-
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
C. elegans Secreted Lipid-Bi
Current Biology 22, 1–9, July 24, 2012 ª2012 Elsevier Ltd All
rights reserved DOI 10.1016/j.cub.2012.06.004
Articlending
Protein NRF-5 Mediates PS Appearanceon Phagocytes for Cell
Corpse Engulfment
Yan Zhang,1,3 Haibin Wang,2,3 Eriko Kage-Nakadai,4
Shohei Mitani,4 and Xiaochen Wang3,*1Graduate Program in Chinese
Academy of Medical Sciencesand Peking Union Medical College,
Beijing 100730, China2College of Biological Sciences, China
Agriculture University,Beijing 100094, China3National Institute of
Biological Sciences, No. 7 Science ParkRoad, Zhongguancun Life
Science Park, Beijing 102206, China4Department of Physiology, Tokyo
Women’s MedicalUniversity, School of Medicine and Institute for
IntegratedMedical Sciences, 8-1, Kawada-cho, Shinjuku-ku,
Tokyo162-8666, Japan
Summary
Background: During programmed cell death, apoptotic cellsare
rapidly removed by phagocytes. How dying cells arerecognized
remains poorly understood.Results: Here we identify a secreted
lipid transfer/LPS-binding family protein, NRF-5, which is required
for efficientclearance of cell corpses. We observed that
phosphatidylser-ine (PS), which is externalized to the outer
leaflet of plasmamembranes in apoptotic cells, is also detected on
the surfaceof engulfing cells. Loss of NRF-5 function completely
blocksPS appearance on engulfing cells but causes accumulationof PS
on apoptotic cells, a phenotype observed in bothced-7(lf) and
ttr-52(lf)mutants. TheNRF-5 protein is expressedin and secreted
from body wall muscle cells and clustersaround apoptotic cells in a
CED-7-dependent manner. NRF-5associates with TTR-52, binds PS, and
displays lipid transferactivity in vitro.Conclusion:Our data
suggest that NRF-5 may act with CED-7and TTR-52 to mediate PS
transfer from apoptotic cells to en-gulfing cells and thus promotes
engulfment by phagocytes.
Introduction
Apoptotic cells generated by programmed cell death arerapidly
removed to prevent inflammatory and autoimmuneresponses and
maintain tissue homeostasis [1, 2]. Phosphati-dylserine (PS), which
is normally restricted to the inner leafletof plasma membranes, is
exposed on the surface of apoptoticcells, thus distinguishing them
from normal living cells andserving as an ‘‘eat me’’ signal for
engulfment [3, 4]. Surfaceexpression of PS also occurs in
nonapoptotic macrophagesand differentiating monocytes and appears
to be related tothe acquisition of the ability to engulf
PS-positive target cells[5–7]. However, it is unknown whether this
is characteristic ofprofessional phagocytes like macrophages or a
general andconserved feature of all phagocytes. It is also unclear
howPS expression on phagocytes is regulated and contributes
toengulfment.
PS on apoptotic cell surfaces can be recognized directly
byphagocyte receptors or indirectly through extracellular
*Correspondence: [email protected]
bridging molecules that cross-link apoptotic cells to
phago-cytes [8]. Although the latter mechanism was only found tobe
important for apoptotic cells cleared by remote phagocytes[9, 10],
we recently identifiedC. elegans TTR-52 as an extracel-lular
bridging molecule that links PS on apoptotic cells withreceptors on
neighboring phagocytes [11].TTR-52 functions in the ced-1/6/7
pathway, which is one of
two partially redundant signaling pathways that control
cellcorpse engulfment in C. elegans (Figure 1E). In the
parallelpathway, multiple receptors recognize apoptotic cells
andtransduce the signal to the conserved intracellular
moleculesCED-2(CRKII), CED-5(DOCK180), and CED-12(ELMO),
whichactivate the small guanosine triphosphatase (GTPase)
CED-10(RAC1), leading to the cytoskeleton rearrangement requiredfor
engulfment [12–15]. In the ced-1/6/7 pathway, TTR-52,
anextracellular protein secreted from nonapoptotic intestinecells,
mediates in part the recognition of apoptotic cells bybinding to
both surface-exposed PS on cell corpses and theextracellular domain
of the phagocytic receptor CED-1[11, 16]. ced-7, which encodes an
ATP-binding cassette(ABC) transporter, is also involved in cell
corpse recognitionand functions in both apoptotic and engulfing
cells [16, 17].It has been proposed that CED-7 is required for
exposure ofPS in apoptotic cells, whereas ABCA1, the mammalian
coun-terpart of CED-7, is thought to play a role in exposing PS
inapoptotic cells and macrophages [7, 18, 19]. However,
recentstudies in cells from ABCA12/2 mice and individuals
withTangier disease or in ABCA1-expressing HeLa cells suggestthat
ABCA1 is dispensable for the transbilayer movement ofPS in
macrophages and apoptotic cells [20]. Therefore, therole of the ABC
transporter in apoptotic cell recognitionremains elusive. It also
remains to be determined whetheradditional extracellular proteins
are involved in cell corpseengulfment.Here we identify a secreted
lipid transfer/LPS (lipopolysac-
charide)-binding family protein, NRF-5, which acts withCED-7 and
TTR-52 to mediate PS appearance on engulfingcells, thus promoting
phagocytic engulfment.
Results
nrf-5 Is Required for Cell Corpse Clearance
In a forward genetic screen for regulators of cell
corpseengulfment, we isolated a recessive mutant qx16, which
accu-mulated significantly more embryonic cell corpses than
wild-type (WT) (Figure 1A). No excessive cell death occurred inqx16
mutants, but cell corpses persisted significantly longerin qx16
embryos than in WT (53 versus 28 min on average),indicating that
cell corpse clearance is defective (Figures 1Band 1C).The gene
affected in qx16 mutants is a previously identi-
fied gene, nrf-5, loss of which causes a defect in nosemuscle
contraction induced by the antidepressant Fluoxe-tine [21] (see
Figure S1A available online). NRF-5 is homolo-gous to proteins of
the LPS-binding/lipid transfer protein(LTP) family including human
bactericidal/permeabilityincreasing protein (BPI), LPS-binding
protein (LBP), choles-teryl ester transfer protein (CETP), and
phospholipid transfer
http://dx.doi.org/10.1016/j.cub.2012.06.004http://dx.doi.org/10.1016/j.cub.2012.06.004mailto:[email protected]
-
Figure 1. nrf-5 Is Required for Cell Corpse Clearance
(A) Time-course analysis of cell corpses during embryonic
development was performed in wild-type (N2, open bar), nrf-5(qx16)
(black bar), or nrf-5(sa513)
(gray bar). At least 15 embryos were scored at every stage. Data
are shown as mean6 SEM. Data derived from WT and nrf-5(qx16) or WT
and nrf-5(sa513)
were compared by unpaired t test. **p < 0.0001; all other
points had p > 0.05.
(B) Embryonic cell deaths were followed inWT (N2, n = 3) and
nrf-5(qx16) (n = 3) embryos. The average number of total cell
deaths (6SEM) is shown in paren-
thesis.
(C) The duration of 33 corpses fromWT (N2, open bar, n = 3) and
61 corpses from nrf-5(qx16) (black bar, n = 4) wasmonitored. The
average duration (6SEM)
is in parenthesis.
(D) nrf-5(sa513) does not enhance the engulfment defect in
ttr-52 and ced-7mutants. Cell corpses were scored in the head
region of 4-fold embryos. At least
15 animals were scored in each strain. Data are shown as mean6
SEM. The number of cell corpses in WT was compared with that in
ttr-52(tm2078) or nrf-
5(sa513). For other points, single ced mutants were compared
with ced;nrf-5 double mutants, and ttr-52;nrf-5 double mutants were
compared with nrf-5
single mutants. **p < 0.0001; all other points had p >
0.05.
(E) Schematic diagram of the cell corpse engulfment pathway.
See also Figure S1.
Current Biology Vol 22 No 142
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
protein (PLTP) [21] (Figure S1B). nrf-5(sa513), a
previouslyisolated Fluoxetine-resistant mutant with a stop
mutationat Ser159, showed a persistent cell corpse phenotypesimilar
to qx16, whereas an incompletely penetrant embry-onic lethality and
yolk trafficking defect reported previouslyin nrf-5(sa513) mutants
was also observed in qx16 worms[21, 22] (Figure 1A; Figures
S1D–S1H). Sequencing of nrf-5in qx16 mutants revealed two G to A
transitions that resultedin replacement of Val446 by Met and of
Trp476 by a prema-ture stop codon (Figures S1A and S1B).
Overexpression ofNRF-5(V446M) but not NRF-5(W476-stop) rescued
qx16phenotypes, indicating that the W476-stop mutation causesloss
of nrf-5 function (Figure S1A).
NRF-5 Is a Secretory Protein that Clusters around
CellCorpses
Like other LTP/LPS-binding family proteins, NRF-5 has
anN-terminal secretion signal (Figure S1B) [21]. To test
whether
NRF-5 is secreted, we expressed a NRF-5::mCHERRY fusion,driven
by heat-shock promoters, or a ssGFP::mNRF-5 fusion,driven by either
heat-shock promoters or the engulfingcell-specific ced-1 promoter.
In ssGFP::mNRF-5, GFP wasfused to the amino terminus of mature
NRF-5 (NRF-5[21–551]) and the secretion signal was added to the
aminoterminus of GFP. Apoptotic cells were specifically labeledby
both NRF-5 fusion proteins, which formed ring-likestructures
surrounding cell corpses and accumulated withinthe extra-embryonic
cavity (Figures 2A, 2A0, 2D, and 2D0).Because heat-shock promoters
induce global gene expres-sion in C. elegans, these results suggest
that NRF-5 is asecretory protein that can recognize apoptotic
cells. Re-porters lacking secretion signals (GFP::mNRF-5 or
NRF-5[21–551]::mCHERRY), displayed diffuse or punctate
stainingpatterns but failed to surround cell corpses or accumulate
inthe extra-embryonic cavity (Figures 2B, 2B0, 2E, and
2E0).Expression of secreted but not nonsecretory NRF-5 fully
-
Figure 2. NRF-5 Is a Secretory Protein fromBody
Wall Muscle Cells and Clusters around Apoptotic
Cells
(A–F0) Differential interference contrast (DIC)and fluorescent
images of WT (A–B0, D–E0) orced-3(n717) embryos (C, C0, F, F0) that
carryPhspNRF-5::mCHERRY (A, A
0, C, C0), PhspNRF-5(21-551)::mCHERRY (B, B0),
Pced-1ssGFP::mNRF-5 (D, D0, F, F0), or Pced-1GFP::mNRF-5(E, E0).
Arrows indicate apoptotic cells, andarrowheads point to accumulated
fluorescent
proteins in the extraembryonic cavity. Cell corp-
ses indicated by yellow arrows are shown at 33
magnification in the insets.
(G–H00 0) DIC and fluorescent images of WTanimals transgenic for
both Pnrf-5GFP and Pmyo-3mCHERRY.
Scale bars in all panels represent 5 mm. See also
Figure S2.
NRF-5 Regulates Cell Corpse Engulfment3
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
rescued the persistent cell corpse, embryonic lethality,
anddefective yolk trafficking phenotypes of nrf-5(qx16)
mutants,indicating that NRF-5 must be secreted to function (Fig-ure
S1A). Cell corpse labeling by NRF-5::mCHERRY orssGFP::mNRF-5 was
abolished by a ced-3 loss-of-functionmutation that blocks almost
all apoptosis in worms, confirmingthat the cells labeledbyNRF-5were
apoptotic (Figures 2C, 2C0,2F, and 2F0).
Using transcriptional reporters, we found that the nrf-5promoter
(Pnrf-5) drove expression in body wall muscle cells,in a pattern
similar to the myo-3 promoter (Pmyo-3) (Figures2G–2H00 0) [23], but
distinct from the ttr-52 and ges-1promoters, which are active only
in the intestine (Fig-ures S2A–S2D00 0) [11, 24]. We also generated
NRF-5 trans-lational fusions with or without the secretion
signal(Pnrf-5ssGFP::mNRF-5 and Pnrf-5GFP::mNRF-5).
SecretedGFP::mNRF-5 (ssGFP::mNRF-5) displayed a diffuse
stainingpattern and associated with the basal surface of
pharyngealcells and the basal-lateral membranes of the
intestine,whereas nonsecretory GFP::mNRF-5 labeled body wallmuscle
cells but not the intestine (Figures S2E–S2H00 0).These results
suggest that NRF-5 is synthesized in andsecreted from muscle cells
to recognize apoptotic cellsand associate with the basal surface of
pharyngeal andintestine cells.
NRF-5 and TTR-52 Recognize Apoptotic Cells in Different
MannersNRF-5 behaves very like TTR-52, a secretory protein
fromintestine cells which specifically recognizes apoptotic cellsby
binding to surface-exposed PS [11]. In tat-1(qx30)mutants, which
ectopically expose PS on the surface of livingcells due to loss of
TAT-1 P4-ATPase function, TTR-52 stainsvirtually all cells (Figures
3J and 3J0) [11]. In contrast,ssGFP::mNRF-5 was still enriched on
apoptotic cells in
tat-1(qx30) embryos, indicating thatcell corpse recognition by
NRF-5 isunaffected by ectopic surface expo-sure of PS by living
cells (Figures 3E,3E0, 3K, and 3L). Loss of function ofced-1,
ced-6, ced-2, ced-12, or ced-5did not disrupt clustering of
NRF-5around apoptotic cells (Figures 3B,3B0, 3K, and 3L). However,
labeling of
cell corpses by ssGFP::mNRF-5 was significantly reducedin ced-7
mutants, suggesting that ced-7 is required forcell corpse
recognition by NRF-5 (Figures 3C, 3C0, 3K,and 3L). None of the
above ced mutants, including ced-7(lf), affected binding of
apoptotic cells by TTR-52 (Figures3F–3H0, 3M, and 3N). These
results suggest that TTR-52binds apoptotic cells through
surface-exposed PS, whereasrecognition of cell corpses by NRF-5
requires CED-7. Weobserved slightly increased labeling of cell
corpses by TTR-52 in nrf-5(qx16) mutants and enhanced apoptotic
celllabeling by NRF-5 in ttr-52(lf) embryos (Figures 3K–3N;Figure
S1C).
nrf-5 Is Required for the Appearance of PS on the Engulfing
Cell SurfaceExposure of PS on apoptotic cells distinguishes them
fromnormal living cells and serves as an ‘‘eat me’’ signal for
clear-ance. We also detected PS on the surface of living cells
adja-cent to cell corpses. In WT embryos expressing the
secretedPS-binding protein Annexin V::GFP, >50% of
GFP-positivecell corpses were surrounded by neighboring living
cells,which were also labeled by Annexin V::GFP, albeit moreweakly
(Figures 4A, 4A0, and 4I). No GFP signal was detectedon the surface
of living cells that did not directly contactdying cells.
Similarly, TTR-52::mCHERRY, which bindsspecifically to PS, labeled
the surfaces of both the apoptoticcell and the surrounding living
cell (Figure 6C). These resultssuggest that PS appears on the
surface of both dying cellsand neighboring engulfing cells. Loss of
function of ced-6,ced-5, ced-12, or ced-2 did not significantly
affect theappearance of PS on engulfing cell surfaces (Figure 4I).
Bycontrast, in nrf-5(lf) animals, only 2% of cell corpses
weresurrounded by Annexin V::GFP-positive neighboring cells(Figures
4B, 4B0, and 4I). Annexin V::GFP staining of engulfingcells was
also completely blocked by loss of function of
-
Figure 3. ced-7 Is Required for the Clustering of NRF-5 around
Cell Corpses
(A–J0) DIC and fluorescent images of WT (A, A0, F, F0),
ced-1(e1735) (B, B0, G, G0), ced-7(n2094) (C, C0, H, H0),
ttr-52(tm2078) (D, D0), nrf-5(qx16) (I, I0), or tat-1(qx30) (E, E0,
J, J0) embryos expressing ssGFP::mNRF-5 (A–E0) or TTR-52::mCHERRY
(F–J0). Arrows indicate apoptotic cells. Cell corpses indicated
byyellow arrows are shown at 33 magnification in the insets. Scale
bars represent 5 mm.
(K–N) Thepercentageof cell corpses labeledbyssGFP::mNRF-5 (K)
orTTR-52::mCHERRY (M)wasquantifiedat the1.5-fold (K) or comma (M)
stage in thestrains
indicated. The ratio of fluorescence intensity on apoptotic
cells versus their living neighbors (X-fold) was determined at the
1.5-fold (L) or comma (N) stage in
indicated strains expressing ssGFP::mNRF-5 (L) or
TTR-52::mCHERRY (N). At least 15 embryos (K, M) or 30 cell corpses
(L, N) were quantified in each strain,
anddata are shownasmean6SEM.Dataderived fromdifferentmutant
backgroundswerecompared toWT. **p< 0.0001, *p< 0.05; all
other points hadp>0.05.
Current Biology Vol 22 No 144
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
-
Figure 4. Loss of nrf-5, ced-7, or ttr-52 Blocks PS Appearance
on Engulfing Cells
(A–H0) DIC and fluorescent images of WT (A, A0), nrf-5(qx16) (B,
B0, G, G0), ced-7(n2094) (C, C0, F, F0), ced-1(e1735) (E, E0), or
ttr-52(tm2078) (D, D0, H, H0)embryos at the early (A–D0) or 4-fold
(E–H0) stage expressing Annexin V::GFP. Arrows indicate Annexin
V::GFP-labeling of living cells that surround cell corp-ses (A0) or
apoptotic cells (E–H0). Yellow boxed regions are shown at 33
magnification in the insets. Scale bars represent 5 mm.(I–K) The
percentage of cell corpses surrounded by Annexin V::GFP-positive
cells (I) or labeled by Annexin V::GFP (J) was quantified at the
comma stage (I) or
the 4-fold embryonic stage (J). The ratio of fluorescence
intensity on apoptotic cells versus their living neighbors (X-fold)
was determined at the 4-fold
embryonic stage in indicated strains expressing Annexin V::GFP
(K). At least 15 animals (I, J) or 30 cell corpses (K) were scored
in each strain and data
are shown as mean 6 SEM. Unpaired t tests were performed to
compare all other data sets with that of WT (I) or ced-1 (J, K).
**p < 0.0001, *p < 0.05; all
other points had p > 0.05. See also Figures S3 and S4.
NRF-5 Regulates Cell Corpse Engulfment5
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
ttr-52 or ced-7, and partially suppressed in ced-1(lf)
mutants(Figures 4C–4D0, and 4I). These data suggest that
NRF-5,CED-7, and TTR-52 are required for PS appearance on
theengulfing cell surface and that CED-1 may play a partialrole in
this process, whereas CED-6, CED-5, CED-12, andCED-2 are likely
dispensable. Consistent with this, loss ofnrf-5 did not affect the
engulfment defect of ttr-52(lf) orced-7(lf) mutants but enhanced
the persistent cell corpse
phenotype in ced-1, ced-6, ced-2, ced-5, or ced-12
mutants(Figure 1D). Expression of nonsecreted NRF-5,
nonsecretedTTR-52 or TTR-52(M5), which is secreted but defective
inPS binding, failed to rescue the engulfing cell PS
labelingphenotype of nrf-5 or ttr-52 mutants, indicating that
bothprotein secretion from phagocytes and PS binding are re-quired
to mediate PS appearance on engulfing cells (FiguresS3A–S3E0)
[11].
-
Figure 5. Loss of nrf-5, ced-7, or ttr-52 Causes
PS Accumulation on Apoptotic Cells
(A–G0) DIC and fluorescent images of WT (A, A0),tat-1(qx30) (B,
B0, F, F0), ced-1(e1735);tat-1(qx30) (C, C0),
ced-7(n2094)tat-1(qx30) (D, D0),tat-1(qx30);nrf-5(sa513) (F, F0),
or tat-1(qx30)ttr-52(tm2078) (G, G0) embryos expressing
TTR-52::mCHERRY (A–E0) or GFP::Lact-C2 (F–G0).Arrows indicate
apoptotic cells. Scale bars repre-
sent 5 mm.
(H) The ratio of fluorescence intensity on
apoptotic cells versus their living neighbors
(X-fold) was determined in indicated strains ex-
pressing TTR-52::mCHERRY (left) or GFP::Lact-
C2 (right). At least 30 cell corpses were quantified
in each strain. Data are shown as mean 6 SEM.
Unpaired t tests were performed to compare all
other data sets with that of tat-1(qx30). **p <
0.0001; all other points had p > 0.05.
See also Figure S3.
Current Biology Vol 22 No 146
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
nrf-5 Mutants Accumulate PS on Apoptotic CellsWe next examined
whether loss of nrf-5, ced-7, or ttr-52affected PS appearance on
apoptotic cells. In earlyembryos, almost all cell corpses were
positive for AnnexinV::GFP in nrf-5(lf), ced-7(lf), or ttr-52(lf)
mutants as in WTor other ced mutants defective in cell corpse
engulfment,indicating that PS exposure on apoptotic cells is
unaffected(Figure S4A). The intensity of Annexin V::GFP labeling
onapoptotic cells in nrf-5(lf), ced-7(lf), or ttr-52(lf) mutantswas
greater than in WT or other ced mutants at both 1.5-and 2.5-fold
stages (Figures S4B and S4C), suggestingthat PS may accumulate on
apoptotic cells in these mutants.We next examined Annexin V
labeling at the 4-fold embry-onic stage when cell deaths have long
been completedand cell corpses are all cleared in WT but persist in
engulf-ment-defective mutants. PS, indicated by Annexin
V::GFPlabeling, was absent from most cell corpses in ced-1,ced-6,
ced-2, ced-5, or ced-12 embryos; a few apoptoticcells (4.6% to 20%)
were weakly positive (Figures 4E, 4E0,4J, and 4K). In ced-7 (60%),
ttr-52 (83%), or nrf-5 (70%)embryos, more corpses were Annexin
V::GFP-positive andthe signal was much stronger, suggesting that PS
accumu-lates on apoptotic cell surfaces in these mutants
(Figures4F–4K). In tat-1(lf) mutants, inward movement of PS
isblocked, and living cells have ectopic surface exposure ofPS,
which can be labeled by the PS binding proteins Lact-C2 or TTR-52
(Figures 5B0 and 5F0) [11, 25]. We found thatTTR-52::mCHERRY or
GFP::Lact-C2, which is distributedmore evenly between apoptotic and
living cells in tat-1(lf)mutants, became re-enriched on apoptotic
cells defectivefor nrf-5, ced-7, or ttr-52 but not ced-1 or ced-5
(Figures5C–5H). Loss of nrf-5, ced-7, or ttr-52 did not alter the
cellsurface labeling by TTR-52::mCHERRY or GFP::Lact-C2
intat-1(lf);ced-3(lf) embryos in which cell death is blocked(Figure
S3F–S3L). This suggests that NRF-5, CED-7, and
TTR-52 do not affect PS externaliza-tion in living cells.
Therefore, re-enrich-ment of PS-binding proteins on cellcorpses in
tat-1(lf) embryos defectivein ced-7, nrf-5, or ttr-52 is
likelycaused by increased PS on apoptoticcell surfaces.
Collectively, these dataindicate that loss of nrf-5, ced-7, or
ttr-52 blocks PS appearance on engulfing cell surfaces
whilecausing PS accumulation on apoptotic cells.
NRF-5 Binds PS and Possesses Lipid Transfer Activity
NRF-5 belongs to the LPS-binding/lipid transfer protein
(LTP)family, members of which bind and transfer various
lipidsbetween lipoprotein particles or lipid bilayers.
EGFP-NRF-5-Flag bound to PS but not other phospholipids, as
previouslyobserved for TTR-52, suggesting that NRF-5 is a
PS-bindingprotein. (Figure 6A) [11]. NRF-5 is required for
trafficking ofthe lipoprotein VIT-2 that associates with
cholesterol (FiguresS1D–S1G0) [22, 26], so we examined cholesterol
transferbetween NRF-5 and liposomes. We found that
[3H]cholesterolcan be transferred fromNRF-5 to liposomes or from
liposomestoNRF-5, suggesting that NRF-5 has lipid transfer activity
(Fig-ure 6B). Because NRF-5, TTR-52, and the ABC transporterCED-7
all mediate PS appearance on engulfing cells, we exam-ined whether
they associate with each other. Myc-taggedNRF-5 (albeit expressed
at a low level), but not the intracellularpart of CED-1
(CED-1[intra]), was specifically coimmunopreci-pitated with
TTR-52-flag, indicating that NRF-5 associateswith TTR-52 (Figure
S5B). Although recognition of apoptoticcells by NRF-5 requires
CED-7 function, no NRF-5/CED-7interaction was detected by
coimmunoprecipitation or cross-linking. Their association may
involve specific modification(s)acquired during apoptosis.
Cell Corpses Are Internalized by PS-PositiveNeighboring
Cells
C. elegans cell corpses are surrounded by multiple living
cellsbut are engulfed by only one of them [16, 27]. How
phagocytesare specified is not known. We found that PS, as
indicated byTTR-52::mCHERRY, first appeared on the apoptotic
cellsurface, forming a bright ring-like structure (Figure 6C). In
about2 min, PS was detected on the surface of one living neighbor
in
-
Figure 6. NRF-5 Binds PS and Possesses Lipid Transfer
Activity In Vitro
(A) NRF-5 binds PS in vitro. Affinity-purified EGFP-NRF-
5-Flag but not EGFP-Flag bound PS spotted on
a membrane strip.
(B) Transfer of [3H]cholesterol from donor proteins to
acceptor liposomes (left) and from donor liposomes to
acceptor proteins (right) was examined as described in
Experimental Procedures. At least two independent
experiments were performed.
(C) Cell corpses are internalized by the PS-positive
neighbor. The clearance of apoptotic cells is followed
in WT (a, b) and nrf-5(qx16) embryos (c, d) carrying
Pttr-52TTR-52::mCHERRY. The apoptotic cells (white arrow-
heads) are quickly internalized and cleared by the PS-
positive neighbor (yellow arrowheads) in WT but persist
much longer in nrf-5(qx16) embryos, which lack PS
labeling on the neighboring cell. Scale bar represents
1 mm.
See also Figure S5 and Movies S1, S2, S3, and S4.
NRF-5 Regulates Cell Corpse Engulfment7
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
WT but not nrf-5(qx16) embryos and was gradually enriched asthis
neighbor internalized the cell corpse (Figure 6C, a and b;Movies S1
and S2). PS always appeared on apoptotic cellsbefore engulfing
cells (n = 106), and most cell corpses (94/106)were eventually
engulfed by the PS-positive neighbor. Becauseall neighboring cells
are capable of engulfing dying cells [16, 27,28], the appearance of
PS may promote engulfment by thePS-positive neighbor, allowing it
to ‘‘outplay’’ other neighboringcells. In support of this, cell
corpses persisted significantlylonger in nrf-5(lf)mutants, inwhich
PSdid not appear on engulf-ing cells (Figures 1C, 4I, 6C, c and d;
Movies S3 and S4).
Discussion
NRF-5 Is a Secreted PS-Binding Protein
Required for Cell Corpse EngulfmentNRF-5 and TTR-52 are the two
secreted PS-binding proteins involved in cell corpse engulf-ment.
NRF-5 is recruited to apoptotic cellsprimarily by CED-7, whereas
TTR-52 bindssurface-exposed PS, cross-linking it withthe phagocytic
receptor CED-1. Both TTR-52and CED-1 are required for removal of
normalcells in tat-1(lf) mutants, in which PS is ectop-ically
exposed on all living cells [11]. However,loss of nrf-5 does not
significantly affectCED-1 binding to apoptotic cells. Neither
isNRF-5 required for the ‘‘missing cell’’ pheno-type in tat-1(lf)
mutants, where a proportionof nonapoptotic cells are absent,
presumablyhaving been engulfed after ectopically ex-pressing
surface PS (data not shown) [29].Therefore, NRF-5 does not seem to
act simplyas a bridge between surface-exposed PS onapoptotic cells
and the phagocytic receptorCED-1 like TTR-52.
CED-7, NRF-5, and TTR-52 May Act
Together to Mediate PS Transfer fromApoptotic Cells to Engulfing
Cells
We observed PS appearance on the surfaceof engulfing cells, a
phenomenon observedpreviously in macrophages, suggesting thatthis
is a conserved event during phagocytosis
of apoptotic cells. Our data show that loss of the ABC
trans-porter CED-7, or either of the two extracellular
PS-bindingproteins NRF-5 and TTR-52, completely blocks PS
appear-ance on engulfing cells and causes accumulation of PS
onapoptotic cells without affecting PS externalization in
eitherliving or dying cells. These results are consistent with
aproposed model in which CED-7, NRF-5, and TTR-52 maycooperate to
transfer PS from apoptotic to engulfing cells (Fig-ure S5C). NRF-5
belongs to a secreted LPS binding/LTP familyincluding CETP and
PLTP, which regulate lipoprotein metabo-lism by exchanging neutral
lipids like cholesteryl ester and
-
Current Biology Vol 22 No 148
Please cite this article in press as: Zhang et al., C. elegans
Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on
Phago-cytes for Cell Corpse Engulfment, Current Biology (2012),
doi:10.1016/j.cub.2012.06.004
phospholipids between lipoproteins [30], and LBP and BPI,which
mediate innate immunity by transferring LPS to themacrophage
surface for cell activation [31]. Our finding thatNRF-5 can
transfer cholesterol to or extract it from liposomesindicates that
NRF-5 has lipid transfer activity, consistent withits role in yolk
protein trafficking and PS transfer duringengulfment.
Apoptotic cell recognition by NRF-5 requires CED-7, anABC
transporter previously thought to promote PS external-ization in
apoptotic cells [18]. We observed that loss ofced-7 caused
increased PS on apoptotic cell surfaces anddid not affect ectopic
exposure of PS on living cells causedby tat-1 mutation, indicating
that ced-7 is not responsiblefor initial exposure of PS on
apoptotic cells or living cells.These results are consistent with
the finding that ABCA1,the mammalian counterpart of CED-7, is
dispensable fortransbilayer PS movement in apoptotic cells or
macro-phages [20]. ABCA1 loads cholesterol and phospholipidsonto
apoA1, a process promoted by PLTP, which bindsABCA1 on the cell
surface [32]. We found that CED-7recruits NRF-5 specifically to
dying cells despite beingpresent in both dying and engulfing cells,
suggesting theinvolvement of other factors or modifications of
CED-7 inapoptotic cells [17]. Because PS is exposed on apoptoticbut
not living cells, it is possible that CED-7, when itreceives PS on
the apoptotic cell surface, recruits andloads NRF-5 with PS, which
is then transferred to engulfingcells (Figure S5C). The requirement
of PS and/or modifica-tions of CED-7 acquired during apoptosis may
explainwhy we failed to detect associations between CED-7
andNRF-5.
The extracellular PS-binding protein TTR-52 is also requiredfor
mediating PS appearance on engulfing cells. TTR-52 asso-ciates with
NRF-5 and may act like soluble CD14 (sCD14)(which receives LPS or
phospholipids from LBP and transferthem to target cells or HDL
particles [33–35]) to relay PSfrom the NRF-5-PS complex to
engulfing cells.
PS-Positive Neighbors Outplay the Others to Engulf Cell
CorpsesMammalian apoptotic cells are cleared mainly by
mobileprofessional phagocytes like macrophages. In C. elegans,
allthe cells neighboring a somatic cell corpse can recognizedying
cells and initiate engulfment, but only one
completesinternalization [16, 27, 28]. We found that most cell
corpseswere internalized by the PS-positive neighbor,
suggestingthat PS may help to specify engulfing cell fate.
Appearanceof PS on one neighboring cell may promote engulfment
bythis cell, thereby excluding other neighbors from
completingengulfment. Consistent with this, loss of nrf-5 blocks
PSappearance on engulfing cells, severely delaying
engulfment.Macrophage surface expression of PS is important for
phago-cytosis of PS-positive targets, suggesting that PS
appearanceon phagocytes is a conserved mechanism for
removingapoptotic cells [5, 6]. How PS appearance on
phagocytespromotes engulfment requires further investigation. The
pres-ence of PS on both dying and engulfing cells may stabilize
thepairing between the eater and the prey; alternatively, PS
maymodify the engulfing cell membrane so that it is in a
morefavorable state for internalization. In addition, TTR-52
maytransfer PS to cell surface proteins to further activate
engulf-ment, like the sCD14-LPS complex, which induces cell
activa-tion of macrophages or neutrophils [34] (Figure S5C). CED-1
isa potential candidate to test because loss of ced-1 partially
blocked PS appearance on engulfing cells but did not causePS
accumulation on cell corpses, suggesting that CED-1 isnot involved
in transferring PS from dying cells but may havea role in anchoring
PS on the engulfing cell surface.
Experimental Procedures
Genetic Analysis
Strains of C. elegans were cultured using standard protocols
[36]. The WT
strain was N2 Bristol. Details of strain usage, mapping, and
cloning are
provided in the Supplemental Experimental Procedures.
Quantification of Cell Corpses, Cell Corpse Duration, PS
Appearance,
and PS Accumulation
Cell corpses were counted in the head region of living embryos
either at
the six different embryonic stages for a time course analysis or
at the
4-fold embryonic stage as described before [37]. Embryonic cell
deaths
and cell corpse duration were examined as described
previously
[14, 38]. Detailed procedures for quantifying PS appearance and
accu-
mulation are provided in the Supplemental Experimental
Procedures.
In Vitro Lipid Binding and Lipid Transfer Assay
EGFP-NRF-5-Flag and EGFP-Flag expressed in and secreted from
293T
cells were affinity purified from the culture medium and used
for in vitro lipid
binding and transfer assays as described in the Supplemental
Experimental
Procedures.
Statistical Analysis
The SEMwas used as y error bars for bar charts plotted from
themean value
of the data. Data derived from different genetic backgrounds
were
compared by Student’s two-way unpaired t test. Data were
considered
statistically different at p < 0.05. p < 0.0001 is
indicatedwith double asterisks
and p < 0.05 with single asterisks.
Supplemental Information
Supplemental Information includes five figures, Supplemental
Experimental
Procedures, and four movies and can be found with this article
online at
doi:10.1016/j.cub.2012.06.004.
Acknowledgments
We thank P. Liu, T. Wei, and K. Zhao for help with the lipid
transfer assay, D.
Xue for providing the Annexin V::GFP reporter, and the
Caenorhabditis
Genetic Center (CGC) for strains.We thank Jing Lin,Mingxing Lu,
andDong-
feng Zhao for technical support and Isabel Hanson for editing
services. This
work was supported by a grant from theMinistry of Science and
Technology
to X.W. (2010CB835201).
Received: January 9, 2012
Revised: April 10, 2012
Accepted: June 1, 2012
Published online: June 21, 2012
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C. elegans Secreted Lipid-Binding Protein NRF-5 Mediates PS
Appearance on Phagocytes for Cell Corpse
EngulfmentIntroductionResultsnrf-5 Is Required for Cell Corpse
ClearanceNRF-5 Is a Secretory Protein that Clusters around Cell
CorpsesNRF-5 and TTR-52 Recognize Apoptotic Cells in Different
Mannersnrf-5 Is Required for the Appearance of PS on the Engulfing
Cell Surfacenrf-5 Mutants Accumulate PS on Apoptotic CellsNRF-5
Binds PS and Possesses Lipid Transfer ActivityCell Corpses Are
Internalized by PS-Positive Neighboring Cells
DiscussionNRF-5 Is a Secreted PS-Binding Protein Required for
Cell Corpse EngulfmentCED-7, NRF-5, and TTR-52 May Act Together to
Mediate PS Transfer from Apoptotic Cells to Engulfing
CellsPS-Positive Neighbors Outplay the Others to Engulf Cell
Corpses
Experimental ProceduresGenetic AnalysisQuantification of Cell
Corpses, Cell Corpse Duration, PS Appearance, and PS AccumulationIn
Vitro Lipid Binding and Lipid Transfer AssayStatistical
Analysis
Supplemental InformationAcknowledgmentsReferences