Annie-Carole Tosello-Trampont

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.

Unconventional Rac-GEF activity is mediated through the Dock180–ELMO complex

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180–ELMO1 interaction is required. We can also detect a trimeric ELMO1–Dock180–Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180–ELMO complex functions as an unconventional two-part exchange factor for Rac.

CED-12/ELMO, a Novel Member of the CrkII/Dock180/Rac Pathway, Is Required for Phagocytosis and Cell Migration

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Dock180 and ELMO1 Proteins Cooperate to Promote Evolutionarily Conserved Rac-dependent Cell Migration

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180·ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO·Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.

CXCR4 acts as a costimulator during thymic [beta]-selection

Passage through the β-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-β gene (Tcrb) and formation of a pre-TCR on the surface of CD4−CD8− thymocytes. How other receptors influence ββ-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced β-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during β-selection.

The Role of Macrophage Polarization in Infectious and Inflammatory Diseases

Macrophages, found in circulating blood as well as integrated into several tissues and organs throughout the body, represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis. Additionally, macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation. Studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli, and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease. This review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology.

Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling.

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.

Evidence for a Conserved Role for CrkII and Rac in Engulfment of Apoptotic Cells

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream ofced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.

Distinct Roles for Intracellular and Extracellular Lipids in Hepatitis C Virus Infection

Hepatitis C is a chronic liver disease that contributes to progressive metabolic dysfunction. Infection of hepatocytes by hepatitis C virus (HCV) results in reprogramming of hepatic and serum lipids. However, the specific contribution of these distinct pools of lipids to HCV infection remains ill defined. In this study, we investigated the role of hepatic lipogenesis in HCV infection by targeting the rate-limiting step in this pathway, which is catalyzed by the acetyl-CoA carboxylase (ACC) enzymes. Using two structurally unrelated ACC inhibitors, we determined that blockade of lipogenesis resulted in reduced viral replication, assembly, and release. Supplementing exogenous lipids to cells treated with ACC inhibitors rescued HCV assembly with no effect on viral replication and release. Intriguingly, loss of viral RNA was not recapitulated at the protein level and addition of 2-bromopalmitate, a competitive inhibitor of protein palmitoylation, mirrored the effects of ACC inhibitors on reduced viral RNA without a concurrent loss in protein expression. These correlative results suggest that newly synthesized lipids may have a role in protein palmitoylation during HCV infection.