RESEARCH ARTICLE 1069
Development 135, 1069-1080 (2008) doi:10.1242/dev.016063
C. elegans Rab GTPase 2 is required for the degradation of
apoptotic cells
Qun Lu1,3,*, Yan Zhang2,3,*, Tianjing Hu3,*, Pengfei Guo3, Weida Li3 and Xiaochen Wang3,†
During apoptosis, the dying cell activates an intrinsic mechanism that quickly dismantles itself. The apoptotic cell corpses are then
recognized and removed by neighboring cells or professional phagocytes. How dying cells are degraded after internalization is
poorly understood. Here, we report the identification and characterization of unc-108, the Caenorhabditis elegans homolog of the
human Rab GTPase 2, as a novel component involved in the degradation of apoptotic cells. unc-108 is expressed and functions in
the engulfing cells and is likely to affect the degradation rather than the internalization of cell corpses. Similar to other Rab
GTPases, unc-108 also affects endocytosis, acting in the endosomal trafficking from early to late endosome and late endosome to
lysosome. UNC-108 co-localizes with RAB-5, RAB-7 and LMP-1 to the phagosome and promotes cell corpse degradation, possibly by
mediating phagosome maturation.
INTRODUCTION
Apoptotic cells generated by programmed cell death are recognized
and cleared by neighboring cells or professional phagocytes.
Efficient clearance of apoptotic cell is crucial for tissue homeostasis
and the regulation of immune responses. Defects in this process
contribute to persistent inflammatory diseases and autoimmune
disorders (Savill et al., 2002; Savill and Fadok, 2000). Phagocytosis
of apoptotic cell is regulated by mechanisms that are highly
conserved from the nematode C. elegans to humans (Fadeel, 2003).
For example, cells that undergo programmed cell death in C. elegans
are quickly removed by neighboring cells (Sulston and Horvitz,
1977). Two partially redundant pathways have been identified in
worm that regulate the cell corpse engulfment process, with ced-1,
ced-6, ced-7 and dyn-1 functioning in one pathway and psr-1, ced2, ced-5, ced-12 and ced-10 in the other (Reddien and Horvitz, 2004;
Wang et al., 2003; Yu et al., 2006).
CED-1 is a transmembrane receptor that mediates the recognition
of the dying cell by engulfing cells, but the ligand of CED-1 has not
been identified (Zhou et al., 2001b). CED-7, a homolog of the
mammalian ABC transporter, functions in both dying cells and
engulfing cells to promote cell corpse engulfment and is required for
the recognition of the dying cell by CED-1 (Wu and Horvitz, 1998;
Zhou et al., 2001b). CED-6 is an adaptor protein that contains a
phosphotyrosine-binding (PTB) domain and may interact directly
with CED-1 to transduce the engulfment signal (Liu and Hengartner,
1998; Su et al., 2002). Recently, C. elegans Dynamin 1 (DYN-1)
was found to function downstream of CED-1, CED-7 and CED-6 to
promote vesicle delivery to the phagocytic cup for the internalization
of the cell corpse (Yu et al., 2006).
In the other pathway, surface-exposed phosphatidylserine (PS),
an ‘eat me’ signal, is recognized by PSR-1, the C. elegans homolog
of the human phosphatidylserine receptor (Fadok et al., 2000; Wang
1
College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Graduate Program in Chinese Academy of Medical Sciences and Peking Union
Medical College, China. 3National Institute of Biological Sciences, No. 7 Science Park
Road, Zhongguancun Life Science Park, Beijing, 102206, China.
2
*These authors contributed equally to this work
†
Author for correspondence (e-mail: [email protected])
Accepted 18 January 2008
et al., 2003), which transduces the signal through a ternary signaling
complex consisting of CED-5/DOCK180 (DOCK1), CED12/ELMO and CED-2/CRKII to activate CED-10/RAC1, a small
GTPase. The activation of CED-10 then triggers the reorganization
of cytoskeleton needed for engulfment of apoptotic cells (Reddien
and Horvitz, 2004). A recent study suggests that CED-10 might also
act downstream of CED-1, CED-6 and CED-7 to promote cell
corpse engulfment (Kinchen et al., 2005). Despite the identification
of these genes involved in cell corpse engulfment, many crucial
components required for this process are still missing, including
genes involved in the degradation of cell corpses. In addition, how
phagosomes form and mature, and how internalized cell corpses are
degraded remain unclear.
Phagocytosis is a receptor-mediated, actin-dependent process
that results in internalization of foreign particles or apoptotic
cells. The internalized vesicle, the phagosome, matures through
interaction with organelles of the endocytic pathway to generate
the phagolysosome, which is capable of degrading particles or
apoptotic cells (Desjardins et al., 1994; Henry et al., 2004; Vieira
et al., 2002). In C. elegans, internalized cell corpses are enclosed
by the phagosome, which may undergo a similar maturation
process. Although phagosome composition and maturation have
been extensively studied in mammalian cells using latex-beadcontaining phagosomes (Garin et al., 2001; Stuart et al., 2007),
the formation and maturation of the phagosome that lead to
the degradation of apoptotic cells in vivo, remain poorly
understood.
In the present study, we have identified C. elegans UNC-108 as
a novel component involved in the degradation of apoptotic cells.
Both loss-of-function by RNA interference (RNAi) and a gain-offunction mutant of unc-108, sm237, resulted in accumulation of
cell corpses. Furthermore, we showed that cell corpses persisting
in the unc-108(sm237) mutant or unc-108(RNAi) animal are
internalized, but not degraded. UNC-108 co-localizes with the
endolysosomal markers RAB-5, RAB-7 and LMP-1 to the
phagosome in C. elegans embryos. We also present evidence that
unc-108 is required for endosomal trafficking, affecting the
transition from the early to the late endosome, the recycling
endosome and the maturation of lysosome. Our results suggest
that UNC-108 promotes cell corpse degradation, possibly by
DEVELOPMENT
KEY WORDS: C. elegans, Apoptotic cell, Degradation, unc-108, Rab GTPase 2, Endocytosis, Phagosome
1070 RESEARCH ARTICLE
MATERIALS AND METHODS
C. elegans strains
Strains of C. elegans were cultured at 20°C using standard
procedures (Brenner, 1974). The N2 Bristol strain was used as the wildtype strain, except for polymorphism mapping that used Hawaiian strain
CB4856.
Mutations used are described in C. elegans II (Riddle et al., 1997) unless
otherwise indicated. Linkage group I (LGI): dpy-5(e61), unc-29(e403), unc11(e47), ced-1(e1735), ced-12(n3261) (Zhou et al., 2001), n501, n777,
hT2(bli-4(e937)let-?(q782)qIs48)/sep-1(e2406) and ok1246 (Wormbase:
www.wormbase.org), sm237 (this study). LGIII: ced-6(n2095), ced7(n2094), ced-4(n1162). LGIV: ced-3(n717), psr-1(tm469) (Wang et al.,
2003), ced-2(n1994), ced-5(n1812), ced-10(n3246).
The following strains carrying integrated transgenes were kindly provided
by Dr Hanna Fares: bIs34 (RME-8::GFP) (Zhang et al., 2001); cdIs73
(RME-8::mRFP) (Treusch et al., 2004); cdIs40 (pcc1:GFP::CUP-5)
(Treusch et al., 2004); cdIs97 (pcc1:mCHERRY::CUP-5); cdIs39
[pcc1:RME-1(271a1)] (Poteryaev et al., 2007); bIs46 (GFP::RME-1; pRF4)
(Grant et al., 2001); cdIs141 (pcc1:mCHERRY::RAB-7); and cdIs113
(pcc1:mCHERRY::RAB-5).
Other endocytosis markers used were: arIs37 (Pmyo-3ssGFP) (Fares and
Greenwald, 2001a); pwIs50 (LMP-1::GFP) (Treusch et al., 2004); and
bIs1(VIT-2::GFP) (Grant and Hirsh, 1999).
45% of embryos (n=105) transgenic for Punc-108unc-108::gfp had bright GFP
fluorescence before injection, but none of the embryos from the same
transgenic line (n=119) showed any visible GFP fluorescence 24 hours after
injection.
Four-dimensional microscopy
Four-dimensional (4D) microscopy analysis of cell corpse duration was
performed as described (Wang et al., 2003) using a Zeiss Axioimager M1
coupled with an AxioCam monochrome digital camera. Images were
processed and viewed using Axiovision Rel. 4.5 software.
Time-lapse fluorescence microscopy
C. elegans embryos (300 minutes old) were mounted on slides with an agar
pad in egg salt (118 mM NaCl and 48 mM KCl) and a cover slip was placed
on top and sealed with beeswax and Vaseline (1:1). Images in a 24 ␮m zseries (1.2 ␮m/section) were captured every 3 or 3.5 minutes for 180 minutes
using a Zeiss LSM 5 Pascal inverted confocal microscope. Images were
processed and viewed using LSM Image Browser software.
Acridine Orange staining
Acridine Orange (AO; Sigma, USA) staining in embryos was performed as
described (Hersh et al., 2002) with a few modifications. Briefly, embryos
were collected from the gravid adults treated with 1.6 M NaOH/12%
hypochlorite until dissolved. Embryos were washed several times in M9
buffer and then incubated in 50 ␮g/ml AO in M9 buffer for 1 hour before
observation by epifluorescence microscopy. For AO staining in germ cells,
aged adults were soaked in 50 ␮g/ml AO in M9 buffer for 2 hours and
recovered on OP50-seeded plates for 3 hours before observation.
Mapping and cloning of unc-108
Endocytosis assay
sm237 was mapped very close to unc-11 on the left arm of linkage group I.
From unc-11 dpy-5/sm237 mothers, 60 of 77 Dpy non-Unc recombinants
contained sm237, whereas 0 of 44 Unc non-Dpy recombinants contained
sm237. We then performed single nucleotide polymorphism (SNP) mapping
to locate sm237 between SNP markers snp-Y47G6A (–3.21) and snp-R12E2
(–1.64). Transformation rescue experiments showed that one fosmid in this
region, WRM0636aD05, rescued the persistent cell corpse phenotype of the
sm237 mutant. Several deletion clones of WRM0636aD05 were generated
and one subclone that contains the MluI-SacII fragment of WRM0636aD05
possessed the rescue activity. Only one intact open reading frame, F54F10.4,
was found in this region, which corresponds to a previously identified gene,
unc-108. We determined the sequence of unc-108 in the sm237 mutant and
identified a missense mutation that caused the substitution of Gly18 with
Glu.
The number of somatic cell corpses in the head region of living embryos or
L1 larvae and the number of germ cell corpses in one gonad arm from
animals at various adult ages were scored using Nomarski optics as
described (Gumienny et al., 1999; Wang et al., 2002).
In vivo pulse-chase experiments were performed as described (Zhang et al.,
2001). Briefly, Texas Red-conjugated BSA (TR-BSA; Sigma, USA) was
injected at 1 mg/ml into the body cavity in the pharyngeal region. Injected
worms were transferred to a seeded NGM plate at room temperature and the
coelomocyte uptake was monitored at different time points (5, 10, 15, 20, 30
and 60 minutes; 6, 12 and 24 hours). At each time point, the injected worms
were transferred to an ice-cold NGM plate to stop the intracellular trafficking
of endocytosed molecules before examination by epifluorescence
microscopy.
The apical uptake of fluid-phase material in the intestine was analyzed by
soaking L4/young adults in 1 mg/ml TR-BSA in M9 buffer for 8 hours in the
dark at room temperature. Animals were recovered on a seeded NGM plate
for 2 hours before observation. For examining the apical uptake of lipophilic
dye in the intestine, L4/young adults were soaked in 40 ␮M FM4-64
(Invitrogen, USA) for 30 minutes in the dark at room temperature and
recovered on a seeded NGM plate for 30 minutes before observation. The
basolateral uptake in the intestine was analyzed by injecting 1 mg/ml TRBSA or 40 ␮M FM4-64 into the body cavity. The injected worms were
transferred to a seeded NGM plate and recovered at room temperature for
30 minutes before observation.
unc-108 RNAi
Plasmid construction
Sense and antisense RNA were in vitro transcribed from the T7- and SP6flanked PCR template (unc-108 cDNA nucleotides 5-599) using RiboMAX
Large Scale RNA Production System (Promega, USA). Double-stranded
RNA (dsRNA) was generated by annealing the sense and antisense RNA for
10 minutes at 65°C, followed by incubating at 37°C for 15 minutes. dsRNA
of unc-108 (550 ng/␮l) was injected into the gonad or the body cavity of
wild-type animals, which were then transferred to fresh OP50-seeded plates
every 12 hours. Embryos laid between 36 and 48 hours post-injection were
used for analyzing the somatic cell corpse phenotype. To determine the
engulfment phenotype in germ cells, wild-type animals were transferred to
the fresh plates 24 hours post-injection. The F1 progeny were aged and
scored at 12, 24, 36, 48 and 60 hours post L4/adult molt. For examining the
endocytosis phenotypes, wild-type animals carrying different endocytic
markers were injected with dsRNA of unc-108 and transferred to the fresh
plate 24 hours post-injection. The F1 progeny were examined at 24 to 48
hours post L4/adult molt for endocytosis defects. We found unc-108 RNAi
significantly diminished the expression of unc-108 in C. elegans embryos:
To construct Punc-108unc-108::gfp and Punc-108unc-108::mcherry, we inserted
a 4 kb fragment containing the genomic sequence of the unc-108 gene
including 2 kb promoter region into the pPD95.77 or pPD95.77-mcherry
vector (generated from pPD95.77 by replacing the gfp fragment with
mcherry) via its SphI-BamHI sites. To construct Pced-1unc-108 and Pegl-1unc108, the full-length unc-108 cDNA was amplified from a C. elegans cDNA
library (Invitrogen, USA) and cloned into the Pced-1 vector via its KpnI site
or into Pegl-1 through its NheI-NcoI sites. To generate Punc-108unc-108, we
first amplified a fragment containing the 2 kb DNA region upstream of the
start codon of the unc-108 gene and cloned it into a pPD49.26 vector via its
SphI-BamHI sites to create the construct Punc-108. The full-length cDNA of
unc-108 was then cloned to Punc-108 at the NheI-EcoRV sites to generate
Punc-108unc-108. To construct the endosomal and lysosomal markers driven
by the ced-1 promoter, the mcherry fragment was amplified from plasmid
pPD95.77-mcherry and cloned into the pPD49.26 vector through its NheIKpnI sites to yield pPD49.26-mcherry. The full-length genomic sequence of
the rab-5 and rab-7 genes were then amplified using N2 genomic DNA as
Quantification of cell corpses
DEVELOPMENT
mediating phagosome maturation, and is a novel component
crucial for the post-engulfment/cell corpse degradation process in
C. elegans.
Development 135 (6)
template and cloned into the pPD49.26-mcherry at the KpnI-EcoRV sites to
obtain the N-terminally tagged mCHERRY fusions. Finally, the 5 kb
promoter region of the ced-1 gene was ligated to the 5⬘ end of mCHERRY
at the BamHI site. To obtain Pced-1lmp-1mcherry, the ced-1 promoter was
cloned to the pPD95.77-mcherry at the BamHI site followed by the ligation
of full-length genomic sequence of the lmp-1 gene through the KpnI site. To
express unc-108 specifically in the coelomocyte, an 800 bp fragment
upstream of the start codon of the unc-122 gene was amplified (Fares and
Greenwald, 2001b) and cloned into the pPD49.26-gfp or pPD49.26-mcherry
vector through its SphI-BamHI sites, which was then ligated with the fulllength unc-108 genomic sequence at the KpnI-EcoRV sites to produce
Punc-122gfp::unc-108 and Punc-122mcherry::unc-108. The N-terminally
GFP/mCHERRY-tagged UNC-108 fusions driven by the unc-108 promoter
(Punc-108gfp::unc-108 and Punc-108mcherry::unc-108) were then generated
from these two constructs by replacing the promoter of unc-122 with that of
unc-108 through the SphI-BamHI sites. The full-length mouse Rab2 cDNA
was obtained by reverse transcription PCR from mouse liver and cloned into
the C. elegans heat-shock vectors pPD49.78 and pPD49.83 via their NheINcoI sites or into the Pced-1 vector at its KpnI site.
RESULTS
sm237 is a new allele of unc-108 that contains
many persistent cell corpses
The sm237 mutant was isolated from a psr-1 enhancer screen (X.W.
and D. Xue, unpublished), but its phenotype is not dependent on or
enhanced by the psr-1 deletion mutant tm469 (data not shown). The
sm237 animal contains many persistent cell corpses at late
embryonic stages that would normally have very few cell corpses
(Fig. 1A). The appearance of cell corpses in the sm237 mutant is
totally blocked by strong loss-of-function mutations in the ced-3 and
RESEARCH ARTICLE 1071
ced-4 genes that are required for almost all apoptosis in C. elegans,
indicating that the persistent cell corpses observed in sm237 are
indeed apoptotic cells (data not shown). We cloned the gene affected
by sm237 and found that it corresponds to a previously identified
gene, unc-108 (see Materials and methods; see Fig. S1A in the
supplementary material). unc-108 encodes a small GTPase that
shares high sequence homology with human RAB2 (RAB2A) (87%
sequence identity and 93% similarity; see Fig. S1B in the
supplementary material), a member of the Rab small GTPase family
(Pereira-Leal and Seabra, 2000). We determined the sequence of
unc-108 in the sm237 mutant and identified a missense mutation that
results in substitution of Gly18 with Glu (G18E), which affects a
conserved nucleotide-binding motif (PM1) present in all members
of the Ras GTPase superfamily (see Fig. S1B in the supplementary
material) (Pereira-Leal and Seabra, 2000). Expression of the fulllength unc-108 cDNA under the control of its own promoter
(Punc-108unc-108) efficiently rescued the persistent cell corpse
phenotype of the sm237 mutant (see Fig. S1A in the supplementary
material). Expression of mouse Rab2 cDNA driven by heat-shock
promoters (Phspmrab2) or the ced-1 promoter (Pced-1mrab2) also
rescued the corpse phenotype of the sm237 mutant (see Fig. S1A in
the supplementary material), indicating that mouse Rab2 can
substitute for UNC-108 in removing apoptotic cells in C. elegans.
The unc-108(sm237) mutant is defective in cell
corpse removal
To determine whether accumulation of cell corpses in sm237
animals is due to a defect in cell corpse clearance, we performed a
time-course analysis of cell corpse appearance during development
Fig. 1. unc-108 is important for cell
corpse clearance in C. elegans. (A) Timecourse analysis of cell corpse appearance
during development in the wild type (N2,
black), the unc-108(sm237) mutant (white)
and in wild-type animals treated with unc108 RNAi (gray). Cell corpses were scored
at the following embryonic or larval stages:
bean/comma (comma), 1.5-fold (1.5F), 2fold (2F), 2.5-fold (2.5F), 3-fold (3F), 4-fold
(4F) and early L1 larvae (L1). The y-axis
represents the mean number of cell corpses
scored at the head region of embryos or
larvae; at least 15 animals were scored at
each stage. Error bars indicate s.e.m.
(B) unc-108(sm237) mutant contains
persistent germ cell corpses. The number of
germ corpses were scored every 12 hours
after the L4/adult molt from one gonad
arm in wild-type (N2, black), unc108(sm237) mutant (white) and unc108(RNAi) animals (gray). The y-axis
represents the average number of germ cell
corpses. At least 15 animals were scored at
each time point. Error bars indicate s.e.m.
In A and B, data derived from different
genetic backgrounds at multiple
developmental stages were compared by two-way analysis of variance. Post-hoc comparisons were by Fisher’s PLSD (protected least squares
differences). *P<0.05, **P<0.0001. All other points had P values>0.05. (C) Four-dimensional microscopy analysis of cell corpse duration in the unc108(sm237) mutant. The duration of 33 cell corpses from wild-type (N2) embryos (n=3, black), unc-108(sm237) embryos (n=3, white) and unc108(RNAi) embryos (n=3, gray) were followed. The numbers in parentheses indicate the average durations of cell corpses (±s.e.m.). The y-axis
represents the number of cell corpses within a specific duration range as shown on the x-axis. The durations of four cell divisions in the MS cell
lineage from MS cell to MS.aaaa cell were also followed to ensure that the embryos scored had similar development rates. The average duration of
four cell divisions is 93±6 minutes in N2 embryos, 96±1 minutes in unc-108(sm237) embryos and 103±3 minutes in RNAi-treated embryos.
DEVELOPMENT
unc-108 promotes cell corpse engulfment
Development 135 (6)
(Wang et al., 2003). In both somatic and germ cells, significantly
higher numbers of cell corpses were observed in the sm237 mutant
than in wild-type animals at all developmental stages (Fig. 1A,B).
To confirm that the increase in cell corpses in sm237 is caused by a
defect in cell corpse removal, we performed 4D microscopy analysis
to measure the duration of embryonic cell corpses in sm237 animals
(Wang et al., 2003). In wild-type animals, the majority of the cell
corpses persisted from 10 to 50 minutes, whereas in the unc108(sm237) mutant most cell corpses persisted from 30 to 110
minutes (Fig. 1C). On average, the duration of cell corpses in unc108(sm237) embryos was 93% longer than in wild-type embryos
(Fig. 1C), indicating that the removal of apoptotic cells is defective
in the unc-108(sm237) mutant.
sm237 represents a gain-of-function allele of
unc-108
sm237 animals are viable but display a dominant Unc
(uncoordinated) phenotype, which is consistent with the previous
characterization of the unc-108 gene (Park and Horvitz, 1986).
Different from its dominant Unc phenotype, we found that the
persistent cell corpse phenotype of sm237 is semi-dominant and
shows a maternal effect: sm237 homozygous embryos produced by
sm237/+ heterozygous mothers showed a weak Ced (cell death
abnormal) phenotype equivalent to that of the mother (sm237/+),
which was weaker than that of the sm237/sm237 embryos produced
by the homozygous mothers (Table 1). The weak Ced phenotype
observed in sm237/+ embryos from the heterozygous mother could
be explained by the gain-of-function nature of sm237 or haploid
insufficiency of sm237/+. To distinguish between these two
possibilities, we examined an unc-108 deletion mutant (ok1246),
which contains a 2198 bp deletion that removes the whole gene
locus and represents a null allele of unc-108 (Wormbase:
www.wormbase.org; see Fig. S2A in the supplementary material).
Most homozygous ok1246 embryos from the heterozygous mother
(hT2/ok1246) appeared to develop normally during embryogenesis,
but failed to hatch or were arrested at early larval stage, indicating
that UNC-108 is essential for C. elegans development. However, no
obvious Ced phenotype was observed either in hT2/ok1246 animals
or their ok1246 progeny, suggesting that sm237 is likely to be a gainTable 1. sm237 represents a gain-of-function allele of unc-108
Maternal genotype
+/+
sm237/+
sm237/sm237
+/null
Zygotic genotype
No. of cell corpses at 4-fold
+/+
sm237/+
null/+
0.2±0.1
1.5±0.2
0.5±0.1
sm237/+
sm237/sm237
4.4±0.5
6.5±0.5
sm237/+
sm237/null
sm237/sm237 (cross)
sm237/sm237 (self)
5.3±0.4
9.1±0.7
14.6±0.7
14.5±1.4
+/null
null/null
0.4±0.2
0.4±0.1
Cell corpses were scored in 4-fold stage embryos and are shown as means±s.e.m. At
least 15 embryos were scored for each genotype. The complete maternal genotypes
are (from top to bottom): unc-76, hT2/sm237, sm237 and hT2/ok1246. The
complete zygotic genotypes are (from top to bottom): hT2/+;unc-76/+, sm237/+
smIs13 (Psur5:gfp)/+;unc-76/+, red non-green progeny of hT2/ok1246;qxEx58
(Psur5:rfp) males crossed with unc-76, green progeny of hT2/sm237, non-green
progeny of hT2/sm237, green progeny of hT2/+ males crossed with sm237, red nongreen progeny of hT2/ok1246;qxEx58 males crossed with sm237, green progeny of
sm237/+ smIs13/+ males crossed with sm237, progeny of sm237, green progeny of
hT2/ok1246 and non-green progeny of hT2/ok1246.
of-function allele (Table 1). Furthermore, the Ced phenotype in
sm237/ok1246 embryos was weaker than that of sm237/sm237
embryos, but stronger than that of sm237/+ embryos (Table 1),
indicating that sm237 indeed represents a gain-of-function allele of
unc-108 and that the wild-type unc-108 activity antagonizes the unc108(sm237) allele. This result is also consistent with the finding that
overexpression of wild-type unc-108 was able to rescue the
persistent cell corpse phenotype of sm237 animals and that wildtype gene product contributed maternally was able to partially rescue
the Ced phenotype of the homozygous sm237 progeny (see Fig. S1A
in the supplementary material; Table 1).
To confirm this result, we overexpressed the UNC-108(G18E)
mutant product in wild-type animals using C. elegans heat-shock
promoters [PhspUNC-108(G18E)] and found that UNC-108(G18E)
resulted in a similar, albeit slightly weaker, corpse phenotype to that
of the sm237 mutant (see Fig. S2C in the supplementary material).
Since ok1246 larvae do not survive, we could not examine their
progeny for the Ced phenotype and therefore cannot rule out the
possibility that the maternal contribution of the wild-type allele is
sufficient to mediate the normal clearance of cell corpses that we
observed in ok1246 embryos. To determine whether a loss-offunction mutation in the unc-108 gene affects cell corpse removal,
we treated wild-type animals with unc-108 RNAi and examined the
persistent cell corpse phenotype in the progeny. Indeed, we found
that unc-108 RNAi caused similar phenotypes to that of the sm237
mutant in both somatic and germ cells, indicating that the wild-type
unc-108 functions to promote cell corpse clearance (Fig. 1A-C).
Two other alleles of unc-108 (n501 and n777) isolated previously by
dominant Unc phenotype also displayed a weak Ced phenotype (see
Fig. S2B in the supplementary material) (Park and Horvitz, 1986).
UNC-108 is expressed and functions in the
engulfing cells to promote cell corpse removal
To examine the expression pattern of unc-108, we generated UNC108 translational GFP fusions under the control of its own promoter
[Punc-108unc-108::gfp (UNC-108::GFP) and Punc-108gfp::unc-108
(GFP::UNC-108)], which partially rescued the persistent cell corpse
phenotype of sm237 animals (see Fig. S1A in the supplementary
material). unc-108::gfp was ubiquitously expressed in the embryo,
starting from the very early stage of 50 to 100 cells and throughout
the larval and adult stages. The expression of unc-108::gfp was
observed in engulfing cells, such as hypodermal cells, intestine cells
and gonadal sheath cells (see Fig. S3A in the supplementary
material; data not shown). unc-108::gfp was also seen in many head
and tail neurons as well as ventral cord neurons (see Fig. S3A in the
supplementary material). Interestingly, unc-108 is also expressed in
the coelomocytes, the scavenger cells in C. elegans that constantly
uptake macromolecules from the body cavity (see Fig. S3A in the
supplementary material). This expression pattern is consistent with
the function of UNC-108 in endosomal trafficking (see below).
Similar expression patterns with more-vesicular localizations were
observed in animals expressing GFP::UNC-108 fusion protein (see
Fig. S3B in the supplementary material).
To determine whether UNC-108 activity is required in the
engulfing cells or dying cells for cell corpse removal, we expressed
unc-108 under the control of the ced-1 promoter (Pced-1) or egl-1
promoter (Pegl-1), which drives gene expression specifically in the
engulfing cells or dying cells, respectively (Conradt and Horvitz,
1998; Zhou et al., 2001b), and examined whether expression of unc108 in these cells rescued the persistent cell corpse phenotype of the
sm237 mutant. Expression of unc-108 in engulfing cells (Pced-1unc108), but not in dying cells (Pegl-1unc-108), rescued the cell corpse
DEVELOPMENT
1072 RESEARCH ARTICLE
clearance defect of sm237 animals, indicating that unc-108 needs to
function in the engulfing cells to promote cell corpse removal (see
Fig. S1A in the supplementary material).
Abnormal endosomal compartments in unc108(sm237) mutant coelomocytes
The involvement of human RAB2 in vesicular trafficking (Tisdale,
1999; Tisdale and Balch, 1996; Tisdale et al., 1992) and the
coelomocyte localization of UNC-108 promoted us to investigate
whether unc-108 affects endocytosis in C. elegans. In adult
hermaphrodites, there are six coelomocytes acting as scavenger cells
to take up macromolecules from pseudocoelom. We first examined
whether coelomocyte uptake is affected in the unc-108(sm237)
mutant using the Cup assay (coelomocyte uptake) (Fares and
Greenwald, 2001a). We introduced arIs37 [pmyo-3::ssGFP] into
the unc-108(sm237) mutant and examined the uptake of secreted
soluble GFP (ssGFP) by the coelomocytes. Compared with efficient
uptake of ssGFP by coelomocytes of wild-type animals, the initial
uptake of ssGFP by the coelomocytes of unc-108(sm237) animals
decreased and GFP accumulated in the body cavity (Fig. 2A).
Moreover, unlike the GFP pattern in wild-type coelomocytes,
internalized GFP was present in the enlarged vacuoles in unc108(sm237) animals (Fig. 2A). To further investigate the possible
cause of this defect, we examined whether the coelomocytes in unc-
RESEARCH ARTICLE 1073
108(sm237) animals contained normal endosomal and lysosomal
compartments using different markers fused with GFP. RME8::GFP marks endosome membrane, displaying many ring-like
structures representative of endosomes in wild-type coelomocyte
(Zhang et al., 2001). We observed three different patterns of RME8::GFP in the coelomocytes of sm237 animals. Twenty percent of
coelomocytes in unc-108(sm237) animals contained normal
endosomes that showed a similar RME-8::GFP pattern to that in
wild type. Forty percent of coelomocytes contained enlarged
vacuoles that were labeled by RME-8::GFP, half of which were so
large that they almost occupied the whole coelomocyte. Another
40% of coelomocytes showed a punctate pattern of RME-8::GFP
instead of the normal ring structure, and this did not correlate with
the age of hermaphrodites (Fig. 2B; data not shown). Moreover, we
saw very few endosomes of normal morphology in this type of
coelomocyte (Fig. 2B). These data suggest that sm237 animals
contain both damaged and enlarged endosomes.
To confirm the identity of the large vacuole, we introduced LMP1::GFP, an early lysosome marker into sm237 animals (Treusch et
al., 2004). To our surprise, these large vacuoles were also marked by
LMP-1::GFP, suggesting that they might represent aberrant hybrids
of endosome and lysosome (Fig. 2C). In addition, only a few normal
lysosomes with LMP-1::GFP were found in the coelomocytes of
sm237 mutant (Fig. 2C; Fig. 4D). Other endosomal and lysosomal
Fig. 2. The coelomocytes of sm237 mutant
C. elegans contain aberrant endosomes.
(Aa-f) The coelomocyte uptake is reduced in the
unc-108(sm237) mutant. The uptake of ssGFP in
the coelomocyte of a wild-type animal (a,b), unc108(sm237) mutant (c,d) and unc-108(RNAi)
animal (e,f) carrying Pmyo-3ssgfp were examined by
visualizing the GFP accumulation in the body cavity
and coelomocytes. White dashed line indicates the
outline of the coelomocyte and arrows point to the
body cavity with accumulated GFP. (Ba-Df) sm237
animals contain abnormal endosomes. The
coelomocytes of a wild-type animal (a,b), unc108(sm237) mutant (Bc-Bf, c,d in C,D), unc108(RNAi) animal (Bg-Bj, e,f in C,D) carrying
different endosome and lysosome markers were
examined. In wild-type coelomocyte, RME-8::GFP
(B) associates with early and late endosomes; LMP1::GFP (C) mostly stains lysosomes and GFP::RME-1
(D) marks recycling endosomes (arrows). Scale
bars: 2.5 ␮m.
DEVELOPMENT
unc-108 promotes cell corpse engulfment
markers, such as RAB-7, which associates with late endosome and
lysosome, and CUP-5, a lysosomal component, were also found to
be associated with the large vacuole (Poteryaev et al., 2007; Treusch
et al., 2004) (see Fig. S4A,B in the supplementary material). To
further confirm this result, we introduced RME-8::mRFP and LMP1::GFP or RME-8::GFP and mCHERRY::CUP-5 simultaneously
into the sm237 mutant and found that these markers co-localized
to the enlarged vacuoles, rather than localizing separately to
endosomes or lysosomes as in the wild-type coelomocytes (see Fig.
S4C,D in the supplementary material), indicating that the enlarged
vacuoles in the sm237 mutant represent hybrids of endosome and
lysosome.
Taken together, our data showed that sm237 animals contained
damaged endosomes and enlarged vacuoles with both endosomal
and lysosomal components, suggesting that unc-108 is involved in
both an early step of endosomal trafficking and in lysosome
formation from late endosome. To further investigate whether
sm237 affects an early step of endosomal transport, we examined the
localization of GFP::RME-1, an EH-domain-containing ATPase
associated with recycling endosomes (Lin et al., 2001). In wild-type
coelomocytes, RME-1 was mostly found in close proximity to the
plasma membrane (Fig. 2D). By contrast, this pattern was disrupted
in the coelomocytes of sm237 animals as GFP::RME-1 was often
found around the endosomes (Fig. 2D, arrow). We also checked the
pattern of early endosome-associated RAB-5 (Pfeffer and Aivazian,
2004; Poteryaev et al., 2007). Many coelomocytes of sm237 animals
showed a normal GFP::RAB-5 pattern, except for those that
contained no other compartments but one large vacuole that was
labeled by GFP::RAB-5 (data not shown). Therefore, we conclude
that unc-108 functions in both early and late steps of endosomal
trafficking, affecting the transition from early to late endosome, the
recycling endosomes and the late endosome to lysosome transition.
Consistent with this result, using early endosome marker
mCHERRY::RAB-5 and lysosome-associated mCHERRY::CUP-5,
we found that GFP::UNC-108 localized to both endosomes and
lysosomes (Fig. 3).
Lysosome maturation is affected in the unc108(sm237) mutant
In order to examine the endosomal trafficking defect of sm237
animals with higher temporal resolution, we performed in vivo
pulse-chase analysis of endocytosis by injecting TR-BSA (Texas
Red-conjugated BSA) into the body cavity of adult hermaphrodites
and examined the uptake of TR-BSA into the coelomocytes in both
wild-type and sm237 animals carrying different endosomal/
lysosomal markers. In wild-type animals, 5 minutes after injection,
Development 135 (6)
TR-BSA started to appear in the endosomes labeled by RME8::GFP. After 15 minutes, a significant amount of TR-BSA left the
RME-8::GFP ring, and after 30 minutes most of the TR-BSA was
present in the lysosomes lacking RME-8::GFP (Fig. 4A). In the
sm237 mutant, however, TR-BSA appeared in the RME-8::GFPlabeled compartment 5 minutes after injection and stayed there
throughout the time-course of the experiment (Fig. 4B; see Materials
and methods; data not shown). We also monitored the uptake of TRBSA using the early lysosomal marker LMP-1::GFP, and found that
TR-BSA started to accumulate in the compartments lacking LMP1::GFP 5 minutes after injection. After 15 minutes, TR-BSA
appeared in the lysosomes marked by LMP-1::GFP (Fig. 4C). By
contrast, 5 minutes after injection, TR-BSA accumulated in the
vacuole marked by LMP-1::GFP in the sm237 mutant (Fig. 4D).
During the remainder of the time points, most TR-BSA stayed
within the vacuole or enlarged endosomes that were labeled by
LMP-1::GFP and failed to move out even at 24 hours post-injection
(Fig. 4D; data not shown). Therefore, our pulse-chase experiments
showed that lysosome biogenesis was severely affected in the sm237
mutant, suggesting that UNC-108 is required for the formation of
lysosome from late endosome.
Yolk protein trafficking and apical uptake in the
intestine are blocked in unc-108(sm237) animals
In C. elegans, yolk uptake by growing oocytes presents a typical
example of receptor-mediated endocytosis (Grant and Hirsh, 1999).
Using a VIT-2::GFP reporter (Grant and Hirsh, 1999), we examined
whether sm237 affects yolk uptake by oocytes. We did not observe
any defect of initial uptake of yolk protein in unc-108(sm237)
oocytes (Fig. 5A). Consistently, the localization of GFP::RME-1
was also normal in the oocytes of sm237 animals (data not shown).
However, the redistribution of yolk protein to gut primordium in the
embryo or to the intestine in larva was blocked in the mutant (Fig.
5B,C; data not shown). These results indicate that UNC-108 is not
required for the initial uptake step of receptor-mediated endocytosis
in developing oocytes, but is involved in the resecretion and
trafficking of the yolk protein. A similar yolk redistribution defect
has been observed previously in rab-7(RNAi) animals and in the
sand-1 mutant, which might suggest that the yolk needs to reach the
late endosomal compartment for its later resecretion (Grant and
Hirsh, 1999; Poteryaev et al., 2007). Therefore, the yolk
redistribution defect that we observed in sm237 animals could be
due to the disruption of UNC-108 function in the late step of
endosomal trafficking. To test whether UNC-108 is required for
endocytosis in the intestine, animals were fed with TR-BSA (fluidphase material) or with the lipophilic dye FM4-64, and the apical
Fig. 3. UNC-108 localizes to both endosomes and
lysosomes. GFP::UNC-108 was specifically expressed in the
coelomocyte driven by unc-122 promoter in cdIs113, which
carries integrated pcc1:mCHERRY::RAB-5 (A), or in cdIs97
that contains integrated pcc1:mCHERRY::CUP-5 (B).
GFP::UNC-108 was observed on endosomes where it
overlapped with mCHERRY::RAB-5 (A, arrows) and on
lysosomes marked by mCHERRY::CUP-5 (B, arrows). Scale
bars: 2.5 ␮m.
DEVELOPMENT
1074 RESEARCH ARTICLE
(luminal) uptake of the dyes was assayed. Both TR-BSA and FM464 were quickly taken up from the lumen by the intestinal cells in
wild-type animals (see Fig. S5 in the supplementary material).
However, in sm237 animals, most of the TR-BSA or FM4-64
RESEARCH ARTICLE 1075
accumulated in the intestinal lumen, indicating that the apical uptake
was mostly blocked (see Fig. S5 in the supplementary material). We
did not observe any obvious defect in sm237 animals when both
markers were delivered basolaterally (data not shown).
Fig. 4. Endocytic trafficking in the coelomocytes of the unc-108(sm237) mutant is blocked from late endosome to lysosome. (A-D) TRBSA was injected into the body cavity and its transport through endocytic compartments is shown over time in wild-type (A,C) and unc-108(sm237)
mutant (B,D) animals with endosomal marker RME-8::GFP (A,B) or lysosomal marker LMP-1::GFP (C,D). White arrows point to the compartments
that contain TR-BSA. The blue arrow in D indicates the normally sized lysosome that lacks TR-BSA. Scale bars: 2.5 ␮m.
DEVELOPMENT
unc-108 promotes cell corpse engulfment
1076 RESEARCH ARTICLE
Development 135 (6)
Fig. 5. Yolk protein trafficking is
blocked in the unc-108(sm237)
mutant. (Aa-d) Yolk protein
uptake is normal in the developing
oocytes of unc-108(sm237) mutant
C. elegans. The accumulation of
VIT-2::GFP in the developing
ooctyes was examined in wild-type
(bIs1) (a,b) and in the sm237
mutant [unc-108(sm237); bIs1]
(c,d). (Ba-d) The yolk protein is
redistributed to the intestine
primordium (arrow) in the wildtype 1.5-fold (a,b) and 4-fold (c,d)
stage embryos. (Ca-Dd) Yolk
protein trafficking is blocked in the
unc-108(sm237) mutant (C) or
unc-108(RNAi) animal (D). 1.5(a,b) and 4-fold (c,d) stage embryos
were examined for the
redistribution of VIT-2::GFP from
the anterior region to the gut
primordium. Abundant VIT-2::GFP
signal could be observed in the
anterior region of the embryos
(arrow). Scale bars: 1 ␮m in A;
5 ␮m in B-D.
108 RNAi-treated animals showed similar apical uptake defects in
the intestine to the sm237 mutants (data not shown). Taken together,
our data showed that loss of unc-108 function caused various
endocytosis defects that were similar to those of the gain-offunction allele, sm237, demonstrating that the wild-type unc-108
activity is required for endocytosis and is likely to act in both early
and late steps of endosomal trafficking.
unc-108 affects the degradation of cell corpses
Our data indicate that unc-108 plays an important role in
endocytosis. We next examined the role of UNC-108 in cell corpse
clearance. We first examined whether cell corpses accumulating in
the unc-108(sm237) mutant or in animals treated with unc-108
RNAi were internalized, using Acridine Orange (AO), which
preferentially stains engulfed apoptotic cells (Gumienny et al., 1999;
Lettre et al., 2004). Similar to that in wild-type animals, both
persistent somatic cell corpses and germ cell corpses in the sm237
mutant or animals treated with unc-108 RNAi could be labeled by
AO (Fig. 6A-C; data not shown). By contrast, the persistent cell
corpses in the ced-1(e1735) or ced-12(n3261) mutant failed to be
internalized and were not stained (Gumienny et al., 1999; Lettre et
al., 2004; Zhou et al., 2001a; Zhou et al., 2001b) (Fig. 6D; data not
shown). These data suggest that the persistent cell corpses in sm237
mutant or unc-108(RNAi) animals were internalized but not
degraded. Thus, unc-108 is likely to affect the degradation rather
than the internalization of cell corpses.
unc-108 functions downstream of the engulfment
pathway to promote cell corpse degradation
The cell corpse degradation process is compromised in the sm237
mutant and in unc-108(RNAi) animals. Several genes have been
described previously that act in two partially redundant pathways
to regulate cell corpse engulfment in C. elegans (Reddien and
Horvitz, 2004; Wang et al., 2003; Yu et al., 2006). We analyzed
double mutants between sm237 and strong loss-of-function
DEVELOPMENT
Loss-of-function of unc-108 causes similar
endocytosis defects to those of sm237
The data shown above indicate that sm237 affects the transition
from early to late endosome, recycling endosomes as well as
lysosome biogenesis in coelomocytes, and yolk protein trafficking
and apical uptake in the intestine. Since sm237 represents a gainof-function allele of unc-108, we determined whether loss-offunction of unc-108 caused by RNAi affects endocytosis. We found
that treatment with unc-108 RNAi caused similar endocytosis
defects to those of the sm237 mutant. First, in 83% of animals
treated with unc-108 RNAi, the uptake of ssGFP was affected,
among which 75% failed to uptake any ssGFP, a more severe
phenotype than that of the sm237 mutant (Fig. 2A). Second, the
abnormal endosomal compartments were observed in 76% of
coelomocytes after RNAi treatment, including enlarged vacuoles
containing both endosome and lysosome components as revealed
by labeling with endolysosomal markers RME-8, LMP-1 and CUP5 (60%), and damaged endosomes as indicated by the punctate
pattern of RME-8::GFP (16%) (Fig. 2B,C; see Fig. S4C,D in the
supplementary material). These distorted endosomal compartments
were also found in the coelomocytes of sm237 mutants, but at
slightly different frequency (40% each). Third, we found similar
mislocalization of RME-1::GFP around endosomes in animals
treated with unc-108 RNAi (Fig. 2D). Fourth, the endosomal
transport was also carefully examined in pulse-chase experiments
after RNAi treatment. We found that most TR-BSA was trapped
within the endosomes for up to 12 hours after injection, whereas in
the wild-type coelomocytes it was transported to lysosome within
15 to 30 minutes post-injection (see Fig. S6 in the supplementary
material; Fig. 4C; data not shown). However, this blockage was not
as complete as that in the sm237 mutant in which TR-BSA stayed
inside the endosomes even at 24 hours post-injection. Fifth, the
resecretion and trafficking of yolk protein was totally blocked in the
embryos or larvae after RNAi treatment, whereas the uptake of yolk
protein was not affected (Fig. 5D; data not shown). Finally, the unc-
Fig. 6. The persistent cell corpses in the unc-108(sm237) mutant
are labeled by Acridine Orange. AO staining of a 1.5-fold stage
embryo of wild type (A,B), and of a 4-fold stage embryo of unc108(sm237) (C,D), unc-108(RNAi) (E,F) or ced-1(e1735) (G,H) that
contain apoptotic cells (A,B) or persistent cell corpses (C-H). Bright AO
staining was observed in the dying cell of the wild-type embryo and the
persistent cell corpses in the unc-108(sm237) mutant and unc108(RNAi) embryos, but not in the ced-1(e1735) mutant (arrows). Scale
bars: 5 ␮m.
mutations in several other genes acting in the two cell-corpse
engulfment pathways (ced-1, ced-6, ced-7 in one pathway, and ced2, ced-5, ced-10 and ced-12 in the other) and found that sm237 does
not significantly affect or enhance the engulfment defect of mutants
in either pathway (data not shown). Similar results were obtained
with unc-108 RNAi treatment (data not shown), suggesting that
unc-108 does not act in a specific pathway and might function
downstream of both engulfment pathways to promote cell corpse
degradation.
UNC-108 co-localizes to phagosomes with RAB-5,
RAB-7 and LMP-1
UNC-108 is expressed and required in engulfing cells to promote
cell corpse removal. To further investigate its function in cell corpse
degradation, we examined whether UNC-108 associates with
phagosomes that contain internalized cell corpses. CED-1 is a
phagocytic receptor and CED-1::GFP localizes to the extending
pseudopods and nascent phagosomes (Yu et al., 2006; Zhou et al.,
2001b). In unc-108(sm237) mutant or unc-108(RNAi) animals, the
clustering of CED-1::GFP around the cell corpse was not affected
(data not shown), which is consistent with our finding that UNC-108
is not required for the internalization of cell corpses. To find out
whether UNC-108 associates with phagosomes, we first checked if
it clusters around cell corpses and co-localizes with CED-1::GFP to
RESEARCH ARTICLE 1077
the extending pseudopods or nascent phagosomes. In wild-type
embryos carrying Punc-108unc-108::gfp, strong GFP signal was seen
surrounding the cell corpses (Fig. 7A), indicating that UNC-108
might associate with phagosomes. Similar phagosome localization
was observed in embryos expressing N-terminally GFP-tagged
UNC-108 (GFP::UNC-108) (see Fig. S7A in the supplementary
material).
We next examined embryos expressing both Punc-108unc108::mcherry and Pced-1ced-1::gfp and found that both UNC108::mCHERRY and CED-1::GFP clustered around cell corpses,
but we could barely detect any co-localization of these two proteins
around dying cells. As a phagocytic receptor, the localization of
CED-1 on phagosomes is transient and it disappears long before the
complete degradation of cell corpses (Yu et al., 2006). Since UNC108 is likely to be involved in the degradation of cell corpses, one
possible explanation is that UNC-108 is recruited to phagosomes
after CED-1 completes its task and disappears. To test this
hypothesis, we followed the recruitment of CED-1 and UNC-108 to
phagosomes in embryos expressing both Punc-108unc-108::mcherry
and Pced-1ced-1::gfp by time-lapse recording. Consistent with our
hypothesis, we found that CED-1::GFP and UNC-108::mCHERRY
were recruited to the phagosomes at different times during the
engulfment process. We set the time point as 0 min when a clear
CED-1::GFP ring was seen. At +5 minutes, CED-1::GFP formed a
bright ring around the cell corpse, whereas UNC-108::mCHERRY
was not seen (Fig. 7Ba-c). At +8 minutes, CED-1::GFP became
weaker and the UNC-108::mCHERRY signal started to appear (Fig.
7Bd-f). At +11 minutes, almost no CED-1::GFP could be detected
whereas the UNC-108::mCHERRY formed a clear circle around the
cell corpse (Fig. 7Bg-i). At +14 minutes, strong UNC108::mCHERRY signal was seen, while CED-1::GFP completely
disappeared from the phagosome (Fig. 7Bj-l). The UNC108::mCHERRY signal could still be detected at +26 minutes when
the ‘button-like’ morphology of the cell corpse was lost (Fig. 7Bmo). UNC-108::mCHERRY eventually disappeared at +29 minutes
(data not shown). These data indicate that UNC-108 is recruited to
the same engulfment site as CED-1 and its association with the
phagosome is preceded by that of CED-1 and lasts until the
degradation of cell corpses. Similar phagosome recruitment kinetics
were observed with the N-terminally tagged UNC-108
(mCHERRY::UNC-108) (see Fig. S7B in the supplementary
material).
To investigate the potential function of UNC-108 in phagosome
maturation, we examined whether UNC-108 co-localizes with
several other phagosome-associated proteins that function at
different phagosome maturation stages in mammals. Rab5 is an
early endosome marker and has been shown to be associated with
the phagosome and to play an important role in phagosome
maturation in mammals and fruit flies (Desjardins et al., 1994;
Henry et al., 2004; Stuart et al., 2007; Vieira et al., 2002). Rab7, a
late endosome component, is recruited to the phagosome by Rab5
and mediates the fusion of phagosome with lysosome (Henry et al.,
2004; Vieira et al., 2003). Lysosomal protein LAMP1 (vertebrate
ortholog of C. elegans LMP-1) was also found to be associated with
the phagosome and functions in mediating phagosome maturation
(Garin et al., 2001). In wild-type embryos transgenic for
Pced-1mcherry::rab-5 and Punc-108unc-108::gfp, we found that
mCHERRY::RAB-5 and UNC-108::GFP co-localized to the
phagosome, forming a ring-like structure around the cell corpse (Fig.
7Ca-d). Similar phagosome co-localization was observed in
embryos expressing Pced-1mcherry::rab-7 and Punc-108unc-108::gfp
or Pced-1lmp-1::mcherry and Punc-108unc-108::gfp, as well as in
DEVELOPMENT
unc-108 promotes cell corpse engulfment
animals expressing N-terminally GFP-tagged UNC-108
(GFP::UNC-108) (Fig. 7Ce-h,i-l; see Fig. S7C in the supplementary
material). Since Rab5 is recruited to the phagosome at a very early
stage and LAMP1 is likely to be involved in the late step of
generating the phagolysosome in mammals (Vieira et al., 2002), the
co-localization of UNC-108 with both of these markers on the
phagosome suggests that UNC-108 might function in both early and
late stages of phagosome maturation.
Development 135 (6)
DISCUSSION
UNC-108 may promote phagosome maturation
required for cell corpse degradation
Phagosome maturation is a dynamic process that involves a series
of interactions among endocytic compartments, which eventually
fuse with lysosomes to generate phagolysosomes that possess
degradative properties (Henry et al., 2004; Vieira et al., 2002; Vieira
et al., 2003). In many ways, this maturation process resembles the
Fig. 7. UNC-108 associates with
phagosomes. (Aa,b) UNC-108::GFP
clusters around cell corpses (arrow).
DIC (a) and fluorescent confocal (b)
images of a wild-type C. elegans
embryo transgenic for Punc-108unc108::gfp. (Ba-o) UNC108::mCHERRY is recruited to the
phagosome preceded by CED-1::GFP.
DIC (a,d,g,j,m), confocal time-lapse
images of CED-1::GFP (b,e,h,k,n) and
UNC-108::mCHERRY (c,f,i,l,o)
around the same cell corpse in a
wild-type embryo. The time point
was set as 0 minute when the CED1::GFP ring was clearly seen. Images
from five time points after that are
shown. Arrows point to the cell
corpse and to the corresponding
fluorescent signals. (Ca-l) UNC108::GFP co-localizes with
mCHERRY::RAB-5, mCHERRY::RAB-7
and LMP-1::mCHERRY to the
phagosome. DIC and fluorescent
confocal images of a wild-type
embryo transgenic for Punc-108unc108::gfp and Pced-1mcherry::rab-5
(a-d) or Punc-108unc-108::gfp and
Pced-1mcherry::rab-7 (e-h) or
Punc-108unc-108::gfp and Pced-1lmp1::mcherry (i-l). Arrows indicate the
co-localization of UNC-108::GFP
with mCHERRY::RAB-5,
mCHERRY::RAB-7 or LMP1::mCHERRY on the phagosome.
Scale bars: 5 ␮m.
DEVELOPMENT
1078 RESEARCH ARTICLE
progression of endocytic compartments, which undergo a series of
fissions and fusions to modify membrane composition and acquire
new contents (Vieira et al., 2002). Our data indicate that unc-108 is
required for both endocytosis and cell corpse degradation in C.
elegans, suggesting that there might be an intrinsic connection
between these two processes. UNC-108 affects the degradation of
cell corpses, associates with the phagosomes containing internalized
cell corpses, and co-localizes with early endosome protein RAB-5,
late endosomal component RAB-7, and lysosomal protein LMP-1.
Since UNC-108 localizes to both endosomes and lysosomes and
functions in both early and late steps of endosomal trafficking, it is
possible that UNC-108 is recruited to the phagosome during its
fusion with early endosome and regulates phagosome maturation.
The gain-of-function allele, sm237, has a missense mutation that
changes Gly18 to Glu (G18E) within the PM1 motif (GxxxxGKs,
mutation underlined) that is required for the binding of phosphate
and Mg2+ and is conserved in all Ras small GTPase superfamily
members (Valencia et al., 1991). Structural and biochemical studies
indicate that mutations in this motif may affect the catalytic activity
of GTPase (Pai et al., 1989; Reinstein et al., 1990). Therefore, G18E
mutant protein might possess less GTPase activity and stay in the
active GTP-bound form that binds to the effector protein. The
persistent interaction of UNC-108(G18E) with downstream
effectors might block phagosome maturation at a certain
intermediate stage and affect the degradation of cell corpses.
Overexpression of wild-type UNC-108 might increase the chance
of interaction between wild-type UNC-108 and its effectors, which
would promote normal degradation of apoptotic cells. This
competition between wild-type UNC-108 and G18E mutant in
binding to effector proteins might explain the variable rescuing
activities we observed with different unc-108 transgenes, which are
likely to carry different copy numbers of wild-type unc-108. In line
with this competition model, we found that overexpression of the
UNC-108(G18E) mutant in wild-type embryos indeed resulted in a
similar persistent cell corpse phenotype to that of the sm237 mutant.
Further experiments need to be undertaken to understand the
biochemical features of the UNC-108(G18E) protein and to test the
above competition hypothesis.
UNC-108 regulates endosomal trafficking at
different steps in C. elegans
Human RAB2 has been implicated in Golgi-ER retrograde transport
(Stenmark and Olkkonen, 2001), but the mechanism by which
RAB2 controls this transport is unknown. In addition, it is not clear
whether RAB2 is involved in other aspects of endocytosis or vesicle
trafficking. In the present study, we identified a gain-of-function
allele of unc-108, sm237, that affects the uptake of ssGFP by
coelomocytes, transition from early to late endosomes, recycling
endosomes, lysosome formation, yolk protein trafficking and the
apical uptake in the intestine. Importantly, inhibition of unc-108
expression by RNAi caused similar endocytosis defects to those of
the sm237 mutant, indicating that unc-108 is indeed required for this
process. For example, the majority of animals treated with unc-108
RNAi failed to uptake ssGFP, a more severe phenotype than that in
the sm237 mutant, suggesting that unc-108 is required for the
internalization of fluid-phase material in coelomocytes, which might
be partially affected by the UNC-108(G18E) protein. In addition,
various endocytosis defects were observed in animals treated with
unc-108 RNAi which were similar to those in the sm237 mutant,
such as distorted endosomal compartments, mislocalized recycling
endosomes and defects in TR-BSA trafficking, yolk redistribution
and apical uptake in the intestine. These data demonstrate that the
RESEARCH ARTICLE 1079
wild-type unc-108 activity is required for endocytosis and it is likely
to regulate endosomal trafficking at different steps, including the
progression from early to late endosome, cargo recycling and
lysosome maturation. Identification of the downstream effector(s)
or the regulatory proteins that act together with UNC-108 is needed
to understand its exact function at these different steps of
endocytosis.
Rab GTPases function as important regulators in
removing apoptotic cells
Rab proteins are small GTPases that constitute the largest branch
of the Ras GTPase superfamily. Rabs have been implicated in
almost all types of membrane trafficking and have emerged as
central regulators of vesicle budding, docking and fusion with
specific target organelles (Mukherjee et al., 1997; Vieira et al.,
2002). Several Rabs have been found to associate with phagosomes
containing latex beads in mice and fruit flies including Rab1, Rab2,
Rab3, Rab4, Rab5, Rab7, Rab11 and Rab14 (Garin et al., 2001;
Stuart et al., 2007). However, the phagocytosis of foreign particles
and of apoptotic cells involve different phagocytic receptors and
elicit different immune responses. Therefore, the involvement of
Rabs in regulating apoptotic cell clearance was not firmly
established. Our identification and characterization of UNC-108 in
mediating cell corpse degradation and the finding that mouse Rab2
can substitute for its function in removing apoptotic cells indicate
that Rab proteins are potential regulators of apoptotic cell clearance
in vivo and that this function is likely to be conserved in mammals
as well. In addition to UNC-108/Rab2, C. elegans RAB-5 and
RAB-7 also localize to the phagosome and an increased number of
cell corpses was observed in rab-5(RNAi) or rab-7(RNAi) animals,
suggesting that these two Rab GTPases might also be involved in
the clearance of apoptotic cells (Fig. 7C; data not shown).
Consistent with our findings, recent studies showed that
overexpression of Rab5 in NIH3T3 fibroblast cells or bone
marrow-derived macrophages promoted the uptake of apoptotic
thymocytes, whereas the dominant-negative constructs inhibited it
(Nakaya et al., 2006).
We thank Drs Hanna Fares for providing endocytosis markers and A. Fire for
vectors; Dr Ding Xue for his critical reading of the manuscript and Drs
Chonglin Yang, Hong Zhang and members in our laboratory for helpful
discussion and suggestions. The sm237 mutant was isolated in Dr Ding Xue’s
laboratory. Some strains used in this work were obtained from the
Caenorhabditis Genetic Center (CGC), which is supported by a grant from the
NIH. This work was supported by the National High Technology Project 863
from the Ministry of Science and Technology.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1069/DC1
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