eat-me signal" or "come-and-get-me signal") (Reddien and Horvitz, 2000;Lauber et al., 2003;Ravichandran, 2003;Ravichandran, 2010). In addition, the migration of primordial germ cells during the metamorphosis of the ascidian Ciona intestinalis depends on caspases and is correlated with the speed of apoptotic wave propagation during tail regression, suggesting apoptoticdependent migration (Krasovec et al., 2019;Krasovec et al., 2021a). ...
... Formation of the complex induces cell spreading, phagosome formation, cell migration, and Rac activation [10][11][12][13]. Studies in C. elegans demonstrated that the worm versions of Crk (CED-2) and Dock180 (CED-5) interact and that they act upstream of Rac (CED-10) in cells that are engulfing apoptotic corpses [14]. Overexpression of dominant negative Rac, or Crk mutants unable to bind p130 Cas or Dock180, prevents engulfment and cell migration [12,13]. ...
Dock1, originally Dock180, was the first identified member of the Dock family of GTPase Exchange Factors. Early biochemical and genetic studies of Dock180 elucidated the functions and regulation of Dock180 and informed our understanding of all Dock family members. Dock180 activates Rac to stimulate actin polymerization in response to signals initiated by a variety of receptors. Dock180 dependent Rac activation is essential for processes such as apoptotic cell engulfment, myoblast fusion, and cell migration during development and homeostasis. Inappropriate Dock180 activity has been implicated in cancer invasion and metastasis and in the uptake of bacterial pathogens. Here, we give an overview of the history and current understanding of the activity, regulation, and impacts of Dock180.
... We previously reported that CHDP-1 acts through the small GTPase CED-10/Rac1 in the BDU and PLM neurons [16]. To examine whether CHDP-1 regulates dendritic cortical actin assembly in PVD neurons via a similar mechanism, we examined dendrite morphogenesis in n3246, a strong loss-of-function allele of ced-10 [42]. ced-10 (n3246) mutants did not show an increased number of 2 o and 3 o branches or faint staining of the 2 o branches by the myr-GFP reporter. ...
Cortical actin, a thin layer of actin network underneath the plasma membranes, plays critical roles in numerous processes, such as cell morphogenesis and migration. Neurons often grow highly branched dendrite morphologies, which is crucial for neural circuit assembly. It is still poorly understood how cortical actin assembly is controlled in dendrites and whether it is critical for dendrite development, maintenance and function. In the present study, we find that knock-out of C. elegans chdp-1, which encodes a cell cortex-localized protein, causes dendrite formation defects in the larval stages and spontaneous dendrite degeneration in adults. Actin assembly in the dendritic growth cones is significantly reduced in the chdp-1 mutants. PVD neurons sense muscle contraction and act as proprioceptors. Loss of chdp-1 abolishes proprioception, which can be rescued by expressing CHDP-1 in the PVD neurons. In the high-ordered branches, loss of chdp-1 also severely affects the microtubule cytoskeleton assembly, intracellular organelle transport and neuropeptide secretion. Interestingly, knock-out of sax-1, which encodes an evolutionary conserved serine/threonine protein kinase, suppresses the defects mentioned above in chdp-1 mutants. Thus, our findings suggest that CHDP-1 and SAX-1 function in an opposing manner in the multi-dendritic neurons to modulate cortical actin assembly, which is critical for dendrite development, maintenance and function.
... Indeed, av[35 integrin is known to recruit the Crkli/Dock180/Rac1 signaling complex, thereby orchestrating cytoskeleton re-organisation. IVloreover, functional ablation of the C.elegans Crkll and Rac homologs impairs apoptotic cell clearance [60]. The plethora of different molecules involved in apoptotic cell engulfment suggests that his process is tightly regulated and may lead to a variety of different functional outcomes for the IDC, i.e., tolerance induction or priming of a T-cell response. ...
p>Agonistic monoclonal antibodies directed against murine CD40 can be used in experimental settings to induce anti-lymphoma T-cells that are able to affect primary tumour eradication in vivo . These anti-tumour CTL provide both protection against re-challenge with the original vaccinating tumour line and, furthermore, prevent relapse, thereby affecting long-term survival. Such therapeutic strategies are desirable in order to provide durable responses against human lymphomas without evolving adverse side-effects.
Here, we further investigate the mechanism by which anti-CD40 exerts its therapeutic activity and demonstrate that interactions via the TNFR family member 4-1BB contribute to the expansion of anti-tumour CTL and, hence, survival following immunotherapy of lymphoma; indeed, we show that all tumour-specific CTL generated during anti-CD40-induced immunotherapy express this molecule. Furthermore, we demonstrate for the first time that the murine lymphomas used in these studies invoke an immunological response prior to anti-CD40 administration which is characterised by the phenotypic maturation of DCs and the differentiation of CD8<sup>*</sup> T-cells, as well as the accumulation of these cells at the site of tumour. Importantly, the kinetics and magnitude of these responses are augmented by administration of anti-CD40 thereby suggesting that this mAb exerts its therapeutic activity by the boosting of a pre-existing ineffectual adaptive immune response. Data shown here suggest that enhanced DC maturation following administration of anti-CD40 to tumour-bearing animals may contribute to this effect and, hence, support the hypothesis that anti-CD40 operates at the axis of the professional APC in order to affect the rejection of both CD40-positive and CD40-negative tumours.
In addition, we show that both CD8<sup>*</sup> T-cell lines and immortalised hybridomas derived from tumour-reactive CD8<sup>*</sup> CTL can be established in vitro from animals in remission following anti-CD40-induced immunotherapy. These techniques are new to this laboratory and provide a basis for the screening of a lymphoma-derived cDNA library in order to identify the tumour rejection antigens operating in this system.</p
... The RAC GTPases have been known to play a broader role in developmental processes involving cell debris engulfment [64,65], migration, and axon guidance [53]. The RAC GTPases control these developmental processes through various downstream effectors of actin and microtubule cytoskeleton [55,66,67]. ...
Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C . elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects–territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.
... The reported roles in some mammalian cell types for Scribble in controlling cell migration through the small GTPases Rac and Cdc42 [7,28] prompted us to consider a similar mode of action for LET-413. C. elegans Rho-family GTPases have already been shown to be involved in many migratory events, such as long-range migration of the Q neuroblasts and the gonadal distal tip cells [44,52,65], morphogenetic changes in embryonic epidermal cells during dorsal intercalation and ventral closure [49,54,[66][67][68][69], and growth cone migration in axonal pathfinding [52,53,70]. In addition, RNAi-mediated inactivation of cdc-42 was shown to prevent seam cell outgrowth [51]. ...
The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C . elegans , the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.
... The RAC GTPases have been known to play a broader role in developmental processes involving cell debris engulfment (Ellis et al., 1991, Reddien andHorvitz, 2000), migration, axon guidance (Lundquist et al., 2001).The RAC GTPases control these developmental . CC-BY-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Neurons are vulnerable to physical insults which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used PVD neurons in C. elegans having stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites as well as the fusion among the higher-order branches as reported before. We quantified the regeneration pattern in three aspects territory length, number of branches and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signaling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidyl serine exposure/PS in dendrite regeneration. We found that neither regrowth nor fusion is affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase CED-10 and its upstream GEF TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.
... For one, Rho-family GTPases play critical roles in coordinating the cytoskeletal rearrangements that drive cellular protrusions (Ridley, 2011;Schaks et al., 2019;Warner et al., 2019). C. elegans Rho-family GTPases have already been shown to be involved in many migratory events, such as long-range migration of the Q neuroblasts and the gonadal distal tip cells (Lundquist et al., 2001;Reddien and Horvitz, 2000;Rella et al., 2016), morphogenetic changes in embryonic epidermal cells during dorsal intercalation and ventral closure (Ouellette et al., 2016;Patel et al., 2008;Walck-Shannon et al., 2015;Walck-Shannon et al., 2016;Wallace et al., 2018;Zilberman et al., 2017), and growth cone migration in axonal pathfinding (Dyer et al., 2010;Lundquist et al., 2001;Shakir et al., 2006). In addition, RNAi-mediated inactivation of cdc-42 was shown to prevent seam cell outgrowth (Welchman et al., 2007). ...
The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is mediated at least in part by the junctional component DLG-1 discs large, which appears to restrict protrusive activity to the apical domain. Finally, we demonstrate that the Rho-family GTPases CED-10 Rac and CDC-42 can regulate seam cell outgrowth and may also function downstream of LET-413. Our data uncover multiple essential functions for LET-413 in larval development and shed new light on the regulation of polarized outgrowth of the seam cells.
... This prompted us to return to the RNA-seq data and re-analyse the expression of genes that encode engulfment receptors in the Slc12a2-deficient phagocytes. In Slc12a2-deficient LR73 phagocytes, there was an increase in gene expression for integrin α v and integrin β 5 , which have been linked to efferocytosis, as well as molecules linked to integrin-dependent signalling, such as p130Cas (BCAR1), and the small GTPases RhoG and CrkI, which are linked genetically and biochemically to efferocytosis through α v β 5 integrin [48][49][50][51][52][53] (Fig. 6g). When we tested whether integrin-dependent interactions contributed to the increased efferocytosis observed with Slc12a2-deficient phagocytes, an RGDS peptide added before the addition of apoptotic targets partially inhibited the increased corpse binding of Slc12a2-deficient phagocytes (almost similar to the positive control for blocking with annexin V; Fig. 6h). ...
Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte ‘appetite’ and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This ‘switch’ to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK—which function upstream of SLC12A2—had a similar effect on efferocytosis. Collectively, the WNK1–OSR1–SPAK–SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.
... Surprisingly, RAB-35 functions in parallel to both of the canonical apoptotic cell clearance pathways, the ced-1/6/7/dyn-1 pathway and the ced-2/5/10/12 pathway [27]. The former pathway is composed of the ABC transporter homolog CED-7, which facilitates the exposure of the 'eat me' signal phosphatidylserine (PS), the phagocytic receptor CED-1, its adaptor CED-6, and the dynamin ortholog DYN-1 [30][31][32][33][34][35]; the latter pathway is defined by the Rac1 ortholog CED-10, its bipartite GEF composed of CED-5 and CED-12, and the adaptor protein CED-2 [36][37][38][39][40]. Consequently, RAB-35 defines a third and comparatively unexplored genetic pathway involved in phagocytosis [27]. ...
We recently identified the novel function of the small GTPase RAB-35 in apoptotic cell clearance in Caenorhabditis elegans, a process in which dying cells are engulfed and degraded inside phagosomes. We have found that RAB-35 functions in two separate steps of cell corpse clearance, cell corpse recognition and the initiation of phagosome maturation. During the latter process, RAB-35 facilitates the removal of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the membranes of nascent phagosomes and the simultaneous production of phosphatidylinositol-3-P (PI(3)P) on these same membranes, a process that we have coined the PI(4,5)P2 to PI(3)P shift. RAB-35 also promotes the recruitment of the small GTPase RAB-5 to the phagosomal surface. During these processes, the activity of RAB-35 is controlled by the candidate GTPase-activating protein (GAP) TBC-10 and the candidate guanine nucleotide exchange factor (GEF) FLCN-1. Overall, RAB-35 leads a third pathway during cell corpse clearance that functions in parallel to the two known pathways, one led by the phagocytic receptor CED-1 and the other led by the CED-10/Rac1 GTPase. Here, we further report that RAB-35 acts as a robustness factor that maintains the clearance activity and embryonic viability under conditions of heat stress. Moreover, we obtained additional evidence suggesting that RAB-35 acts upstream of RAB-5 and RAB-7. To establish a precise temporal pattern for its own dissociation from phagosomal surfaces, RAB-35 controls the removal of its own GAP. We propose that RAB-35 defines a largely unexplored initial phase of phagosome maturation.
... The first pathway is composed of ced-1, ced-6, and ced-7, the mammalian homologs of which are MEGF10 (multiple EGF-like domains 10), Gulp1 (phosphotyrosine-binding domain-containing engulfment adaptor protein 1), and ABCA1 (ABC binding cassette subfamily A member 1), respectively [57][58][59]. The second pathway comprises ced-2, ced-5, ced-10 and ced-12, for which the corresponding mammalian homologs are CrkII (CRK adaptor protein), DOCK1 (dedicator of cytokinesis 1; also referred to as DOCK180), Rac1 (Rac family small GTPase 1), and ELMO1 (engulfment and cell motility 1), respectively [60][61][62]. Park et al. demonstrated that the Gulp1-dependent signaling pathway is required for stabilin-mediated phagocytosis [63,64]. Gulp1, composed of a phosphotyrosine-binding (PTB) domain, a leucine zipper domain and a proline-rich domain, acts as an adaptor protein to transduce a signal for cytoskeletal rearrangement in several phagocytic receptors, including MEGF10, LRP1 (LDL receptor-related protein 1), PEAR1 (platelet endothelial aggregation receptor 1; also referred to as MEGF12 and JEDI), and SR-BI (scavenger receptor class B member 1) [58,[65][66][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.
... In Caenorhabditis elegans and zebrafish, Crk adapters play important developmental roles in cell migration, axon outgrowth, phagocytosis, and myoblast fusion. C. elegans Ced-2 regulates phagocytic clearance of apoptotic cells, cell migration, and motor neuron development as part of a Crk/DOCK180/Rac pathway (Reddien and Horvitz, 2000;Gumienny et al., 2001;Wu et al., 2001Wu et al., , 2002. In zebrafish, morpholino-mediated knockdown of either Crk or CrkL impairs fusion of fast-twitch myoblasts (Moore et al., 2007), while overexpressing Crk or CrkL enhances fusion. ...
Small SH2/SH3 adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The Crk family has tissue-specific roles in phagocytosis, cell migration and neuronal development, and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk's full role in embryogenesis. We circumvented these limitations with shRNA and CRISPR technology to assess Crk's function in Drosophila morphogenesis. We found Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the Arp2/3 complex, its regulator SCAR, and F-actin to actin caps and pseudocleavage furrows. Crk loss leads to loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.
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... CED-1 Rezeptoren sind an der Erkennung von Zellkörpern beteiligt, außerdem geben sie Signale weiter, die die Ummantelung des Zellkörpers sowie die Bildung des Phagosoms betreffen (Chen, Jian et al. 2013). CED-2 ist Bestandteil eines zweiten Rezeptor-vermittelten Signalweges, der über Aktivierung von CED-10 an der Polymerisierung von Aktinfilamenten beteiligt ist, was für die Internalisierung von Mittelkörpern bei C. elegans benötigt wird (Reddien and Horvitz 2000). Bei Störungen im CED1-oder CED-2 Signalweg, der bei C. elegans wesentlich an der Phagozytose beteiligt ist, ist ein Ausbleiben der Internalisierung von Mittelkörpern zu erwarten. ...
Bei der Teilung einer Zelle werden das Genom und die Zellbestandteile zwischen zwei Tochterzellen aufgeteilt. Dies erfordert verschiedene fein aufeinander abgestimmte Vorgänge. Unter anderem ist eine proteinreiche Struktur beteiligt, die 1891 entdeckt wurde: der Mittelkörper. In vorliegender Arbeit wurden gezielt gekennzeichnete Mittelkörperproteine analysiert und verschiedene Phasen des Transports unterschieden. Es erfolgten erstmals Messungen unter Nutzung der ZF1-Methode. Zudem wird anhand der ZF1-Technik nachgewiesen, dass im Rahmen der Zellteilung die Trennung der interzellulären Brücke zu beiden Seiten des Mittelkörpers stattfindet, woraufhin dieser nach extrazellulär abgegeben wird und über einen der Phagozytose ähnlichen und von Aktin abhängigen Mechanismus von einer Tochterzelle oder unverwandten Nachbarzelle aufgenommen wird.
... Two partly redundant, parallel apoptotic engulfment pathways recognize the PS 'eat-me' signal and mediate the clearance of dying cells by phagocytes (Kloditz et al., 2017) (Fig. 3). In the first, the phosphatidylserine receptor PSR-1/JMJD6 on the phagocytic membrane binds the exposed PS (Fadok et al., 2000;Yang et al., 2015) and activates an intracellular signaling pathway consisting of CED-2/CrkII, CED-5/ DOCK180, CED-12/ELMO, and finally CED-10/Rac1 to orchestrate the actin cytoskeletal rearrangements necessary for engulfing the dying cell (Gumienny et al., 2001;Reddien and Horvitz, 2000;Wang et al., 2003;Wu and Horvitz, 1998a). The second pathway includes the secreted PS- Fig. 2. Molecular changes associated with axonal fusion. ...
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.
... Dock1 is highly evolutionarily conserved across species and can specifically bind and activate Rac1 [13][14][15]. In vitro and in vivo studies in various model organisms have shown that Dock1 influences a variety of cytoskeletal-related cell processes such as phagocytosis and cell migration [16][17][18][19]. Thus, Dock1 represents an ideal intracellular candidate to study for a role in cell shape regulation. ...
Background:
In the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood.
Methods:
Through a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes.
Results:
We show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development.
Conclusions:
Together, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.
... Accumulating evidence demonstrated that the dying cells are able to reveal their presence to phagocytes (33). Specifically, based on the acquired data from Caenorhabditis elegans, it was realized that recruitment of phagocytes to the area of cell death can happen before cell apoptosis is completed (34,35). Expression of various "find me" signals by apoptotic cells can attract phagocytic cells through a chemotactic gradient (36). ...
Apoptosis happens continuously for millions of cells along with the active removal of apoptotic debris in order to maintain tissue homeostasis. In this respect, efferocytosis, i.e., the process of dead cell clearance, is orchestrated through cell exposure of a set of “find me,” “eat me,” and “tolerate me” signals facilitating the engulfment of dying cells through phagocytosis by macrophages and dendritic cells. The clearance of dead cells via phagocytes is of utmost importance to maintain the immune system tolerance to self-antigens. Accordingly, this biological activity prevents the release of autoantigens by dead cells, thus potentially suppressing the undesirable autoreactivity of immune cells and the appearance of inflammatory autoimmune disorders as systemic lupus erythematous and rheumatoid arthritis. In the present study, the apoptosis pathways and their immune regulation were reviewed. Moreover, efferocytosis process and its impairment in association with some autoimmune diseases were discussed.
The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase ( ced-10 ) and its GEF complex ( ced-5 /DOCK180, ced-2 /CrkII, ced-12 /ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation—the now-smaller DTC and the membranous patch of cellular material—localize important stem cell niche signaling (LAG-2/DSL ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long-term. Prior work found that Rac1 -/- ;Rac2 -/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.
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.
Programmed cell death (PCD) is crucial for normal development and homeostasis. Our first insights into the genetic regulation of apoptotic cell death came from in vivo studies in the powerful genetic model system of C. elegans. More recently, novel developmental cell death programs occurring both embryonically and post-embryonically, and sex-specifically, have been elucidated. Recent studies in the apoptotic setting have also shed new light on the intricacies of phagocytosis in particular. This review provides a brief historical perspective of the origins of PCD studies in C. elegans, followed by a more detailed description of non-canonical apoptotic and non-apoptotic death programs. We conclude by posing open questions and commenting on our outlook on the future of PCD studies in C. elegans, highlighting the importance of advanced imaging tools and the continued leveraging of C. elegans genetics both with classical and modern cutting-edge approaches.
Cell death in response to physiological cues is a feature of many normal tissues, and often occurs due to tissue damage or disease. Regardless of the reason, dying cells require their rapid removal from the tissue in order to maintain homeostasis in the tissue, prevent potential cytotoxic immunity against self-antigens, and to promote wound healing. Phagocytic removal of dying cells is referred to as efferocytosis, a process necessary for sterile wound healing. Interestingly, malignant tumor progression has long been compared to wound healing, and recent evidence demonstrates a molecular link between efferocytosis and tumor a ‘wound healing’ phenotype. We discuss the impact of efferocytosis in the normal breast and in breast cancers.
DOCK proteins are a family of multi-domain guanine nucleotide exchange factors (GEFs) that activate the RHO GTPases CDC42 and RAC1, thereby regulating several RHO GTPase-dependent cellular processes. DOCK proteins are characterized by the catalytic DHR2 domain (DOCKDHR2 ), and a phosphatidylinositol(3,4,5)P3 -binding DHR1 domain (DOCKDHR1 ) that targets DOCK proteins to plasma membranes. DOCK-family GEFs are divided into four sub-families (A to D) differing in their specificities for CDC42 and RAC1, and the composition of accessory signalling domains. Additionally, the DOCK-A and DOCK-B subfamilies are constitutively associated with ELMO proteins that auto-inhibit DOCK GEF activity. We review structural studies that have provided mechanistic insights into DOCK-protein function. These studies revealed how a conserved nucleotide sensor in DOCKDHR2 catalyses nucleotide exchange, the basis for how different DOCK proteins activate specifically CDC42 and RAC1, and sometimes both, and how up-stream regulators relieve the ELMO-mediated auto-inhibition. We conclude by presenting a model for full-length DOCK9 of the DOCK-D subfamily. The involvement of DOCK GEFs in a range of diseases highlights the importance of gaining structural insights into these proteins to better understand and specifically target them.
The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.
Development of the C. elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.
In the central nervous system (CNS), execution of programmed cell death (PCD) is crucial for proper neurodevelopment. However, aberrant activation of these pathways in adult CNS leads to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). How a cell dies is critical, as it can drive local immune activation and tissue damage. Classical apoptosis engages several mechanisms to evoke “immunologically silent” responses, whereas other forms of programmed death such as pyroptosis, necroptosis, and ferroptosis release molecules that can potentiate immune responses and inflammation. In ALS, a fatal neuromuscular disorder marked by progressive death of lower and upper motor neurons, several cell types in the CNS express machinery for multiple PCD pathways. The specific cell types engaging PCD, and ultimate mechanisms by which neuronal death occurs in ALS are not well defined. Here, we provide an overview of different PCD pathways implicated in ALS. We also examine immune activation in ALS and differentiate apoptosis from necrotic mechanisms based on downstream immunological consequences. Lastly, we highlight therapeutic strategies that target cell death pathways in the treatment of neurodegeneration and inflammation in ALS.
The sections in this article are
Introduction
Trichome Morphogenesis
Arabidopsis Trichome Development
Arabidopsis Trichome Morphogenesis
Genetics of Trichome Morphogenesis
Concluding Remarks
Acknowledgements
MicroRNAs (miRNAs) associate with argonaute (AGO) proteins to post-transcriptionally modulate the expression of genes involved in various cellular processes. Herein, we show that loss of the Caenorhabditis elegans AGO gene alg-2 results in rapid and significantly increased germ cell apoptosis in response to DNA damage inflicted by ionizing radiation (IR). We demonstrate that the abnormal apoptosis phenotype in alg-2 mutant animals can be explained by reduced expression of mir-35 miRNA family members. We show that the increased apoptosis levels in IR-treated alg-2 or mir-35 family mutants depend on a transient hyperactivation of the C. elegans ERK1/2 MAPK ortholog MPK-1 in dying germ cells. Unexpectedly, MPK-1 phosphorylation occurs downstream of caspase activation and depends at least in part on a functional cell corpse-engulfment machinery. Therefore, we propose a refined mechanism, in which an initial proapoptotic stimulus by the core apoptotic machinery initiates the engulfment process, which in turn activates MAPK signaling to facilitate the demise of genomically compromised germ cells.
Small SH2/SH3 adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The Crk family has tissue-specific roles in phagocytosis, cell migration and neuronal development, and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crks full role in embryogenesis. We circumvented these limitations with shRNA and CRISPR technology to assess Crks function in Drosophila morphogenesis. We found Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates levels of the Arp2/3 complex, its regulator SCAR, and F-actin in actin caps and pseudocleavage furrows. Crk loss leads to loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.
List of mutant alleles used in this study.
(PDF)
Intestinal epithelial cells play a critical role in nutrient absorption as well as in protection against infection by pathogenic microorganisms. The cells drop out in a few days, and regeneration occurs subsequently; cells are eliminated by apoptosis. Clearance of dead cells frequently occurs in the intestinal tract, and apoptotic cells and phagocytes cooperate to facilitate cell clearance quickly and efficiently. The complex signaling network for cell clearance is well-understood. In recent years, the mechanism of programmed cell death accompanied by autophagy has been elucidated, and it has become clear that autophagy is involved in inflammation and intestinal tract diseases. In this review, we discuss intestinal regeneration and intestinal diseases through phagocytic clearance and autophagy of apoptotic cells.
Purpose:
Rupture of lens cataract (RLC) is a hereditary mouse model that shows spontaneous rupture of the lens at the posterior pole at 45-100 days of age. The responsible gene for this phenotype was identified as Dock5, a guanine nucleotide exchange factor for small GTPase Rac1. This study was performed to elucidate the pathway initiating this phenotype.
Methods:
We examined the RNA expression by microarray in lens epithelial cells (LECs) from wild-type and RLC mice at the pre-rupture age of 21 days. We applied the list of altered genes to an Ingenuity Pathway Analysis (IPA) to predict the pathways that are altered upon dedicator of cytokinesis-5 (Dock5) protein loss. The activation status of the predicted pathways was examined by western blotting in the cultured epithelial cells treated with a Dock5 inhibitor.
Results:
The highest-scored network was "Antimicrobial Response, Inflammatory Response, Dermatological Diseases and Conditions." In that network, it is predicted that extracellular signal-regulated kinase (Erk) is activated in LECs from RLC mice. Our test confirmed that Erk was more phosphorylated in the LECs at the equator in both Dock5-knockout mice and RLC mice. In an in vitro experiment of the cultured epithelial cells, the inhibition of Dock5 activity significantly induced Erk activation. It was also confirmed that Akt (cellular homolog of murine thymoma virus akt8 oncogene, also called protein kinase B) and nuclear factor-kappa B (NFκB), predicted to be the key molecules in two other high-scoring networks by IPA, were activated upon Dock5 inhibition in the cultured epithelial cells.
Conclusions:
Dock5 participates in epithelial cell maintenance by regulating gene expression.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms.
There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly
conserved genes provides evidence for a regional organization of the chromosomes.
The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen in Drosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbc mutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotide-independent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes in Drosophila embryogenesis.
DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. Here, we have studied the
involvement of DOCK180 in integrin signaling. DOCK180 was neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells
and 3Y1 cells. We found that DOCK180 was phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also
in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlated with the binding of CrkII to p130Cas, which is a major CrkII SH2 domain-binding protein at focal adhesions. In a reconstitution experiment, expression of DOCK180
induced hyperphosphorylation of p130Cas and a concomitant increase in the amount of CrkII bound to p130Cas. Similarly, binding of DOCK180 to CrkII was also enhanced by the coexpression of p130Cas. Finally, we found that coexpression of p130Cas and CrkII with DOCK180 induced local membrane spreading and accumulation of DOCK180-CrkII-p130Cas complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130Cas complexes at focal adhesions.
During programmed cell death, cell corpses are rapidly engulfed. This engulfment process involves the recognition and subsequent phagocytosis of cell corpses by engulfing cells. How cell corpses are engulfed is largely unknown. Here we report that ced-5, a gene that is required for cell-corpse engulfment in the nematode Caenorhabditis elegans, encodes a protein that is similar to the human protein DOCK180 and the Drosophila melanogaster protein Myoblast City (MBC), both of which have been implicated in the extension of cell surfaces. ced-5 mutants are defective not only in the engulfment of cell corpses but also in the migrations of two specific gonadal cells, the distal tip cells. The expression of human DOCK180 in C. elegans rescued the cell-migration defect of a ced-5 mutant. We present evidence that ced-5 functions in engulfing cells during the engulfment of cell corpses. We suggest that ced-5 acts in the extension of the surface of an engulfing cell around a dying cell during programmed cell death. We name this new family of proteins that function in the extension of cell surfaces the CDM (for CED-5, DOCK180 and MBC) family.
Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.
We tested 42 tetrapeptides for their ability to bind to the rat brain p21ras protein farnesyltransferase as estimated by their ability to compete with p21Ha-ras in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-A1-A2-X, where positions A1 and A2 are occupied by aliphatic amino acids and position X is occupied by a COOH-terminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the A1 position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CAIL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein gamma subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21Ki-rasB was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltransferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.
A new member of the ras superfamily, designated CErac1 has been identified. The CErac1 cDNA clone was isolated from a Caenorhabditis elegans mixed stage library and encodes a protein of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. The CErac1 cDNA maps to a position on C. elegans chromosome IV in close proximity to cha-1, a choline acetyltransferase gene. The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kilobases). Their expression is developmentally regulated, that of the more abundant 1.7 kilobases being highest at the embryonic stage and decreasing dramatically during development with 10% of the embryonic level in adult nematodes. The glutathione-S-transferase/CErac1 fusion protein expressed in Escherichia coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity of the CErac1 protein is stimulated by human n-chimaerin, a GTPase-activating protein for p21 rac1. These data suggest a role of CErac1 in C. elegans early development. The conserved biochemical properties indicate that further characterization of CErac1 by genetic analysis will be helpful in elucidating not only its role in the signal transduction, but also the biological function of its mammalian homologues.
CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.
The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast fusion (Rushton et al., 1995. Development [Camb.]. 121:1979-1988). We describe herein the isolation and characterization of the mbc gene. The mbc transcript and its encoded protein are expressed in a broad range of tissues, including somatic myoblasts, cardial cells, and visceral mesoderm. It is also expressed in the pole cells and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a role in signal transduction from focal adhesions. Taken together, these results suggest the possibility that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal closure.
Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.
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.
At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.
DOCK180 is involved in integrin signaling through CrkII-p130(Cas) complexes. We have studied the involvement of DOCK180 in Rac1 signaling cascades. DOCK180 activated JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increased the amount of GTP-bound Rac1 in 293T cells. Coexpression of CrkII and p130(Cas) enhanced this DOCK180-dependent activation of Rac1. Furthermore, we observed direct binding of DOCK180 to Rac1, but not to RhoA or Cdc42Hs. Dominant-negative Rac1 suppressed DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling.
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.
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.
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.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
The ancestry of the cells in the hermaphrodite and male gonadal somatic structures of C. elegans has been traced from the two gonadal somatic progenitor cells (Z1 and Z4) that are present in the newly hatched larvae of both sexes. The lineages of Z1 and Z4 are essentially invariant. In hermaphrodites, they give rise to a symmetrical group of structures consisting of 143 cells, and in males, they give rise to an asymmetrical group of structures consisting of 56 cells. The male gonad can be distinguished from the hermaphrodite gonad soon after the first division of Z1 and Z4. However, the development of Z1 and Z4 in hermaphrodites shares several features in common with their development in males suggesting that the two programs are controlled by similar mechanisms. In the hermaphrodite lineage, a variability in the positions of two cells is correlated with a variability in the lineages of four cells. This variability suggests that cell-cell interaction may play a more significant role in organisms that develop by invariant lineages than has hitherto been considered. None of the somatic structures (e.g., uterus, spermatheca, vas deferens) develops as a clone of a single cell. Instead, cells that arise early in the Z1–Z4 lineage generally contribute descendants to more than one structure, and individual structures consist of descendants of more than one lineage.
Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.
GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.
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.
Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.
While determining the 5' ends of C. elegans actin mRNAs, we have discovered a 22 nucleotide spliced leader sequence. The leader sequence is found on mRNA from three of the four nematode actin genes. The leader also appears to be present on some, but not all, nonactin mRNAs. The actin mRNA leader sequence is identical to the first 22 nucleotides of a novel 100 nucleotide RNA transcribed adjacent, and in the opposite orientation, to the 5S ribosomal gene. The evidence suggests that the actin mRNA leader sequence is acquired from this novel nucleotide transcript by an intermolecular trans-splicing mechanism.
Mutations in two nonessential genes specifically block the phagocytosis of cells programmed to die during development. With few exceptions, these cells still die, suggesting that, in nematodes, engulfment is not necessary for most programmed deaths. Instead, these deaths appear to occur by cell suicide.
The mRNAs of six C. elegans genes are known to be trans-spliced to SL2. We report here that a similarly oriented gene is located 100-300 bp upstream of each. We present evidence that the genes in these clusters are cotranscribed and downstream mRNAs are formed by cleavage at the polyadenylation site and trans-splicing. From one three-gene cluster we isolated cDNA clones representing both polycistronic RNAs and mRNAs polyadenylated at the free 3' end created by trans-splicing, suggesting that polycistronic RNAs can be processed by trans-splicing. Several experiments indicate that SL2 trans-splicing is a consequence of a gene's downstream location in an operon. In particular, when an SL1-accepting gene was moved to a downstream location, its mRNA was trans-spliced largely to SL2. The possible regulatory significance of cotranscription of C. elegans genes is discussed.
Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.
We have identified and partially characterized a gene located on chromosome 22, band q11, centromeric of the chronic myelogenous leukemia breakpoint region. A number of overlapping cDNAs were isolated from this locus and the largest of 1.8 kb was sequenced. Its deduced amino acid sequence shows homology to the SH2 domains of protein tyrosine kinases such as FER, and is strikingly similar to the cellular part of the v-crk oncogene product. We identified one SH2 and two SH3 domains within the 303 amino acid open reading frame of this crk-like gene, CRKL. The CRKL gene product is predicted to have a molecular mass of 36 kDa. In addition, we demonstrate that this gene does not represent the human homolog of v-crk but rather a novel gene potentially capable of mediating the transduction of intracellular signals.
The authors are grateful for financial support from the National Institutes of Health (grants GM23244 and GM53905), and to very helpful comments on the manuscript from Elliot Elson, Vlodya Gelfand, Paul Matsudaira, Julie Theriot, and Sally Zigmond. D. A. L. and A. F. H. would also like to thank Alan Wells, and Anna Huttenlocher and Rebecca Sandborg, respectively, for stimulating conversations on this subject, and Sean Palecek for Figure 2Figure 2. Finally, we extend our apologies to all our colleagues in the field whose work we were unable to cite formally because of imposed reference limitations.
Using a novel strategy that enables the isolation of previously unknown genes encoding selectable recessive phenotypes, we identified a gene (tsg101) whose homozygous functional disruption produces cell transformation. Antisense RNA from a transactivated promoter introduced randomly into transcribed genes throughout the genome of mouse 3T3 fibroblasts was used to knock out alleles of chromosomal genes adjacent to promoter inserts, generating clones that grew in 0.5% agar and formed metastatic tumors in nude mice. Removal of the transactivator restored normal growth. The protein encoded by tsg101 cDNA encodes a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated previously in tumorigenesis. Overexpression of tsg101 antisense transcripts in naive 3T3 cells resulted in cell transformation and increased stathmin-specific mRNA.
Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).
The gene unc-76 (unc, uncoordinated) is necessary for normal axonal bundling and elongation within axon bundles in the nematode Caenorhabditis elegans. The UNC-76 protein and two human homologs identified as expressed sequence tags are not similar to previously characterized proteins and thus represent a new protein family. At least one of these human homologs can function in C. elegans, suggesting that it, like UNC-76, acts in axonal outgrowth. We propose that the UNC-76 protein, which is found in cell bodies and processes of all neurons throughout development, either has a structural role in the formation and maintenance of axonal bundles or transduces signals to the intracellular machinery that regulates axonal extension and adhesion.
The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
The C. elegans gene ced-7 functions in the engulfment of cell corpses during programmed cell death. We report that the CED-7 protein has sequence similarity to ABC transporters, is broadly expressed during embryogenesis, and is localized to the plasma membrane. Mosaic analysis revealed that ced-7 functions in both dying cells and engulfing cells during the engulfment process. We propose that CED-7 functions to translocate molecules that mediate homotypic adhesion between the cell surfaces of the dying and engulfing cells. Like CED-7, the mammalian ABC transporter ABC1 has been implicated in the engulfment of cell corpses, suggesting that CED-7 and ABC1 may be functionally similar and that the molecular mechanism underlying cell corpse engulfment during programmed cell death may be conserved from nematodes to mammals.
Although apoptotic cell death is widespread, dying cells are rarely seen in situ because of their rapid clearance by neighbouring phagocytes. Phagocytic recognition of apoptotic cells is less well understood than the death programme itself, but an increasing number of recent studies are highlighting its importance. This review discusses the nature of the receptors that have been implicated in apoptotic cell phagocytosis, the mechanisms of uptake and the immunological consequences of apoptotic cell ingestion.
REVIEW
Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, the cell suicide program critical for development,
tissue homeostasis, and protection against pathogens. Those most similar to Bcl-2 promote cell survival by inhibiting adapters
needed for activation of the proteases (caspases) that dismantle the cell. More distant relatives instead promote apoptosis,
apparently through mechanisms that include displacing the adapters from the pro-survival proteins. Thus, for many but not
all apoptotic signals, the balance between these competing activities determines cell fate. Bcl-2 family members are essential
for maintenance of major organ systems, and mutations affecting them are implicated in cancer.
Cells that die by apoptosis need to be removed before lysis to preserve tissue integrity and function. Recent studies have identified components of the uptake machinery used by phagocytes, but much remains to be learnt, particularly about the recognition mechanisms and their coupling to the uptake machinery.
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.
The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent
uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular
switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis.
Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42
and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the
different biological consequences that are associated with phagocytosis.
The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.
Crk family adapter proteins including Crk-II, Crk-I, and Crk-L consist mostly of SH2 and SH3 domains. Through the interactions between SH2 domain and phosphotyrosine residues and/or between SH3 domain and proline-rich motifs, they are involved in a variety of signaling cascades. Despite their essential roles in the signal transductions, knock-out mice of these molecules have not been reported yet. We performed the gene trap insertional mutagenesis with a trap vector, pU-Hachi, and generated a mutant mice line, Ayu 8104, in which the trap vector was inserted into the c-crk gene. Homozygous Ayu 8104 mice lacked Crk-II and Crk-I transcripts but expressed the truncated Crk proteins retaining one SH2 and one SH3 domain. Since the structure of the truncated proteins was similar to that of Crk-I, the insertion was considered to cause Crk-II-specific disruption. Homozygous mutant mice, however, did not exhibit any obvious abnormalities, suggesting that Crk-family adapters, Crk-II, Crk-I, and Crk-L would redundantly function in the signaling cascades and Crk-II was not apparently essential for embryonic development.