Lisa B. Haney

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.

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and Armadillo repeats of CED-12/ELMO

BACKGROUND Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.

ELMO1 and Dock180, a Bipartite Rac1 Guanine Nucleotide Exchange Factor, Promote Human Glioma Cell Invasion

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.

PH domain of ELMO functions in trans to regulate Rac activation via Dock180

The members of the Dock180 superfamily of proteins are novel guanine nucleotide exchange factors (GEF) for Rho family GTPases and are linked to multiple biological processes from worms to mammals. ELMO is a critical regulator of Dock180, and the Dock180–ELMO complex functions as a bipartite GEF for Rac. We identified a mechanism wherein the PH domain of ELMO, by binding the Dock180–Rac complex in trans, stabilizes Rac in the nucleotide-free transition state. Mutagenesis studies reveal that this ELMO PH domain–dependent regulation is essential for the Dock180–ELMO complex to function in phagocytosis and cell migration. Genetic rescue studies in Caenorhabditis elegans using ELMO and its homolog CED-12 support the above observations in vivo. These data reveal a new mode of action of PH domains and a novel, evolutionarily conserved mechanism by which a bipartite GEF can activate Rac.

Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans

Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily conserved, survey mechanism that identifies and removes unfit cells within a tissue.

Characterization of a Novel Interaction between ELMO1 and ERM Proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.

Cooperation between Noncanonical Ras Network Mutations in Cancer

Cancer develops after the acquisition of a collection of mutations that together create the ‘cancer phenotype’. How collections of mutations work together within a cell, and whether there is selection for certain combinations of mutations, are not well understood. Using a Ras signaling network mathematical model we tested potential synergistic combinations within the Ras network. Intriguingly, our modeling, including a “computational random mutagenesis” approach, and subsequent experiments revealed that mutations of the tumor suppressor gene NF1 can amplify the effects of mutations in multiple other components of the Ras pathway, including weakly activating, noncanonical, Ras mutants. Since conventional wisdom holds that mutations within the same pathway do not co-occur, it was surprising that modeling and experiments both suggested a functional benefit for co-occurring Ras pathway mutations. Furthermore, we analyzed >3900 sequenced cancer specimens from the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) and we uncovered an increased rate of co-occurrence between mutations the model predicted could display synergy. Overall, these data suggest that selective combinations of Ras pathway mutations could serve the role of cancer “driver”. More generally, this work presents a mechanism by which the context created by one mutation influences the evolutionary trajectories of cancer development, and this work suggests that mutations that result in “network instability” may promote cancer in a manner analogous to genomic instability.