3230 RESEARCH ARTICLE
Development 140, 3230-3243 (2013) doi:10.1242/dev.093732
© 2013. Published by The Company of Biologists Ltd
Phagocytic receptor signaling regulates clathrin and epsinmediated cytoskeletal remodeling during apoptotic cell
engulfment in C. elegans
Qian Shen1, Bin He1, Nan Lu1, Barbara Conradt2, Barth D. Grant3 and Zheng Zhou1,*
SUMMARY
The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently
removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat
protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic
cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is
partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that
EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with
EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like
EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to
CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling.
Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking
clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.
INTRODUCTION
In multicellular organisms, efficient removal of apoptotic cells
through an evolutionarily conserved engulfment and degradation
process is crucial for sculpting organs and establishing correct body
asymmetry, and, furthermore, for preventing inflammatory and
autoimmune responses induced by the contents of dying cells
(Elliott and Ravichandran, 2010). Engulfing cells use distinct cellsurface receptors to recognize the surface features of apoptotic cells
and initiate the extension of pseudopods around their targets (Elliott
and Ravichandran, 2010). The fusion of the extending pseudopods
and the ensuing scission of the apoptotic cell-containing membrane
vacuole (phagosome) from the plasma membrane completes the
engulfment process. The assembly and growth of actin filaments
underneath the plasma membrane provide the driving force for
pseudopod extension around dying cells (Caron, 2001). In addition,
plasma membrane expansion at the phagocytic cup region is also
essential (Touret et al., 2005). Despite extensive studies, however,
how a phagocytic receptor orchestrates both actin assembly and
membrane expansion to accomplish apoptotic-cell engulfment is
not fully understood.
1
Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. 2Center for
Integrated Protein Science, Department of Biology II, Ludwig-Maximilians-University,
Munich, 82152 Planegg-Martinsried, Germany. 3Department of Molecular Biology
and Biochemistry, Rutgers, the State University of New Jersey, NJ 08854-5638, USA.
*Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution
and reproduction in any medium provided that the original work is properly attributed.
Accepted 22 May 2013
The Rho family small GTPases are important regulators for actin
rearrangement during phagocytosis (Ravichandran and Lorenz,
2007). However, besides the Rho GTPases and their modulators,
whether there are additional regulatory pathway(s) for actin
polymerization for apoptotic-cell engulfment is not well known.
More specifically, how a phagocytic receptor establishes the spatial
cue that attracts actin molecules to the region of engulfment needs
thorough investigation.
Traditionally, particles less than 0.5 μm in diameter are believed
to be internalized through endocytosis, whereas particles of 0.5 μm
in diameter or more, such as parasites and apoptotic or senescent
host cells, are thought to be internalized through an actin-dependent,
clathrin-independent phagocytosis (engulfment) process (Caron,
2001). However, this stereotypical belief that clathrin and actin each
acts independently in separate cell internalization events has been
seriously challenged. Accumulating evidence has established the
important roles of actin-clathrin crosstalk during endocytosis
(Kaksonen et al., 2006). Here, we report that apoptotic-cell
engulfment requires the functions of clathrin and its adaptor epsin,
two endocytic factors, in actin remodeling.
During C. elegans embryogenesis, 113 somatic cells undergo
apoptosis; most apoptosis events occur during mid-embryogenesis
[200-450 minutes post-first embryonic cell division (first cleavage)]
(Sulston et al., 1983). Genetic screens have identified many genes
that regulate apoptotic-cell engulfment or degradation or both; in
mutant embryos, cell corpses accumulate during mid- and late
embryonic stages (Lu and Zhou, 2012). Epistasis analyses have
placed genes acting in the engulfment process into two partially
redundant and parallel pathways (Reddien and Horvitz, 2004;
Mangahas and Zhou, 2005; Yu et al., 2006). One pathway is led by
CED-1, a single-pass transmembrane protein and the prototype of
a phagocytic receptor family for apoptotic cells, which also includes
Drosophila Draper, mouse Jedi and human mEGF10 and mEGF11
DEVELOPMENT
KEY WORDS: Actin cytoskeleton, Apoptotic cell engulfment, C. elegans, Clathrin, Epsin, Membrane remodeling
RESEARCH ARTICLE 3231
Clathrin and its adaptor in engulfment
MATERIALS AND METHODS
Reagents
C. elegans strains were raised at 20°C as described (Brenner, 1974). The N2
Bristol strain was the reference wild-type strain, whereas the Hawaii strain
CB4856 was the single nucleotide polymorphism (SNP)-mapping strain.
Mutations and integrated transgenes used are described elsewhere (Riddle
et al., 1997), except when otherwise noted (supplementary material Table
S1). Transgenic lines are generated using germline transformation protocol
(Jin, 1999) and previously established selection strategy (He et al., 2010).
chc-1(b1025ts) worms were raised at 15°C. Hermaphrodites laid egg for 1
hour at 20°C and then were removed. Eggs were kept in 20°C until scoring.
Plasmids and primers are listed in supplementary material Tables S2 and
S3.
Molecular cloning of epn-1
We mapped en47 to chromosome X, between unc-3 and lin-15
(supplementary material Fig. S1A). We further located en47 to a region
between SNP markers Haw109990 and Haw110228 (supplementary
material Fig. S1A) (Wicks et al., 2001). Cosmids and fosmids covering this
region (supplementary material Fig. S1A) were injected individually (at 10
ng/μl) and transgenic animals were scored for the larval-lethal and Ced
phenotypes. In cosmid T04C10, epn-1 is the gene defined by en47
(supplementary material Fig. S1).
RNAi treatment
RNAi of epn-1 and unc-60 individually and together were performed by
microinjection. dsRNAs were synthesized and injected into the gonads of
adult hermaphrodites at 20 hours post mid-L4 at 500 ng/μl (Gönczy et al.,
2000). RNAi of all other clathrin adaptors, chc-1 and chc-1/unc-60
combination were performed by feeding (Fraser et al., 2000), starting at
mid-L4 stage. F1 embryos were scored 24 hours later.
Microscopy
Both somatic cell corpses in staged embryos and germ-cell corpses in adult
gonads under DIC microscope were scored as described (Lu et al., 2009).
Time-lapse recording of the DIC morphology of embryonic cell corpses
and of C1, C2 and C3 engulfment events using fluorescence markers were
performed using the DeltaVision Imaging System (Applied Precision) as
described previously (Lu et al., 2009). The recording of C4 and C5
engulfment started at ~220-250 minutes after first cleavage, and lasted 120140 minutes. Images were deconvolved and analyzed using the SoftWoRx
4.0 software (Lu et al., 2009).
RESULTS
EPN-1 is important for cell-corpse removal
In a screen for mutants containing excessive cell corpses in fourfold
stage embryos, we isolated two recessive alleles, en47 and en48
(supplementary material Fig. S1). We cloned the gene defined by the
en47 and en48 mutations using standard genetic techniques and
found it was epn-1 (supplementary material Fig. S1A) (see
Materials and methods) because: (1) epn-1 genomic DNA or cDNA
rescued the lethal phenotype and cell-corpse removal defective
(Ced) phenotype of the en47 and en48 mutants (supplementary
material Figs S1, S2); (2) nonsense and missense mutations were
identified in the epn-1-coding sequence in en47 and en48 mutants,
respectively (Fig. 1A,F); (3) knocking down epn-1 by RNA
interference (RNAi) reproduced the Ced phenotype of en47 and
en48 mutants (Fig. 1D); and (4) tm3357, an epn-1 deletion allele
(National BioResource Project in Japan), displayed en47 and en48
mutant phenotypes (Fig. 1D; supplementary material Fig. S1B).
en47(m+z–) (m, maternal gene product; z, zygotic gene product)
homozygous embryos descended from en47/+ heterozygous
mothers display relatively modest Ced phenotype, containing 5.1
cell corpses in late fourfold stage embryos (11-13 hours post-first
cleavage) and undergo 100% penetrant L1 larval developmental
arrest (supplementary material Fig. S2A,B). We constructed a strain
that generated homozygous en47(m–z–) progeny that lost both the
maternal and zygotic epn-1(+) (supplementary material Fig. S2A).
Approximately one-third of en47(m–z–) progeny underwent
embryonic developmental arrest, whereas the rest underwent L1
larval arrest (Fig. 1B; supplementary material Fig. S2B,C).
Furthermore, the number of persistent cell corpses retained in late
fourfold stage en47(m–z–) embryos was over threefold of that
retained in en47(m+z–) embryos (Fig. 1B,D; supplementary material
Fig. S2A). These results indicate that depleting both the maternal
and zygotic epn-1 products resulted in strong loss-of-function
phenotypes. Meanwhile, RNAi of epn-1, which is known to
inactivate both maternal and zygotic gene activities (Grishok et al.,
2000), resulted in a Ced phenotype as severe as that displayed by the
en47(m–z–) embryos (Fig. 1D). We thus further followed phenotypes
of en47(m–z–) or epn-1(RNAi) embryos.
Expression of epn-1 cDNA in cell types that can function as
engulfing cells, under the control of the ced-1 promoter (Pced-1)
(Zhou et al., 2001b), resulted in efficient rescue of the Ced
phenotype in epn-1 mutants at all embryonic stages (supplementary
material Fig. S1B,C), suggesting that EPN-1 acts in engulfing cells
to promote cell-corpse removal.
epn-1 encodes a C. elegans homolog of epsin
epn-1 encodes an epsin family member. Epsins possess a
conserved ENTH (Epsin N-terminal homology) domain that
displays high affinity for the membrane phosphoinositide species
PtdIns(4,5)P2 and is able to induce membrane curvature (Horvath
et al., 2007). In addition, epsins contain ubiquitin interacting
DEVELOPMENT
(Lu et al., 2012). Multiple lines of evidence suggest that
phosphatidylserine (PS) serves as an ‘eat-me’ signal that directly or
indirectly stimulates CED-1 (Zhou et al., 2001b; Venegas and Zhou,
2007; Wang et al., 2010). The intracellular domain of CED-1 bears
conserved binding sites for SH2 and PTB domains (Zhou et al.,
2001b). CED-6, a PTB-domain protein, is a candidate adaptor for
CED-1 (Liu and Hengartner, 1998; Su et al., 2002). CED-1 and
CED-6 recruit a downstream mediator DYN-1 (dynamin), a
conserved large GTPase, to budding pseudopods (Yu et al., 2006).
During phagocytosis, instead of promoting membrane fission as in
endocytosis, DYN-1 and mammalian dynamin 2 play
unconventional roles in promoting ‘focal exocytosis’, the
recruitment and fusion of intracellular vesicles to the plasma
membrane, which supports local plasma membrane expansion and
the consequential pseudopod extension (Yu et al., 2006; Gold et al.,
1999). These findings define membrane expansion as one particular
event regulated by CED-1. Here, we further identified actin
rearrangement as another CED-1-regulated event.
In the second pathway, CED-5/Dock180 and CED-12/ELMO1
form a bipartite nucleotide exchange factor to activate CED10/Rac1 GTPase to promote cytoskeleton reorganization (Reddien
and Horvitz, 2004). CED-2/CrkII, an SH2-SH3-domain protein, is
proposed to connect a phagocytic receptor with the CED-5/CED-12
complex. A previous report proposes that both pathways converge
at CED-10 and that CED-10 mediates the actin-reorganization
activity of CED-1 (Kinchen et al., 2005). Our results lead to a
different conclusion.
Here, we have discovered that C. elegans CHC-1 and EPN-1
(epsin) play crucial roles in actin remodeling during apoptotic-cell
engulfment, under the regulation of the CED-1 pathway. Our
findings identify a new event regulated by CED-1 and a new
mechanism that promotes actin remodeling during the engulfment
of apoptotic cells.
3232 RESEARCH ARTICLE
Development 140 (15)
motifs (UIMs), and motifs for binding other endocytic factors,
such as clathrin and clathrin adaptors AP2 and Eps15 (Fig. 1A)
(De Camilli et al., 2002; Horvath et al., 2007). Epsins contribute
to multiple subcellular events that require membrane remodeling,
and are best known for acting as clathrin adaptors during
endocytosis (Horvath et al., 2007). However, whether any epsin
family member(s) contribute to phagocytosis of apoptotic cells
has not been explored previously.
EPN-1 possesses all of the above domains and motifs (Fig. 1A).
In EPN-1, the nine α-helices that are crucial for the tertiary structure
of the ENTH domain and residues that are crucial for binding to
PtdIns(4,5)P2 are all highly conserved (Fig. 1F) (Ford et al., 2002;
Itoh et al., 2001). The en47 allele alters residue Q94 to a stop codon
and creates a presumptive null mutation (Fig. 1F). en48 carries a
missense mutation, converting an invariant residue (R63) that is
crucial for PtdIns(4,5)P2-binding to alanine (Fig. 1F). The Ced
phenotypes displayed by the en48 and en47 mutants are
quantitatively similar, indicating that PtdIns(4,5)P2-binding is likely
to be essential for EPN-1 function (Fig. 1D).
Inactivating C. elegans clathrin heavy chain
causes cell-corpse accumulation
We further examined whether chc-1, the only C. elegans clathrin
heavy chain (CHC-1)-coding gene (Fig. 1C), was involved in
DEVELOPMENT
Fig. 1. Inactivating epn-1 or chc-1 results in cell-corpse removal defects. (A) EPN-1 domain structure (top) and gene structures of epn-1 (middle)
and epn-1(tm3357) deletion allele (bottom), which causes a frameshift after amino acid 258 and generates a premature stop codon. ENTH, epsin Nterminal homology domain; U, ubiquitin-interaction motif; AP2, Cla and Eps15, AP2-, clathrin- and EPS15-binding motifs. (B) DIC images of 11-13 hours
post-first cleavage (late fourfold stage) embryos (a-c) and L1 larvae (d,e). (b) An arrested embryo; (c) an embryo that elongates normally. Scale bars: 10
μm in a-c; 20 μm in d,e. Arrows indicate cell corpses. (C) CHC-1 domain structure (top) and gene structures of chc-1 (middle) and the chc-1(bc376) allele
(bottom). T, trimerization domain. chc-1(b1025ts) carries a 9 bp deletion (red letters) around the stop codon and adds 22 novel amino acids to the C
terminus (Sato et al., 2009). chc-1(bc376) carries a 2002 bp deletion (dashed line), creating a premature stop codon (underlined). (D) Ced phenotype
quantification. Data are mean±s.d. n, numbers of embryos scored. (RNAi)(1) and (RNAi)(2) correspond to two different regions of epn-1 cDNA. (E) DIC
images of embryos aged 11-13 hours post-first cleavage to indicate elongation arrest and persistent cell corpses (arrows). (b,c) Arrested embryos. Scale
bars: 10 μm. (F) Alignment of ENTH domains. Gray bars underline α-helices. Arrowheads indicate residues crucial for Ins(1,4,5)P3 binding in epsin 1.
Clathrin and its adaptor in engulfment
RESEARCH ARTICLE 3233
apoptotic cell removal. chc-1(RNAi) resulted in severe defects in
the uptake of yolk by oocytes (supplementary material Fig. S3A)
and in embryonic development (Fig. 1Eb), as previously reported
(Grant and Hirsh, 1999). Although embryonic elongation was
arrested, the head appeared to develop normally, allowing us to
visualize developmental apoptosis events (Fig. 1Eb). chc-1(RNAi)
caused cell-corpse accumulation in embryos (Fig. 1D,E). The Ced
and lethal phenotypes were also observed from chc-1(bc376)(m–z–)
deletion mutant embryos (Fig. 1C-E). In adult hermaphrodite
gonads, where apoptotic germ cells should be swiftly removed by
gonadal sheath cells, chc-1(RNAi) also resulted in the accumulation
of germ-cell corpses (supplementary material Fig. S3B,C).
ced-3(n717), a loss-of-function mutation of the C. elegans CED3 caspase, blocks all apoptosis (Yuan et al., 1993). In ced-3(n717);
chc-1(RNAi) embryos, no cell corpse-like objects were observed
(Fig. 1D), indicating that the button-shaped objects observed from
chc-1(RNAi) embryos (Fig. 1E) were indeed cell corpses.
Inactivating chc-1 or epn-1 specifically impairs
cell-corpse engulfment
We first found that chc-1(RNAi) did not induce any extra apoptosis
events (supplementary material Fig. S4A). By contrast, frequent
Fig. 2. Inactivating multiple clathrin adaptors other than EPN-1 or
the actin-clathrin coupling protein HIPR-1 failed to cause cell-corpse
removal defects in embryos. (A) Number of cell corpses in embryos 1113 hours post-first cleavage. Data are mean±s.d. n, number of embryos
scored. (B) DIC images of 11- to 13-hour-old embryos that develop
normally (a) or undergo elongation arrest (b-d). Arrows indicate
pharyngeal lumen. Scale bars: 10 μm.
delays in cell-corpse removal were observed from chc-1(RNAi)
embryos (supplementary material Fig. S4B). These observations
indicate that the Ced phenotype observed in chc-1(RNAi) animals
is caused by impairing cell-corpse removal rather than excessive
cell deaths.
An engulfing cell-specific CED-1ΔC::GFP reporter, in which
the intracellular domain of CED-1 is replaced by GFP, allows us
to track both the engulfment and degradation of cell corpses,
because this transmembrane reporter is capable of recognizing
neighboring apoptotic cells and is enriched on extending
pseudopods; moreover, it is subsequently retained on the surface
of a phagosome until total degradation (Zhou et al., 2001b) (N.L.
and Z.Z., unpublished). We scored the number of engulfed
(labeled with a CED-1ΔC::GFP circle) and unengulfed (not
labeled) cell corpses in epn-1(en47)(m–z–) mutants and in chc1(b1025) mutants, a temperature-sensitive allele (Fig. 1C) (Sato et
al., 2009). Unlike in wild-type embryos, in chc-1 and epn-1 mutant
embryos, <13% of cell corpses were engulfed by mid- (1.5-fold)
or late (fourfold) embryonic stages (Fig. 3C-D). These results
clearly indicate an engulfment defect.
To obtain further detail, we monitored the engulfment of three
particular cell corpses (C1, C2 and C3), which undergo highly
reproducible apoptosis at ~330 minutes post-first cleavage
(Fig. 3A,B), in living embryos following our previously established
DEVELOPMENT
The cell-corpse removal activity of EPN-1 is unique
among clathrin adaptors
The functions of clathrin as a coat protein in different sorting events
are specified by distinct adaptors (Robinson, 2004). Major clathrin
adaptors include the AP (adaptor protein) 1 and 3 complexes for
sorting at the trans-Golgi network (TGN), the AP-2 complex, which
is localized to the plasma membrane and facilitates clathrindependent endocytosis, and several monomeric clathrin adaptors,
including epsins and AP180 (Robinson, 2004). To examine whether
any additional clathrin adaptors regulate cell-corpse removal, we
scored the Ced phenotype in embryos in which C. elegans homologs
of particular adaptors were individually inactivated. We found that
inactivation of subunits of AP-1, AP-2 and AP-3 complexes or
AP180 (Grant and Hirsh, 1999; Boehm and Bonifacino, 2001;
Nonet et al., 1999; Gu et al., 2008) by either RNAi or loss-offunction mutations failed to cause abnormal accumulation of cell
corpses (Fig. 2A). RNAi of apb-1 and apg-1 both resulted in highly
penetrant embryonic arrest as previously reported (Fig. 2B) (Grant
and Hirsh, 1999; Simmer et al., 2003; Sönnichsen et al., 2005),
indicating effective gene inactivation by RNAi. Interestingly,
inactivating AP-1, AP-2 or AP-3 complex subunits or epn-1 failed
to affect germ cell-corpse removal (supplementary material Fig.
S3B). These results suggest that among the clathrin adaptors
examined, EPN-1 is uniquely involved in cell-corpse removal in
embryos.
Previously, through comparative analysis of two distinct kinds of
DYN-1 mutations, we learned that endocytosis and cell-corpse
engulfment were independent events (Yu et al., 2006). Inactivating
each of the AP2 subunits APA-2, APB-1, DPY-23 or APS-2
individually is known to impair endocytosis (Grant and Hirsh, 1999;
Gu et al., 2008; Gu et al., 2013), but not cell-corpse removal (Fig.
2A). Likewise, inactivating yolk receptor gene rme-2 resulted in
severe yolk endocytosis defect and embryonic lethality (Grant and
Hirsh, 1999) (Fig. 2Bd) but did not affect cell-corpse removal
(Fig. 2A). Our observation that inactivating AP2 subunits or rme-2
does not result in Ced phenotype (Fig. 2) again confirms that
impairing clathrin-mediated endocytosis does not necessarily
inactivate cell-corpse removal; they further underline the unique
function of EPN-1 in apoptotic-cell removal.
3234 RESEARCH ARTICLE
Development 140 (15)
protocol (Lu et al., 2009). In wild-type embryos, the engulfment
process, starting with the budding of pseudopods and ending when
the phagocytic cup is fully closed, took ~7 minutes (Fig. 3E,Fa-e).
In chc-1(b1025) mutants raised at the restrictive temperature and in
epn-1(en47)(m–z–) embryos, the initiation of pseudopod budding
towards C1, C2 and C3 occurred at normal time points (data not
shown), yet pseudopod extension took a significantly longer time to
complete (Fig. 3E,Fh-x). By contrast, phagosomes containing C1,
C2 or C3, once formed, took relatively normal time to degrade the
contents in chc-1 or epn-1 mutants (Fig. 3E,F). These results
revealed that pseudopod extension, but not phagosome maturation,
is specifically affected by chc-1 or epn-1.
epn-1 and chc-1 both act in the ced-1 pathway
To determine whether epn-1 and chc-1 act in either one of the two
known cell-corpse engulfment pathways (see Introduction), we
performed epistasis analysis, following the same principle that
governs the original analyses placing the engulfment genes into two
pathways (Mangahas and Zhou, 2005). We found that chc-1(RNAi)
in ced-5(n1812) null or ced-12(n3261) strong loss-of-function
mutants (Wu and Horvitz, 1998b; Zhou et al., 2001a), which belong
to the ced-10 pathway, increased the number of persistent cell
corpses by 32% and 49%, respectively (Fig. 4Aa). By contrast, chc1(RNAi) in ced-1(e1735) or dyn-1(n4039) null mutants (Zhou et
al., 2001b; Yu et al., 2006), and in ced-6(n2095) strong loss-of-
DEVELOPMENT
Fig. 3. The chc-1 and epn-1 mutations impair cell-corpse engulfment. (A) The identity of the dying cells and their engulfing cells monitored here.
(B) The positions of the dying (dots) and corresponding engulfing cells (open circles or petals) on the ventral side at stages when engulfment occurs.
Anterior is towards the top. (C) DIC and GFP images of 1.5-fold embryos expressing Pced-1ced-1ΔC::gfp. Arrows and arrowheads indicate cell corpses
labeled or not labeled with GFP circles, respectively. Embryos were raised at 20°C, the restrictive temperature for chc-1(b1025) mutants. Scale bars: 5 μm.
(D) The number of cell corpses (DIC morphology) and phagosomes (CED-1ΔC::GFP circles). Normally, cell corpses lose their DIC morphology ~30
minutes after phagosome maturation initiates; hence, more phagosomes are observed than button-like corpses in fourfold stage wild-type embryos.
Data are mean±s.d. n, number of embryos scored. (E) The average time periods for engulfing or degrading C1, C2 or C3. Data are mean±s.d. n, numbers
of events analyzed. (F) Time-lapse images of CED-1ΔC::GFP around C3 in ~330-minute embryos. 0 minutes: when pseudopods (arrowheads) are first
generated. Arrows indicate nascent phagosomes. Scale bars: 2 μm.
RESEARCH ARTICLE 3235
Fig. 4. CHC-1 and EPN-1 act in the CED-1 pathway. (A) Epistasis analysis between chc-1 (a) or epn-1 (b) and existing engulfment mutants. The
numbers of cell corpses in at least 15 embryos (11-13 hours old). Data are mean±s.d. *P<0.0001 (Student’s t-test). ns, no significant difference (P>0.05).
(B) Images of wild-type embryos expressing Pced-1epn-1::gfp. Arrows indicate the plasma membranes of eight intestinal precursor cells (a,b) and a few
hypodermal cells (c,d). Open arrows (b) indicate cytoplasmic puncta. An arrowhead (c,d) marks a phagosome containing C3. Anterior is towards the top.
Scale bars: 5 μm. (C,D) Time-lapse images of reporter enrichment on phagocytic cups (closed arrowheads) and nascent phagosomes (closed arrows). 0
minutes: when pseudopods are first generated. Open arrowheads indicate pseudopods lacking reporter enrichment. The open arrow in C(a) indicates
the engulfing cell for C3. At least nine engulfment events were monitored for each condition. Scale bars: 2 μm.
function mutants (Liu and Hengartner, 1998), which all belong to
the ced-1 pathway, did not significantly enhance the Ced phenotype
of any single mutants (Fig. 4Aa).
We further constructed double mutants in which the epn1(en47)(m–z–) allele was coupled to six ced mutants, three from
each pathway. epn-1(en47) enhanced the Ced phenotype of ced-5,
ced-12 or ced-10 mutants by 47%, 69% and 122%, respectively,
but failed to significantly enhance the ced-1, ced-6 or ced-7 single
mutant phenotypes (Fig. 4Ab). chc-1(RNAi) did not significantly
enhance the Ced phenotype of epn-1(en47)(m–z–) (Fig. 4Aa)
either, indicating that they act in the same pathway. Together, the
above results indicate that both chc-1 and epn-1 act in the ced-1,
DEVELOPMENT
Clathrin and its adaptor in engulfment
ced-6 and ced-7 pathway, and in parallel to ced-5, ced-12 and
ced-10.
EPN-1 is enriched on extending pseudopods in a
CED-1 pathway-dependent manner
We detected a broad expression pattern of Pepn-1epn-1::gfp, which
is functional in rescuing the epn-1(en47) mutant phenotype
(supplementary material Fig. S1B) in many cell types, including
cells that can act as engulfing cells (supplementary material Fig.
S5). In adults, EPN-1::GFP was observed in the pharynx, the nerve
ring, body wall muscles, the intestine, vulva, hypodermis and
coelomocytes (supplementary material Fig. S5c-j) (data not shown).
In embryos, EPN-1::GFP was observed in most cells, in particular
in hypodermal and intestinal cells, major embryonic engulfing cell
types (supplementary material Fig. S5a-b). EPN-1::GFP, regardless
expressed from Pepn-1 or Pced-1, is localized to both the cytoplasm
and plasma membrane (Fig. 4B; supplementary material Fig. S5). In
the cytoplasm, EPN-1::GFP is either evenly distributed or
associated with puncta that likely represent intracellular organelles
(Fig. 4Bb,d).
In embryos, EPN-1::GFP is enriched on extending pseudopods
and remains on the surfaces of nascent phagosomes for 4-6 minutes
before disappearing (Fig. 4Cb; supplementary material Fig. S5a-b).
PtdIns(4,5)P2, which binds to many clathrin adaptors [including
epsin (Itoh et al., 2001)], is a plasma membrane-enriched lipid
essential for actin cytoskeleton remodeling in many cellular events,
including phagocytosis (Botelho et al., 2004). We constructed a
reporter for PtdIns(4,5)P2 by tagging the pleckstrin homology (PH)
domain of human phospholipase C δ1, a high-affinity PtdIns(4,5)P2binding domain, with GFP, and expressed it in engulfing cells
(Pced-1). In embryos, PH::GFP is transiently enriched on extending
pseudopods during engulfment and disappears once a nascent
phagosome forms (Fig. 4Ca). The temporal enrichment patterns of
EPN-1::GFP and PtdIns(4,5)P2 are almost identical (Fig. 4Ca,b),
suggesting that PtdIns(4,5)P2 might be a key factor recruiting
EPN-1.
When the point mutation en48(R63A) was introduced into the
EPN-1::GFP reporter, EPN-1(R63A)::GFP was neither localized to
the plasma membrane nor enriched on phygocytic cups or
phagosomes (Fig. 4Cc), indicating that PtdIns(4,5)P2 plays an
important role in attracting EPN-1 to pseudopods.
In ced-1, ced-5, ced-6 and ced-12 mutants, the speed of
pseudopod extension was severely reduced; however, in ced-1 and
ced-6 mutants, but not in ced-5 or ced-12 mutants, EPN-1::GFP
failed to enrich on pseudopods (Fig. 4Cd-g). Interestingly, in ced-5
and ced-12 mutants, EPN-1::GFP persisted on pseudopods whose
extension was blocked (Fig. 4Cd,e). These results indicate that the
recruitment of EPN-1 to pseudopods is dependent on ced-1 and ced6 but not on ced-5 or ced-12. By contrast, the dynamic clustering of
CED-1::GFP on extending pseudopods is normal in epn-1(m–z–)
mutants (Fig. 4D). Together, our studies indicate that EPN-1 acts
downstream of the CED-1 signaling pathway.
EPN-1 recruits CHC-1 to extending pseudopods in
response to CED-1 signaling
A CHC-1::GFP reporter expressed in embryos from Pced-1 is
localized to the plasma membrane, the cytoplasm and the
perinuclear region (Fig. 5A). Part of CHC-1::GFP was enriched on
cytoplasmic puncta (Fig. 5A), which might correspond to early
endosomes and/or Trans-Golgi network. CHC-1::GFP is transiently
enriched on pseudopods and nascent phagosomes (Fig. 5A-C),
frequently appearing as puncta (Fig. 5B), indicating that CHC-1
Development 140 (15)
might form either traditional clathrin-coated pits or flat patches
through self-oligomerization (Brodsky, 2012). In embryos coexpressing CHC-1::GFP and EPN-1::mRFP in engulfing cells, they
exhibit colocalization on extending pseudopods and nascent
phagosomes, with significant co-enrichment on puncta (Fig. 5B),
indicating that EPN-1 is associated with oligomerized clathrin
coating specific regions of the pseudopod membrane.
In epn-1(RNAi) embryos, the recruitment of CHC-1::YFP to
pseudopods and phagosomes was defective (Fig. 5D,E). [In cells
lacking enrichment of CHC-1 on pseudopods (Fig. 5Dc), a
phagosome was distinguishable as a dark sphere embedded inside
the green engulfing cell cytoplasm.] By contrast, the enrichment
pattern of EPN-1::GFP on pseudopods and phagosomes remained
relatively normal in chc-1(RNAi) embryos (Fig. 4Ch). These results
indicate that enrichment of CHC-1 to pseudopods and nascent
phagosomes relies on EPN-1, but not vice versa.
Like EPN-1::GFP, in ced-5, ced-10 and ced-12 mutants, CHC1::YFP was persistently enriched on pseudopods and nascent
phagosomes (Fig. 5C,E), indicating that the ced-10 pathway is not
involved in recruiting CHC-1 to pseudopods. By contrast, in ced-1,
ced-6 or dyn-1 mutants, the enrichment of CHC-1 to pseudopods
was severely reduced or completely blocked (Fig. 5Cb,c,Dd,E),
indicating that the CED-1 pathway recruits CHC-1 to the
engulfment site. The enrichment of DYN-1::GFP to pseudopods
and nascent phagosomes remained normal when epn-1 or chc-1 was
inactivated (supplementary material Fig. S6), again suggesting that
DYN-1 acts upstream of both EPN-1 and CHC-1.
EPN-1 and CHC-1 promote actin polymerization
during pseudopod extension
Actin rearrangement underneath the phagocytic cup provides a
mechanical driving force for pseudopod extension (Caron, 2001).
Using a GFP-tagged actin-binding domain of Drosophila moesin
as a reporter for polymerized actin filaments (F-actin) expressed in
engulfing cells (Lu et al., 2011), we monitored the dynamics of actin
polymerization during pseudopod extension around C1, C2 and C3.
In wild-type embryos, F-actin first appeared at the site where
pseudopods budded (Fig. 6A, 0 minutes), and subsequently
extended around the cell corpse in a zipper-like manner until a
phagocytic cup was closed, in a period lasting 4-6 minutes
(Fig. 6A,D). Afterwards, F-actin disassembled asymmetrically from
the base of a phagosome, completing within ~6 minutes (Fig. 6A,
6-9 minutes). Furthermore, GFP::moesin was simultaneously coenriched with EPN-1::mRFP on extending pseudopods and nascent
phagosomes, frequently on patches and puncta that might be actin
organizing centers (Fig. 6A).
When epn-1 or chc-1 was RNAi inactivated, the extension of Factin was often slowed down (Fig. 6B, 0-8 minutes and 6C, 0-16
minutes). Furthermore, we observed repeated extension, retraction
and re-extension of actin filaments (Fig. 6B, 8-14 minutes and 6C,
16-26 minutes and 28-34 minutes). As a consequence, engulfment
took two to four times as long as in wild-type embryos (Fig. 6D).
These phenotypes indicate that EPN-1 and CHC-1 are essential for
the extension and stability of F-actin underneath pseudopods.
To explore further the variety of actin rearrangement defects in
engulfing cells of different identities, we monitored the actin
behavior during the engulfment of two additional dying cells, C4
and C5, by their sister cells ABplpppapa and ABprpppapa,
respectively, during early embryogenesis (Fig. 3A,B) (Sulston et
al., 1983). Unlike C1, C2 or C3, which lacked detectable
GFP::moesin expression, C4 and C5 inherited a faint GFP::moesin
signal from their mother cells (Fig. 6F, 0 minutes). However, the
DEVELOPMENT
3236 RESEARCH ARTICLE
Clathrin and its adaptor in engulfment
RESEARCH ARTICLE 3237
Fig. 5. CHC-1 enrichment to phagocytic cups depends on EPN-1, DYN-1, CED-6 and CED-1. (A) GFP images of wild-type embryos expressing
Pced-1chc-1::gfp. Yellow arrows indicate the nuclear membranes of eight intestinal precursor cells (a) and the plasma membrane of hypodermal cells (b,c).
Yellow arrowheads indicate cytoplasmic puncta. A white arrow (c) marks a nascent phagosome. Anterior is towards the top. Scale bars: 5 μm. (B) Timelapse images of EPN-1::mRFP and CHC-1::GFP on the phagocytic cup engulfing C3. 0 minutes: when engulfment just completes. Arrowheads mark
puncta on phagocytic cups and phagosomes. Scale bars: 2 μm. (C) Images of ~330-minute stage embryos expressing Pced-1chc-1::yfp. Phagosomes
labeled or not labeled with YFP are marked with closed or open arrows, respectively. Scale bars: 5 μm. Insets: the region surrounding each arrow, with
2.5-fold magnification. (D) Time-lapse images monitoring CHC-1::YFP enrichment on phagocytic cups (arrowheads) and phagosomes (white arrows)
internalizing C1, C2 or C3. 0 minutes: when engulfment just completes. Closed and open arrowheads mark pseudopods with or without YFP signal,
respectively. Black arrows indicate cell corpses. Scale bars: 2 μm. (E) The frequency of CHC-1::YFP enrichment on phagocytic cups. n, the number of
engulfment events analyzed.
which was not observed from C1, C2 or C3, suggests defects in
another actin-related event: cell adhesion (Cougoule et al., 2004).
Impairing actin depolymerization partially
suppresses epn-1 and chc-1 phenotypes
C. elegans unc-60 encodes two isoforms that are members of the
ADF/cofilin family of actin-depolymerization factors (McKim et al.,
1994; Ono and Benian, 1998). These isoforms exhibit differential
actin-remodeling activities (Ono et al., 2008). UNC-60A, a nonmuscle isoform, strongly inhibits actin polymerization in vitro (Ono
and Benian, 1998). Inactivation of unc-60 results in actin organization
defects in multiple tissues (Ono et al., 2003; Ono et al., 2008). We
found that inactivating unc-60, through either gk239, a null mutation,
DEVELOPMENT
extension of engulfing cell F-actin around them could still be easily
recognized because of the further enriched GFP signal within
phagocytic cups (Fig. 6F; supplementary material Movie 1). In
wild-type embryos, the time needed for engulfing C4 and C5 varied
over a bigger range than for engulfing C1, C2 and C3 (Fig. 6E,F),
perhaps influenced by factors such as the different identities of the
corresponding engulfing cells. In epn-1(RNAi) and chc-1(RNAi)
embryos, delayed formation and closure of phagocytic cups
occurred frequently and over a large time span (Fig. 6E). In certain
extreme cases, despite the attempt of engulfing cells to extend actin
filaments around them, C4 and C5 were detached from their
engulfing cells and eventually from the embryo (Fig. 6G;
supplementary material Movie 2). This detachment phenotype,
3238 RESEARCH ARTICLE
Development 140 (15)
Fig. 6. EPN-1 and CHC-1 promote actin polymerization underneath pseudopods. (A-C) Time-lapse images of GFP::moesin along C2 and C3.
0 minutes: when pseudopods are first generated. Yellow or white arrowheads indicate pseudopods shorter or longer than one-quarter of a phagosome
(white arrows), respectively. Red boxes highlight F-actin retraction events. Scale bars: 2 μm. (A) Co-expression of Pced-1epn-1::mRFP and Pced-1gfp::moesin.
Open arrows indicate GFP and mRFP co-enriched puncta. (D,E) Histograms of the time needed for engulfing C1, C2 and C3 (D) or C4 and C5 (E). n, total
numbers of engulfment events monitored. (F,G) Time-lapse images of normal (F) and failed engulfment (G) of C4 and C5 (white arrows). 0 minutes:
when the refractile DIC morphology of C5 first appears. Open arrowheads mark the engulfing cells for C4 and C5. Yellow arrows mark F-actin
connecting C5 with its engulfing cell. Scale bars: 5 μm.
suggest that inactivation of epn-1 and chc-1 directly influences the
extension and stability of actin filaments.
Budding yeast Sla2 and its mammalian homolog Hip1R are
proteins that couple actin with clathrin-coated pits during
endocytosis (Boettner et al., 2012). We examined the involvement
of HIPR-1, the only full-length homolog of Hip1R in C. elegans, in
engulfment. We found that hipr-1(ok1081), a deletion that
eliminates the C-terminal two-thirds of HIPR-1 and is likely a null
mutation (www.wormbase.org), does not affect embryonic cellcorpse engulfment (Fig. 2A). This result does not support a role of
HIPR-1 in clathrin-mediated engulfment.
ced-1, ced-6 and dyn-1 mutants are defective in
promoting actin polymerization for cell-corpse
engulfment
If, as indicated by the above results, the CED-1 pathway regulates
CHC-1 localization during engulfment through EPN-1,
inactivating CED-1, CED-6 or DYN-1 should impair actin
rearrangement around cell corpses. In ced-1 mutant embryos, the
extension of F-actin along C1, C2 or C3 was often much slower
DEVELOPMENT
or RNAi, reduced the number of persistent cell corpses generated by
epn-1(RNAi) or chc-1(RNAi) (Fig. 7A). We further monitored
engulfment in unc-60 single RNAi and unc-60/epn-1 and unc-60/chc1 double RNAi embryos using GFP::moesin (Fig. 7B-F). Inactivation
of unc-60 partially reverted the delayed actin filament extension
phenotype caused by inactivating epn-1 or chc-1 (Fig. 7C,D). As a
result, engulfment took much shorter time than in epn-1 or chc-1
single RNAi backgrounds (Fig. 7E,F). In unc-60(RNAi)-treated adult
hermaphrodites, instead of forming long filaments, actin formed short
aggregates and puncta in gonadal sheath cells (supplementary
material Fig. S7), similar to a previous report (Ono et al., 2008). This
phenotype indicates the important function of UNC-60 in establishing
F-actin structures in multiple cell types. Interestingly, unc-60(RNAi)
alone resulted in a modest delay of pesudopod extension (Fig. 7E),
whereas in wild-type embryos, the engulfment of C1, C2 and C3 was
finished within 6 minutes in 100% of cases, and in unc-60(RNAi)
embryos, 27% of engulfment events lasted 7-10 minutes. Together,
our results thus indicate that the organization and extension of actin
filaments along the engulfment targets requires the balanced action of
dynamic actin polymerization and depolymerization. They further
Clathrin and its adaptor in engulfment
RESEARCH ARTICLE 3239
than in wild-type embryos (Fig. 8A). In addition, along the path of
pseudopod extension, F-actin repeatedly retracted and re-grew
(Fig. 8A, red boxes). Similar slow extension and repeated
retraction phenotypes of F-actin were also observed in ced6(n2095) and dyn-1(n4039) mutant embryos (Fig. 8B,C). These
defects significantly contributed to the overall slow speed of
engulfment (Fig. 8D).
During the engulfment of C4 and C5, we also observed retraction
of actin filaments around the target in ced-1(e1735) background
(Fig. 8F, 0-23 minutes). In addition, persistent C4 and C5 were often
seen partially or fully detached from their engulfing cells (Fig. 8F,
47-55 minutes) and remained eventually unengulfed (Fig. 8F, 55131 minutes). The severe engulfment defects are shared by dyn-1
mutants (Fig. 8E,G; supplementary material Movie 3).
The distinct actin-related defects observed from ced-1, ced-6 and
dyn-1 mutants, such as the repeated retraction of actin filaments in
the phagocytic cups, and the loss of cell-cell attachment, are similar
to that observed in epn-1(RNAi) and chc-1(RNAi) embryos (Figs 6,
8), indicating that the CED-1 pathway regulates actin assembly
underneath the growing pseudopods through EPN-1 and CHC-1.
DISCUSSION
Clathrin and EPN-1 form an actin-organizing
center to facilitate pseudopod extension
During endocytosis, clathrin forms triskelions that further assemble
into polyhedron cages surrounding endocytic vesicles (Young,
2007). The diameter of a canonical clathrin cage is usually less than
150 nm (Harrison and Kirchhausen, 1983; Fotin et al., 2004). The
clathrin coat was thus long thought to be only involved in the
internalization of small particles, not large ones such as dying cells.
Although, traditionally, endocytosis was regarded as an actinindependent event, recent discoveries have established that actin is
recruited to clathrin cages to facilitate vesicle invagination, applying
the membrane-bending force generated by F-actin (Mooren et al.,
2012).
Here, we reveal the essential functions of clathrin heavy chain and
its adaptor epsin in C. elegans apoptotic-cell engulfment. These
functions establish an engulfment mechanism mediated by clathrin,
implicating multiple novel aspects. First, CHC-1::GFP form puncta
decorating phagocytic cups. These puncta might represent clusters of
clathrin-coated buds as intermediates of endocytosis, or, alternatively,
larger lattices that are not subject to being pinched off the plasma
membrane. If they represent clathrin-coated buds, inactivating
dynamin, a fission factor that pinches off clathrin-coated vesicles
(Schmid and Frolov, 2011), should result in their membrane retention.
However, inactivating dyn-1 prevents CHC-1 from being recruited to
phagocytic cups instead of causing its retention, indicating that the
CHC-1-puncta are unlikely to represent endocytic intermediates. By
contrast, EPN-1 colocalizes with F-actin underneath pseudopods;
furthermore, inactivating chc-1 or epn-1 impairs multiple aspects of
actin rearrangement underneath the phagocytic cup, reducing the
growth speed and stability of pseudopods and weakening the
DEVELOPMENT
Fig. 7. Inactivating unc-60 partially rescue the actin-polymerization defects caused by inactivating epn-1 or chc-1. (A) unc-60 null mutation or
RNAi partially suppressed the Ced phenotype caused by epn-1 or chc-1 RNAi. (B-D) Time-lapse images of actin polymerization along pseudopods
engulfing C2 and C3. 0 minutes: when pseudopods are first generated. Yellow or white arrowheads indicate pseudopods shorter or longer than onequarter of a phagosome (white arrows), respectively. Scale bars: 2 μm. (E,F) Histograms of the time needed for engulfing C1, C2 or C3 in wild-type
embryos treated with various RNAi constructs. n, total numbers of C1, C2 and C3 monitored.
3240 RESEARCH ARTICLE
Development 140 (15)
engulfing-dying cell adhesion. Based on these observations, we
propose that underneath a phagocytic cup, CHC-1 oligomerizes into
a scaffolding structure that facilitates actin remodeling. This structure
might resemble the flat clathrin patches that coat specific membrane
domains on endosomes or the trans-Golgi network (Young, 2007;
Williams and Urbé, 2007), or the clathrin plaques that facilitate the
entry of bacterial pathogens into non-phagocytic mammalian cells
through organizing actin polymerization (Bonazzi et al., 2011). Our
observations further suggest that, unlike in endocytosis, where actin
primarily facilitates the invagination of clathrin-coated membrane
and the generation of relatively small endocytic vesicles, during the
engulfment of apoptotic cells, which are much larger (at least 3 μm),
the clathrin-actin crosstalk not only induces membrane curvature, but,
more importantly, directs actin polymerization and drives pseudopod
extension around apoptotic cells.
Further study is needed to determine, during phagocytosis of
apoptotic cells or other targets, how a flat clathrin scaffold promotes
actin assembly and whether traditional coated pits play any role in
actin remodeling.
Second, our study suggests that HIPR-1, the only worm homolog
of mammalian actin-clathrin coupling protein Hip1R in endocytosis,
is either not involved in clathrin-mediated engulfment, or,
alternatively, is involved in a manner completely redundant with
another unknown protein. Either way, this finding implies that a yetto-be-identified non-HIPR-1 protein couples clathrin with actin, and
thereby might regulate F-actin organization via a novel mechanism.
Third, this work reveals that DYN-1 regulates clathrin-actin
crosstalk in engulfment. DYN-1 acts to recruit clathrin to
phagocytic cups through first recruiting EPN-1. This
unconventional activity of dynamin might be used in multiple
cytoskeleton remodeling events.
Moreover, this work identifies the novel physiological role of
EPN-1 in apoptotic-cell engulfment. Epsins are known to recruit
clathrin to the plasma membrane during endocytosis and to the
trans-Golgi network during vesicle trafficking from the trans-Golgi
network to endosomes (Wendland, 2002; Mills et al., 2003). C.
elegans EPN-1 was reported to promote the endocytosis of Notch
ligands and LRP-1/Megalin, a low-density lipoprotein receptor
family member (Tian et al., 2004; Kang et al., 2013). We find that
EPN-1 and CHC-1 act in the same engulfment pathway, and,
furthermore, EPN-1 promotes the recruitment of CHC-1 to
phagocytic cups, supporting the model that EPN-1 serves as a
clathrin adaptor (Fig. 9B).
In addition to recruiting clathrin, epsin might also contribute its
membrane curvature-inducing activity to promote efficient
engulfment. Pseudopod extension around a prey requires continuous
DEVELOPMENT
Fig. 8. ced-1, ced-6 and dyn-1 mutants display multiple defects in actin polymerization during engulfment. (A-C) Time-lapse images monitoring
engulfment of C1 and C3. White arrowheads and arrows indicate F-actin underneath pseudopods and nascent phagosomes, respectively. Red boxes
highlight F-actin retraction events. 0 minutes: when pseudopods are first generated. (D,E) Histograms of the time period for engulfing C1, C2 and C3 (D)
or C4 and C5 (E). n, total numbers of engulfment events monitored. (F,G) Time-lapse images monitoring C5 (arrows). Open and closed arrowheads
indicate the engulfing cell for C5 and the F-actin partially surrounding or attached to C5, respectively. Red boxes highlight F-actin retraction events. 0
minutes: when C5 was first observed as a cell corpse. Yellow arrow marks unengulfed C4 (G). Scale bars: 2 μm.
Clathrin and its adaptor in engulfment
RESEARCH ARTICLE 3241
generation of membrane curvature to match the shape of the prey.
The curvature-generating mechanism dedicated to apoptotic-cell
engulfment remains unclear. Epsin is an efficient membrane
generator (Ford et al., 2002; Boucrot et al., 2012). We propose that,
during engulfment, EPN-1 induces membrane curvature, whereas
the associating clathrin scaffold and the subsequently attached Factin further stabilize the curvature. The cooperative actions of
EPN-1, CHC-1 and F-actin thus result in the continuous extension
of pseudopods along apoptotic-cell surfaces (Fig. 9B).
The CED-1 pathway recruits EPN-1 to phagocytic
cups
During apoptotic-cell engulfment, we observe transient enrichment of
lipid second messenger PtdIns(4,5)P2 to extending pseudopods, and
further discover that PtdIns(4,5)P2 is an important signaling molecule
recruiting EPN-1 to pseudopods. Similarly, during the phagocytosis
of opsonized objects by mammalian phagocytes, PtdIns(4,5)P2 is
transiently enriched to extending pseudopods (Botelho et al., 2004).
As CED-1, CED-6 and DYN-1 are all essential for recruiting EPN-1
to pseudopods, we propose that, as a phagocytic receptor, CED-1
might promote the regional burst of a PtdIns(4,5)P2-synthesis activity
through its mediator DYN-1 and an effector PI-kinase(s) (Fig. 9),
similar to how it promotes PtdIns(3)P synthesis on nascent
phagosomes (Yu et al., 2008; Lu et al., 2012).
Epsins are known to interact with transmembrane receptors
through ubiquitin-mediated or ubiquitylation-independent
interactions (Horvath et al., 2007; Kang et al., 2013). Currently, it
is unknown whether CED-1 is ubiquitylated and whether CED-1
and EPN-1 directly interact. If direct interaction occurs, CED-1 and
PtdIns(4,5)P2 might cooperatively control the transient enrichment
of EPN-1 to the site of engulfment initiation (Fig. 9B).
The CED-1 signaling pathway orchestrates the
remodeling of both the plasma membrane and
cytoskeleton during apoptotic-cell engulfment
Previously, we have identified ‘focal exocytosis’ as a membrane
fusion event stimulated by CED-1 and mediated through DYN-1
for facilitating membrane expansion around apoptotic cells (Yu et
al., 2006). Our current study further reveals F-actin assembly as
another crucial pseudopod extension event regulated by CED-1. The
similar category of actin-remodeling defects resulted from
inactivating ced-1, ced-6, dyn-1, epn-1 or chc-1, together with the
essential roles of CED-1, CED-6 and DYN-1 in recruiting EPN-1
and CHC-1, indicate that the CED-1 pathway drives EPN-1 and
CHC-1 to regulate specific aspects of actin remodeling underneath
pseudopod membrane.
Rat CED6 was reported to interact with clathrin heavy chain
(Martins-Silva et al., 2006). Recently, Drosophila Ced-6 was found
to act as a clathrin adaptor that mediated yolk uptake in egg
chambers (Jha et al., 2012). During C. elegans apoptotic-cell
engulfment, however, ced-6(–); chc-1(–) and ced-6(–); epn-1(–)
double mutants display quantitatively similar levels of Ced
phenotype as ced-6(–) single mutants (Fig. 4A,B), suggesting that
CED-6 and EPN-1 are unlikely to act as two parallel clathrin
receptors. Rather, we find CED-6 acts upstream of EPN-1 in a linear
pathway (Fig. 4C). Inactivating ced-1, ced-6 or dyn-1 all resulted in
engulfment defects stronger than that of epn-1 or chc-1, supporting
the model in which actin remodeling is one of the two branches
regulated by the CED-1 pathway.
Previously, the two parallel C. elegans engulfment pathways were
proposed to converge at the point of CED-10, primarily based on
epistasis analysis results (Kinchen et al., 2005). However, the same
type of analysis, performed both previously (Mangahas and Zhou,
2005; Yu et al., 2006) and here, indicates that not only do CED-1,
CED-6, CED-7 and DYN-1 act in parallel to CED-10, CED-2, CED5 and CED-12, but CHC-1 and EPN-1 also belong to the CED-1, but
not the CED-10, pathway (Fig. 9A). Furthermore, the transient
enrichment of EPN-1 and CHC-1 to phagocytic cups is independent
of CED-5, CED-12 or CED-10. These results indicate that the CED1-EPN-1-clathrin pathway regulates actin remodeling in a Rac
GTPase-independent manner (Fig. 9B). Identifying the candidate
protein(s) that links the CED-1 pathway to actin remodeling will
further reveal this Rac-independent mechanism.
Acknowledgements
We thank A. Sokac for suggestions; X. Yu, S. Odera, C. Huber and X. Liu for
technical support; X. He for the Deltavision; and the C. elegans Genetic Center
(CGC) and National BioResource Project in Japan (Shohei Mitani) for strains.
DEVELOPMENT
Fig. 9. CHC-1 and EPN-1 promote actin polymerization in response to CED-1 signaling. (A) Two parallel pathways that regulate apoptotic-cell
engulfment. The mammalian homologs of C. elegans proteins are indicated in parentheses. (B) Model depicting how CHC-1 and EPN-1 regulate cytoskeleton
polymerization and promote pseudopod extension. In response to the ‘eat me’ signal, CED-1 initiates a signaling pathway that recruits EPN-1 to the plasma
membrane at the site of engulfment through PtdIns(4,5)P2 and perhaps direct interaction. EPN-1 further recruits CHC-1 to the same site. CHC-1 oligomerizes
into a scaffold upon which actin molecules assemble into polymers, driving pseudopod extension around the apoptotic cell.
Funding
Z.Z. is supported by the National Institutes of Health (NIH) [GM067848] and
the March of Dimes Foundation. B.C. acknowledges support from the NIH
[GM069950 and GM076651]. B.D.G. acknowledges support from the NIH
[GM067237]. Deposited in PMC for immediate release.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
Z.Z., Q.S. and B.H. designed the experiments. Q.S., B.H. and Z.Z. performed
the experiments. N.L., B.C. and B.D.G. generated critical reagents and
contributed to experimental design and manuscript preparation. Z.Z., Q.S. and
B.H. were responsible for manuscript preparation.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.093732/-/DC1
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Clathrin and its adaptor in engulfment