BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.     

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

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

Phagocytosis promotes programmed cell death in C. elegans.

In the nematode Caenorhabditis elegans programmed cell death requires the killer genes egl-1, ced-4 and ced-3 (refs 1 and 2), and the engulfment of dying cells requires the genes ced-1, ced-2, ced-5, ced-6, ced-7, ced-10 and ced-12 (refs 3,4,5). Here we show that engulfment promotes programmed cell death. Mutations that cause partial loss of function of killer genes allow the survival of some cells that are programmed to die, and mutations in engulfment genes enhance the frequency of this cell survival. Furthermore, mutations in engulfment genes alone allow the survival and differentiation of some cells that would normally die. Engulfment genes probably act in engulfing cells to promote death, as the expression in engulfing cells of ced-1, which encodes a receptor that recognizes cell corpses, rescues the cell-killing defects of ced-1 mutants. We propose that engulfing cells act to ensure that cells triggered to undergo programmed cell death by the CED-3 caspase die rather than recover after the initial stages of death.     

Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.     

Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck

Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. Here we report a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 (p53-inducible messenger RNA with a relative molecular mass (M(r)) of 140,000), Nap125 (NCK-associated protein with an M(r) of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. We therefore propose that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation.     

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.     

Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration

Cell migration is a fundamental biological process involving membrane polarization and cytoskeletal dynamics, both of which are regulated by Rho family GTPases. Among these molecules, Rac is crucial for generating the actin-rich lamellipodial protrusion, a principal part of the driving force for movement. The CDM family proteins, Caenorhabditis elegans CED-5, human DOCK180 and Drosophila melanogaster Myoblast City (MBC), are implicated to mediate membrane extension by functioning upstream of Rac. Although genetic analysis has shown that CED-5 and Myoblast City are crucial for migration of particular types of cells, physiological relevance of the CDM family proteins in mammals remains unknown. Here we show that DOCK2, a haematopoietic cell-specific CDM family protein, is indispensable for lymphocyte chemotaxis. DOCK2-deficient mice (DOCK2-/-) exhibited migration defects of T and B lymphocytes, but not of monocytes, in response to chemokines, resulting in several abnormalities including T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. In DOCK2-/- lymphocytes, chemokine-induced Rac activation and actin polymerization were almost totally abolished. Thus, in lymphocyte migration DOCK2 functions as a central regulator that mediates cytoskeletal reorganization through Rac activation.     

RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo

The small GTPase Rac has a central role in regulating the actin cytoskeleton during cell migration and axon guidance. Elmo has been identified as an upstream regulator of Rac1 that binds to and functionally cooperates with Dock180 (refs 2-4). Dock180 does not contain a conventional catalytic domain for guanine nucleotide exchange on Rac, but possesses a domain that directly binds to and specifically activates Rac1 (refs 5, 6). The small GTPase RhoG mediates several cellular morphological processes, such as neurite outgrowth in neuronal cells, through a signalling cascade that activates Rac1 (refs 7-12); however, the downstream target of RhoG and the mechanism by which RhoG regulates Rac1 activity remain unclear. Here we show that RhoG interacts directly with Elmo in a GTP-dependent manner and forms a ternary complex with Dock180 to induce activation of Rac1. The RhoG-Elmo-Dock180 pathway is required for activation of Rac1 and cell spreading mediated by integrin, as well as for neurite outgrowth induced by nerve growth factor. We conclude that RhoG activates Rac1 through Elmo and Dock180 to control cell morphology.     

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

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

Rac GTPases control axon growth, guidance and branching

Growth, guidance and branching of axons are all essential processes for the precise wiring of the nervous system. Rho family GTPases transduce extracellular signals to regulate the actin cytoskeleton. In particular, Rac has been implicated in axon growth and guidance. Here we analyse the loss-of-function phenotypes of three Rac GTPases in Drosophila mushroom body neurons. We show that progressive loss of combined Rac1, Rac2 and Mtl activity leads first to defects in axon branching, then guidance, and finally growth. Expression of a Rac1 effector domain mutant that does not bind Pak rescues growth, partially rescues guidance, but does not rescue branching defects of Rac mutant neurons. Mosaic analysis reveals both cell autonomous and non-autonomous functions for Rac GTPases, the latter manifesting itself as a strong community effect in axon guidance and branching. These results demonstrate the central role of Rac GTPases in multiple aspects of axon development in vivo, and suggest that axon growth, guidance and branching could be controlled by differential activation of Rac signalling pathways.     

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

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

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

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

Identification of Tim4 as a phosphatidylserine receptor

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

IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling

Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42 (refs 7, 8), which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. Here we demonstrate that IRSp53, a substrate for insulin receptor with unknown function, is the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, we found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex.     

Rac function and regulation during Drosophila development

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, we use loss-of-function mutations to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved.     

Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7

Mutations that enhance the response to double-stranded RNA (dsRNA) have revealed components of the RNA interference (RNAi) pathway or related small RNA pathways. To explore these small RNA pathways, we screened for Caenorhabditis elegans mutants displaying an enhanced response to exogenous dsRNAs. Here we describe the isolation of mutations in two adjacent, divergently transcribed open reading frames (eri-6 and eri-7) that fail to complement. eri-6 and eri-7 produce separate pre-messenger RNAs (pre-mRNAs) that are trans-spliced to form a functional mRNA, eri-6/7. Trans-splicing of eri-6/7 is mediated by a direct repeat that flanks the eri-6 gene. Adenosine to inosine editing within untranslated regions of eri-6 and eri-7 pre-mRNAs reveals a double-stranded pre-mRNA intermediate, forming in the nucleus before splicing occurs. The ERI-6/7 protein is a superfamily I helicase that both negatively regulates the exogenous RNAi pathway and functions in an endogenous RNAi pathway.     

A tension-induced mechanotransduction pathway promotes epithelial morphogenesis

Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins. Mechanotransduction is crucial for the physiology of several organs and for cell migration. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome: p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.     

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.     

Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.