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

Abstract

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.
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.
The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.

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Current Biology, Vol. 14, 2208–2216, December 29, 2004, ©2004 Elsevier Ltd. All rights reserved.DOI 10.1016/j.cub.2004.12.029
Phagocytosis of Apoptotic Cells Is Regulated
by a UNC-73/TRIO-MIG-2/RhoG Signaling Module
and Armadillo Repeats of CED-12/ELMO
mediated Rac activation and cytoskeletal reorgani-
zation.
Conclusions: The combination of in vitro and in vivo
studies presented here identify two evolutionarily con-
served 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 activa-
tion 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.
Colin D. deBakker,1,7Lisa B. Haney,1,7
Jason M. Kinchen,3Cynthia Grimsley,1,2
Mingjian Lu,1Doris Klingele,3Pei-Ken Hsu,4
Bin-Kuan Chou,4Li-Chun Cheng,4Anne Blangy,5
John Sondek,6Michael O. Hengartner,3Yi-Chun Wu,4
and Kodi S. Ravichandran1,*
1Department of Microbiology
Beirne Carter Center for Immunology Research and
2Department of Pharmacology
University of Virginia
Charlottesville, VA 22908
3Institute of Molecular Biology
University of Zurich
8057 Zurich,
Switzerland
4Institute of Molecular and Cellular Biology
National Taiwan University
Taipei
Taiwan
5Centre de Recherches de Biochimie
Macromole ´culaire
Centre National de la Recherche Scientifique Unite ´
Propre de Recherche 1086
1919 route de Mende
34293 Montpellier Cedex 5
France
6Department of Pharmacology
University of North Carolina, Chapel Hill
Chapel Hill, NC 27599
Introduction
Engulfment of apoptotic cells is intimately tied to the
apoptoticprogramandislinkedtodevelopment,cellular
homeostasis, and woundhealing [1–4]. Phagocytes rec-
ognize and engulf dying cells at an early stage in the
apoptoticprocesstopreventleakageofpotentiallytoxic
and inflammatory contents from the dying cells [5]. Fail-
ure to clear dying cells has been linked to several auto-
immune disorders in mice and humans [6–9]. Thus, un-
derstanding the specific molecular pathways regulating
engulfment is an issue of fundamental importance.
Studies in C. elegans and mammalian cells have iden-
tified a number of proteins involved in the recognition
and uptake of apoptotic cells [1]. To date, at least seven
genes have been identified in C. elegans and catego-
rized into two functional genetic pathways [1, 10]. In one
pathway, two membrane proteins, CED-1 and CED-7
(representing the mammalian homologs LRP and ABCA1,
respectively), have been shown to function upstream of
the intracellular adaptor protein CED-6/GULP [11–14].
In a second pathway, three cytosolic proteins, CED-2,
CED-5, and CED-12 (representing the mammalian ho-
mologs CrkII, Dock180, and ELMO, respectively), have
beenshowntoleadtotheactivationofthesmallGTPase
CED-10/Rac [15–22]. Recently, ELMO and Dock180
havebeenshowntofunctionasanovel,bipartitenucleo-
tide exchange factor for Rac, thereby leading to the
reorganization of the actin cytoskeleton during engulf-
ment [23].
ELMO is represented by a single gene in the worm
(ced-12) and fly (dced-12) and by three genes (elmo 1,
2, and 3) in mammals [18, 19, 22]. ELMO1 encodes a
novel, highly evolutionarily conserved protein of 727
amino acids with no obvious catalytic domains. With
the exception of a C-terminal PH domain, a predicted
leucine zipper motif, and a Proline-rich motif, no other
domains or motifs have been recognized within ELMO/
CED-12 [18, 19, 22]. The C-terminal ?195 amino acids
ofELMO1(denotedC-term)arenecessaryandsufficient
to interact with Dock180 and for promoting Rac activa-
tion and membrane ruffling when overexpressed in cells
[24]. The specific role of the N-terminal two-thirds of
ELMO1(?530aminoacids;denotedN-term)isunknown
but might play a role in targeting the ELMO1/Dock180
complex to the membrane [24]. The goal of this study
Summary
Background: Phagocytosis of cells undergoing apo-
ptosisisessentialduringdevelopment,cellularturnover,
and wound healing. Failure to promptly clear apoptotic
cells has been linked to autoimmune disorders. C. ele-
gans 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 fac-
tor TRIO, which functions upstream of ELMO/Dock180/
Rac during engulfment. Complementary studies in C. ele-
gansshow thatMIG-2 (whichwe identifyas thehomolog
of mammalian RhoG) and UNC-73 (the TRIO homolog)
also regulate corpse clearance in vivo, upstream of
CED-12. Atthe molecular level,we identify a novelset of
evolutionarily conservedArmadillo (ARM)repeats within
CED-12/ELMO that mediate an interaction with acti-
vated MIG-2/RhoG; this, in turn, promotes Dock180-
*Correspondence: ravi@virginia.edu
7These authors contributed equally to this work.

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TRIO/RhoG/ELMO Signaling Module in Engulfment
2209
amino acids in length [25, 26] and are involved in pro-
tein:protein interactions in other signaling proteins. In
all, 5–7 ARM repeats were predicted within CED-12/
ELMO (Figure 1A; Figure S1). To test the functional re-
quirement of the ARM repeats, we individually mutated
two of the repeats (Figure 1A). Two residues in each
repeat were chosen for mutation, the choice being
based upon their high degree of conservation among
CED12/ELMO proteins as well as other known ARM re-
peat-containingproteins[25,26].WhereasELMO1wtwas
able to functionally synergize with Dock180 in an in vitro
phagocytosis assay, individual mutations in the ARM
repeats of ELMO (ELMO1ARM1and ELMO1ARM2) failed to
efficientlycooperatewithDock180(Figure1B).Whereas
transient coexpression of ELMOwtand Dock180 in LR73
fibroblasts induced lamellipodia formation and colocali-
zation of the proteins at the ruffles, the ELMO1ARM2mu-
tant was deficient in promoting lamellipodia formation
and was not enriched at the cell periphery (Figure 1C).
This suggested a role for the ARM repeats of ELMO in
Dock180/ELMO1-mediated induction and/or localiza-
tion of this complex at membrane ruffles. The failure of
the ARM mutants to cooperate with Dock180 in en-
gulfment or in the induction of lamellipodia was not due
to a defect in Dock180 binding by the ARM mutants
because themutants efficientlycoprecipitated Dock180
(Figure 1D). Moreover, in an in vitro GEF assay, where
the presence of ELMO increases the Dock180-depen-
dent GEF activity toward Rac, ELMO1ARM2was still able
to enhance the Dock180-mediated Rac-GEF activity
(Figure S2). Thus, the requirement for the ARM repeats
of ELMO during engulfment appeared distinct from its
interactions with Dock180.
Table 1. Intact ARM 2 Repeat of CED-12/ELMO1 Is Required
for DTC Migration and Cell Corpse Removal
ced-12 ARM2 Repeat Is Required for Efficient Corpse Removal
Genotype Corpse Numbern
Wild-type
ced-12(k149)
ced-12(k149);[Peft-3::ced-12(WT)::yfp]
ced-12(k149);[Peft-3::ced-12N-term::yfp]
ced-12(k149);[Peft-3::ced-12C-term::yfp]
ced-12(k149);[Peft-3::ced-12ARM1::yfp]
ced-12(k149);[Peft-3::ced-12ARM2::yfp]
0
16.8 ? 5.0
2.8 ? 1.9
17.5 ? 4.2
19.5 ? 4.5
2.6 ? 2.0
17.6 ? 5.0
10
20
10
30
30
30
30
ced-12/ELMO ARM2 Mutation Affects DTC Migration
Genotype Mismigration (%)n
Wild-type
ced-12(k149)
mig-2(mu28)
ced-12(k149);[Peft-3::ced-12(WT)::yfp]
ced-12(k149);[Peft-3::ELMO1(WT)::gfp]
ced-12(k149);[Peft-3::ced-12N-term::yfp]
ced-12(k149);[Peft-3::ced-12C-term::yfp]
ced-12(k149);[Peft-3::ELMO1ARM1:gfp]
ced-12(k149);[Peft-3::ELMO1ARM2::gfp]
ced-12(k149);[Peft-3::ced-12ARM1::yfp]
ced-12(k149);[Peft-3::ced-12ARM2::yfp]
0
36
21
9
24
39
34
22
32
6
40
204
305
305
302
302
862
1129
922
919
930
926
Scores shown are an average of three independent transgenic lines.
When the ced-12ARM2mutant was expressed in a wild-type back-
ground,itresultedinaweakDTCmigrationdefectof2.5%mismigra-
tion (average of two independent lines). All constructs have the
marker Plim-7::gfp used to score DTC migration. n is the number of
gonad arms scored in the case of DTC migration or the number of
worms scored for engulfment.
was to examine whether the N-term of ELMO may link
to upstream players during engulfment, to identify such
players, and to better delineate the molecular details of
pathways involved in corpse clearance.
RhoG Binds to ARM Repeats of ELMO
and Promotes Rac-Dependent Engulfment
Recent studies on Rac-like GTPases in mammals have
shown that the GTPase RhoG functions upstream of
Rac, with a role for RhoG demonstrated in neurite out-
growth and integrin-mediated cell adhesion [27–30]. In-
terestingly, ELMO2 was identified in a yeast two-hybrid
screen for proteins that bind active (GTP bound) RhoG
[29, 30]. We hypothesized that ARM repeats of ELMO1
might be involved in binding to activated RhoG and that
RhoG, via the ELMO/Dock180 complex, might regulate
Rac activation during engulfment. We first confirmed
that ELMO1 associated with activated RhoG (RhoGQ61L)
(Figure 2A, lanes 5 and 6). Moreover, a trimeric complex
of ELMO1:Dock180:RhoGQ61Lcould be detected when
the three proteins were coexpressed (lanes 7 and 8).
Consistent with the association of RhoG with the
Dock180/ELMO1 complex, RhoG immunoprecipitates
contained a Rac-GEF activity, but only when both
ELMO1andDock180proteinswerecoexpressed(Figure
S3).ELMO1wasessentialforbridgingRhoGtoDock180
because the latter two became part of a complex only
whenELMO1waspresent(Figure2A,lanes7–10).These
data suggested a link between activated RhoG and
ELMO1/Dock180-mediated Rac activation.
We then asked whether RhoG would affect engulfment
upstream of Rac. We transiently transfected LR73 cells
with various GFP-tagged RhoG constructs and tested
Results and Discussion
ARM Repeats of ELMO1 Are Required
for Engulfment
To better understand CED-12-mediated signaling in vivo,
we performed genetic rescue studies with fragments of
CED-12 into CED-12-deficient worms. We tested whether
the C-term of CED-12/ELMO is sufficient for function
during engulfment in vivo and whether any requirement
exists for the N-term (which does not bind CED-5/
Dock180). We expressed the C-term or N-term fragments
of CED-12 (denoted C-term::YFP or N-term::YFP) as a
transgene in CED-12-deficient worms. However, neither
theC-term::YFPnortheN-term::YFPconstructwasable
to rescue the engulfment defect (Table 1). This sug-
gested that the CED-5 binding via the C-term is insuffi-
cient and that features within the N-terminal 550 amino
acids of CED-12 are required for engulfment in vivo.
No obvious motifs/domains have been recognized
within the N-terminal 550 amino acid region. With sec-
ondary structure-based threading programs, we identi-
fied several conserved Armadillo (ARM) repeats within
CED-12 and ELMO (see Supplemental Experimental
Procedures and Figure S1 in the Supplemental Data
available withthis articleonline). ARMrepeats are35–50

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Figure 1. Functional Requirement for the ARM Repeats of ELMO1
(A) Schematic representation of the various ELMO1 constructs.
(B) Requirement for ELMO1 ARM repeats in phagocytosis. GFP or ELMO1-GFP plasmids were transiently transfected into LR73 cells with or
without Flag-Dock180, and phagocytosis was measured. Cells with comparable GFP expression were analyzed, and the fraction of GFP-
positive cells with engulfed particles is shown (data representative of five independent experiments).
(C) ELMO1ARM2fails to promote ruffling with Dock180 and/or membrane localization. LR73 cells were transiently transfected with the indicated
plasmids and analyzed with confocal microscopy. ELMO1-GFP was visualized by its green fluorescence, Dock180 by anti-Flag and labeled
secondary antibody, and polymerized actin via phalloidin-rhodamine staining. Quantitation of the cells with ruffles (arrows) in the GFP-positive
or GFP-negative untransfected populations were as follows: untransfected cells, 17% (n ? 690); ELMO1wt-GFP ? Flag-Dock180, 70% (n ?
135), and ELMO1ARM2-GFP ? Flag-Dock180, 21% (n ? 114).
(D) Mutations in ARM repeats do not affect ELMO:Dock180 interaction. LR73 cells were transiently transfected and immunoprecipitated with
anti-Flag antibody, and the association of ELMO1 and its mutants with Dock180 was determined by immunoblotting. Immunoblotting of total
lysates indicated equal loading. ELMO1-T677 (which does not bind Dock180) served as a negative control.
their effect on phagocytosis. Expression of RhoGwt
strongly promoted phagocytosis in multiple indepen-
dent experiments (fold increase of 2.7? 0.7; p ? 0.0006;
n ? 5) (Figure 2B). RhoGV12Aor RhoGQ61L, two constitu-
tively active mutants [28], also promoted phagocytosis;
however, RhoG mutants deficient in binding to nucleo-
tide (RhoGT17N) or to downstream effectors (RhoGF37A)
[28]didnotpromoteuptake(Figure2B;datanotshown).
Moreover, coexpression of RhoGIP122, a protein that
specifically sequesters GTP bound RhoG [28, 31], po-
tently inhibited the RhoG-mediated engulfment (Figure
2B). Thus, GTP loading of RhoG and the coupling of
effectors that bind GTP bound RhoG (such as ELMO1)
appeared essential.
RhoG appeared to function upstream of Rac1 be-
cause the enhancement of uptake due to RhoGV12Awas
strongly inhibited by a dominant-negative form of Rac1
(RacT17N). In contrast, the enhanced uptake due to a con-
stitutivelyactiveformofRac1(RacV12A)wasstillobserved
when coexpressed with a dominant-negative mutant of
RhoG (RhoGT17N) (Figure 2B) [28, 30]. Coexpression of
Dock180 and ELMOwtwith RhoG modestly (but consis-
tentlyinmultipleexperiments)enhancedtheuptakeover
that induced by RhoG alone (Figure 2B). Such a synergy

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TRIO/RhoG/ELMO Signaling Module in Engulfment
2211
Figure 2. RhoG and TRIO Promote Rac-Dependent Phagocytosis
(A) RhoGQ61Lassociates with Dock180 via ELMO1. 293T cells were transfected with GFP-RhoGQ61L, His-Dock180, and ELMO1-Flag plasmids
as indicated, immunoprecipitated with anti-GFP, and immunoblotted with the indicated antibodies.
(B) RhoG promotes engulfment and synergizes with ELMO and Dock180. LR73 fibroblasts were transiently transfected with plasmids encoding
GFP, GFP-RhoGwt, or GFP-RhoGF37Ain triplicate with or without coexpression of constitutively active or dominant-negative Rac mutants or
the indicated Dock180 and ELMO1 plasmids. The phagocytosis was measured as in Figure 1B. An additional sample was transfected to check
protein expression of Flag-tagged Dock180 and ELMO proteins. Data are indicative of at least three independent experiments.
(C) TRIO promotes phagocytosis upstream of Rac1 and RhoG. LR73 cells were transiently transfected with GFP-TRIO alone or with the
indicated RhoG or Rac1 mutant plasmids and assayed for phagocytosis (data are representative of three to six independent experiments).

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2212
was not observed when ELMOARMmutants were tested
(Figure 2B). The enhancement with ELMOwtwas not due
to the direct nucleotide exchange on RhoG by Dock180
because the Dock180/ELMO complex displayed little
detectableinvitroGEFactivitytowardRhoG(FigureS4).
Further biochemical studies revealed that ELMO1
boundspecificallytoconstitutivelyactiveformsofRhoG
(Figure 3A). This interaction with RhoG occurred via the
N-term of ELMO1, but not the C-term (Figure 3B, lanes
3 and 4). Importantly, an N-terminal 115 amino acid
ELMO1 fragment comprising only the ARM repeats 1
and 2 (see schematic in Figure 1A) was able to bind
RhoGQ61L(Figure 3B, lane 7); furthermore, ELMOARM1and
ELMOARM2mutants in the context of full-length ELMO1
failed to interact with RhoGQ61L(Figure 3B, lanes 5 and
6). Taken together, the intact ARM repeats of ELMO1
are required for binding GTP-RhoG, and this correlates
withthefailureoftheARMmutantstofunctioninphago-
cytosis.
TRIO Promotes RhoG- and Rac-
Dependent Engulfment
We then sought components of this pathway that may
function upstream of RhoG. The multidomain protein
TRIO has been shown to function as a GEF for RhoG
within cells [28, 32–34] (Figure S4). We examined
whether TRIO would affect engulfment via RhoG activa-
tion. Transient transfection of a GFP-TRIO construct
[34] into LR73 cells strongly increased phagocytosis
compared to GFP-transfected cells in multiple experi-
ments (fold increase of 3.1 ? 0.4; p ? 0.0009; n ? 6)
(Figure 2C). However, a construct with a GEF D1 domain
point mutation that abolishes the GEF activity of TRIO
(GFP-TRIO-AEP) or a deletion mutant lacking the GEF
D1 domain (GFP-TRIO 1-1203) [34] failed to enhance
engulfment; instead, they partially inhibited the basal
engulfment (see Figure S4). The GFP-TRIO-mediated
uptake was inhibited by a dominant-negative form of
RhoG (RhoGT17N) (Figure 2C) and by RhoGIP122 (data
not shown). Moreover, the TRIO-RhoG-mediated phago-
cytosis was inhibited by dominant-negative RacT17N, but
not the constitutively active RacQ61L(Figure 2C). Taken
together, these data suggest that a signaling module of
TRIO → RhoG can function during phagocytosis and
may do so at a step upstream of Rac activation.
Requirement for CED-12 ARM Repeats during
Engulfment in C. elegans
We then determined the evolutionary significance of the
ARM repeats of CED-12 as well as the role of worm
homologs of RhoG and TRIO in corpse clearance in
C. elegans. We engineered independent mutations in
ARM1 and ARM2 repeats of CED-12 (analogous to the
ELMO1 mutants) and tested their ability to rescue the
Figure 3. ELMO/CED-12 Specifically Binds Active RhoG/MIG-2 via
ARM Repeats
(A) ELMO1 interacts specifically with active RhoG. 293T cells were
transiently transfected with the indicated RhoG and Rac plasmids.
The lysates were precipitated with GST-ELMO1, and the bound
proteins were analyzed by anti-GFP immunoblotting. The intensity
of the binding to RhoGwt(presumably GTP bound) was variable
between experiments, whereas the binding to RhoGQ61Lwas con-
sistent.
(B) Intact ARM repeats are required for ELMO/RhoG interaction.
293T cells were transiently transfected with constitutively active
RhoGQ61L,andthelysateswereprecipitatedwiththeindicatedbacte-
rially produced wild-type and mutant GST-ELMO1 proteins. The
presence of the different GST proteins was confirmed (bottom).
(C) CED-12 specifically interacts with active MIG-2, but not CED-10,
in a yeast two-hybrid assay. Constructs expressing the Gal4 DNA
binding domain (DB) or DB-fusion proteins (DB-CED-2 and DB-
CED-12)andtheGal4transcriptionactivationdomain(AD)orAD-fusion
proteins (AD-MIG-2, AD-MIG-2G16V, AD-CED-10, and AD-CED-10G12V)
were transformed into yeast, and the transformants were tested for
growthin theabsence(permissive) orthepresenceof 3AT(selecting
condition). (1) CED-12? activated MIG-2G16V, (2) CED-12 ?MIG-2wt,
(3) CED-2 ? MIG-2G16V, (4) CED-12 ? CED-10G12V, (5) CED-12 ?
CED-10, and (6) DB ? AD (negative control).

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TRIO/RhoG/ELMO Signaling Module in Engulfment
2213
engulfment defects in ced-12 null animals. Whereas
ced-12wtand ced-12ARM1were able to efficiently rescue
the engulfment defect, ced-12ARM2was unable to do so
(Table 1). Notably, the residues in the ARM1 repeat are
relatively less conserved between CED-12 and ELMO,
whereas the ARM2 repeat was highly conserved (Figure
S1). It is likely that the ARM1 repeat of CED-12 may not
play a requisite role in C. elegans.
Table 2. unc-73 and mig-2 Enhance Persistent Cell Corpses
in ced Mutants
Corpse
NumberStrain Changea
t test
Wild-type
unc-73(e936)
mig-2(mu28)
unc-73(e936);mig-2(mu28)
ced-10(n3246)
ced-10(n3246);unc-73(e936)
ced-12(tp2)
unc-73(e936);ced-12(tp2)
ced-12(n3261)
unc-73(e936);ced-12(n3261)
ced-5(n2002)
unc-73(e936);ced-5(n2002)
ced-5(n1812)
unc-73(e936);ced-5(n1812)
ced-2(e1752)
unc-73(e936);ced-2(e1752)
ced-2(n1994)
unc-73(e936);ced-2(n1994)
ced-2(e1752)
ced-2(e1752);mig-2(mu28)
ced-2(n1994)
ced-2(n1994);mig-2(mu28)
ced-5(n2002)
mig-2(mu28);ced-5(n2002);
ced-5(n1812)
mig-2(mu28);ced-5(n1812)
ced-12(tp2)
mig-2(mu28);ced-12(tp2)
ced-12(n3261)
mig-2(mu28);ced-12(n3261)
ced-10(n1993)
mig-2(mu28);ced-10(n1993)
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
24.7 ? 5.3
31.7 ? 3.0
17.3 ? 2.4
23.8 ? 3.5
27.9 ? 3.9
26.6 ? 4.2
37.1 ? 5.9
41.1 ? 4.1
41.9 ? 3.7
40.1 ? 5.3
12.6 ? 4.0
32.1 ? 6.3
35.2 ? 4.4
33.4 ? 6.4
12.6 ? 4.0
43.1 ? 5.4
35.3 ? 4.4
41.5 ? 4.2
37.7 ? 5.9
46.4 ? 7.4
42.8 ? 4.5
44.4 ? 5.9
17.9 ? 3.0
30.5 ? 5.0
27.9 ? 3.9
30.0 ? 3.2
18.3 ? 3.0
23.6 ? 5.0
↑
p ? 0.0001
MIG-2 and UNC-73 Influence Corpse Clearance
In Vivo Upstream of CED-12
Whereas the unc-73 gene in C. elegans has been identi-
fied as the trio homolog [35, 36], the homolog of RhoG
has not been defined. Among the three Rac-like genes
in the worm (ced-10, mig-2, and rac-2), we examined
whether mig-2 might represent rhoG; this stems from
studies suggesting ced-10 as the Rac1 homolog and
mig-2 being placed genetically in the same pathway as
ced-5/dock180 during several migration events [20]. In
yeast two-hybrid assays, CED-12 specifically interacted
with the constitutively active (GTPbound) form of MIG-2
(MIG-2G12V) but not wild-type MIG-2 (Figure 3C). The fail-
ureofMIG-2wttobindCED-12islikelyduetotheabsence
of GEFs that could exchange nucleotides on wild-type
MIG-2 in the yeast (as reported previously; [37]). The
specific binding of CED-12 to active MIG-2G12Vagain
placed CED-12 as a downstream “effector” of GTP
bound MIG-2. It is noteworthy that UNC-73 [38] has
been previously shown to have in vitro GEF activity
toward MIG-2 and likely acts as a GEF for MIG-2 in
C. elegans. Importantly, CED-12 did not interact with a
constitutivelyactiveformofCED-10,consistentwitharole
of CED-12 upstream of CED-10 activation (Figure 3C).
Wethenassessedwhethermig-2orunc-73couldplay
a role in engulfment in vivo. Although worms deficient
in mig-2 or unc-73 alone showed no obvious defects in
corpse clearance, a role for both of these genes was
revealed in double mutants. ced-12;mig-2 and ced-
12;unc-73 double-mutant worms showed a significantly
higher number of unengulfed corpses compared to the
ced-12 single mutant (Table 2). Interestingly, absence
of mig-2 or unc-73 augmented the number of persistent
corpses due to a weak allele of ced-12 (tp2), but the
phenotype due to a strong allele of ced-12 (n3261) was
not further enhanced, suggesting that mig-2 and unc-
73 likely function in the same pathway as ced-12, rather
than being in a parallel pathway (Table 2).
We tested whether unc-73 or mig-2 mutations would
also enhance the corpse clearance defects in mutants
of ced-2, ced-5, or ced-10, three other members of the
same genetic pathway as ced-12 [18, 19, 22]. Loss of
unc-73 significantly enhanced persistent corpses in the
ced-2 (p ? 0.0001), ced-10 (p ? 0.0001), and ced-5 (p ?
0.02) backgrounds (Table 2). Again, the strong alleles
of ced-2 and ced-5 did not show a greater number of
corpses due to loss of unc-73 in the double mutants. In
a similar double-mutant analysis, the mig-2 mutation
also significantly increased corpse number in weak
ced-2, ced-5, or ced-10 backgrounds, but not in the
strong ced-2 or ced-5 mutants (Table 2; [20]). It is note-
worthy that the ced-10 mutant used above is not a null
mutant but a partial loss-of-function mutant (because a
null mutant of ced-10 is embryonic lethal) (J.K. and
↑
p ? 0.0001
—p ? 0.133
↑
p ? 0.019
—p ? 0.232
↑
p ? 0.0001
—p ? 0.087
↑
p ? 0.0001
↑
p ? 0.0001
↑
p ? 0.0001
—p ? 0.377
↑
p ? 0.0001
—p ? 0.052
↑
p ? 0.0001
Number of cell corpses in the head regions of twenty L1 larvae
within 30 min of hatching was scored with Nomarski optics as de-
scribed [40].
a↑ indicates significant increase of cell corpse number in double
mutants when compared to corresponding ced single mutants (p ?
0.05); — indicates no significant change (p ? 0.05).
M.O.H., unpublished data). Thus, consistent with the
enhancedengulfmentduetoRhoGandTRIOinmamma-
lian phagocytosis, mig-2 and unc-73 do play a role in
corpse clearance at the level of a whole organism, and
they genetically link to the same pathway as ced-12,
ced-2, ced-5, or ced-10. These data also revealed a
novel insight on the two Rac homologs ced-10 and
mig-2, in that they can function in the same genetic
pathway as ced-12 during engulfment but appear to do
so at distinct steps.
There are at least two possibilities for the lack of an
engulfment phenotype in single mutants of UNC-73 and
MIG-2. First, there are multiple (and what often appear
redundant) engulfment receptors operational in mam-
mals, and homologs for many of these receptors also
exist in worms. Because the precise receptor(s) up-
streamofUNC-73hasnotyetbeendefined,itispossible
that only some of them go through the UNC-73/MIG-2
module, whereas some others may directly recruit the
CED-12/CED-5/CED-10 pathway. Alternatively, recent
studies suggest that the CED-1/CED-7/CED-6 pathway
of engulfment also can signal to CED-10, although how
this recruitment occurs is unclear (J.K. and M.O.H., un-
published data). Thus, either of these possibilities could

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Current Biology
2214
Figure 4. ARM Repeats of ELMO1 Affect In Vitro Cell Migration
(A) ARM mutants of ELMO1 fail to synergize with Dock180 in migration in vitro. LR73 cells were transiently transfected with the indicated
plasmids, and the migration of transfected cells through a 24-well Transwell chamber filter was assessed. The luciferase alone control was
set at 100%. Aliquots of cells from each transfection were immunoblotted to confirm expression of Dock180 and ELMO mutants.
(B) Intact ARM repeats of ELMO are required for RhoG-mediated in vitro migration. RhoG was expressed alone or together with the indicated
ARM mutant ELMO plasmids in LR73 cells, and the migration was assessed as above.
(C) Working model for the TRIO/RhoG signaling module leading to Rac activation via the ELMO/Dock complex. “Eat-me” signals or migration
cues via an unidentified receptor(s) promote the activation of RhoG through its GEF, TRIO (1, a). Genetic studies in C. elegans suggest the
existence of a parallel pathway independent of UNC-73 and MIG-2 (b), but signals through CED-10. GTP bound RhoG/MIG-2 could target
ELMO1 to the membrane (2) where the Dock180/ELMO complex becomes recruited/activated and can function as a bipartite GEF for Rac1
(3), in turn leading to lamellipodia formation (4).
lead to UNC-73/MIG-2-independent CED-10/Rac acti-
vation to promote engulfment.
[18, 19, 22]. The expression of the ced-12ARM2mutant
failed to rescue the DTC migration defect in the ced-12
null animals (Table 1), suggesting that the second ARM
repeat of CED-12 is essential in both engulfment and
DTC migration. Similarly, the elmoARM2mutant also failed
torescuetheDTCmigrationdefectinced-12nullworms,
whereas the elmowt(as has been reported previously;
[22]) and elmoARM1mutant were able to partially rescue
the defect (Table 1). The requirement of the intact ARM
ARM Repeats of CED-12/ELMO Are Also Critical
for Cell Migration
In addition to the corpse clearance defect, worms defi-
cient in ced-12 also show defects in migration of the
distal tip cells (DTCs), which guide the migration of the
hermaphrodite gonad during development in the worm

Page 8

TRIO/RhoG/ELMO Signaling Module in Engulfment
2215
grants from the National Science Council and National Health Re-
search Institutes (to Y.-C.W.), and grants from the Swiss National
Science Foundation, The Ernst Hadorn Foundation, and the Euro-
pean Union (FP5 project APOCLEAR) (to M.O.H.).
repeats of ELMO was also tested in a mammalian cell
migration assay, where coexpression of ELMO1 and
Dock180 promotes Transwell migration of LR73 cells
[24]. Although wild-type ELMO1 promoted migration
when coexpressed with Dock180, none of the three
ELMO1 ARM mutants were able to promote migration
(Figure 4A).
Because MIG-2 has also been shown to be involved
in DTC migration (see also Table 2), we asked whether
RhoG could promote mammalian cell migration. Low-
level expression of RhoG strongly promoted migration
of LR73 cells in the in vitro Transwell migration assay
(Figure4B).ThisRhoG-mediatedmigrationwaspotently
inhibited by ELMOT625, a mutant that can bind RhoG
but cannot couple to Dock180/Rac [23, 30], and by the
ELMOARM2mutant that cannot bind RhoG; in contrast,
coexpression of Dock180 and ELMO1wtdid not affect
the enhanced migration due to RhoG (Figure 4B). These
data suggest that increased cell migration due to RhoG
requires the intact ARM repeats of ELMO1 as well as
theabilityofELMOtobindDock180andtherebyactivate
Rac.
Received: September 24, 2004
Revised: October 24, 2004
Accepted: October 27, 2004
Published: December 29, 2004
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Althoughseveralmoleculesinvolvedinengulfmenthave
been identified in recent years, the molecular mecha-
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notfullyunderstood[1,39].Here,throughacombination
of in vitro and in vivo approaches, we identify two evolu-
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(Figure 4C).
Supplemental Data
DetailedExperimentalProcedures andseveralsupplementalfigures
are available at http://www.current-biology.com/cgi/content/full/
14/24/2208/DC1/.
Acknowledgments
We are grateful to Krister Wennerberg for HA-tagged RhoG plas-
mids, Ann Debant for GFP-tagged TRIO plasmids, Michiyuki Mat-
suda for the original His-Dock180 plasmid, and Ian Macara for the
His-tagged bacterially produced Rac. We thank members of the
Ravichandran, Hengartner, and Sondek laboratories for helpful sug-
gestions during this work. This work was supported by National
Institutes of Health grant GM-64709 (to K.S.R.), an Infectious Dis-
eases Training grant from the National Institutes of Health (C.G.),

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