Professional Documents
Culture Documents
Phagosome Maturation: Going Through The Acid Test: Jason M. Kinchen and Kodi S. Ravichandran
Phagosome Maturation: Going Through The Acid Test: Jason M. Kinchen and Kodi S. Ravichandran
Phagosome maturation:
going through the acid test
Jason M. Kinchen and Kodi S. Ravichandran
Abstract | Phagosome maturation is the process by which internalized particles (such as
bacteria and apoptotic cells) are trafficked into a series of increasingly acidified
membrane-bound structures, leading to particle degradation. The characterization of the
phagosomal proteome and studies in model organisms and mammals have led to the
identification of numerous candidate proteins that cooperate to control the maturation of
phagosomes containing different particles. A subset of these candidate proteins makes up
the first pathway to be identified for the maturation of apoptotic cell-containing
phagosomes. This suggests that a machinery that is distinct from receptor-mediated
endocytosis is used in phagosome maturation.
Receptor-mediated
Eukaryotic cells internalize a variety of particles during called phagosomes14. These phagosomes sequentially
endocytosis their lifetime. The uptake of particles >0.5 µm in size acquire different proteins (for example, the Rab GTPases15)
The process by which a is termed phagocytosis, whereas particles <0.5 µm are during maturation and eventually fuse with acidic lyso
ligand-bound receptor is taken up by receptor-mediated endocytosis or pinocytosis. some structures. Defects in degradation have a number
internalized into a
Distinct types of phagocytosis tend to be ligand specific: of consequences that depend on the type of particle to
membrane-bound vesicle.
These vesicles sequentially bacteria (~0.5–3 µm) or yeast (~3–4 µm) are internalized be removed. For example, defects in lysosomeassociated
acquire different Rab GTPases. by macrophages through scavenger receptors; micro membrane protein2 (LAMP2; see below) block phago
organisms can also be coated with serum components some maturation and have been linked to periodontal
Pinocytosis (for example, complement) or antibodies and then taken disease, owing to decreased neutrophilmediated bacte
The process by which liquids
and small particles are
up through complement1,2 or Fc receptors3,4, respectively. rial killing16. Defects in apoptotic cell degradation can
internalized by the cell. Cells that undergo apoptosis, which can range in size result in autoimmune disease17. For example, apoptotic
from 5 to 50 µm, must also be removed (BOX 1). In fact, cell removal by dendritic cells has been implicated
Complement in humans ~200 billion cells are turned over each day in the establishment of tolerance, the process by which
A protein complex component
per individual, making apoptotic cell removal one of the immune system does not respond to selfderived
of the innate immune system
that can bind to foreign the most common types of phagocytosis5. Thus, under antigens9,12,13,18,19. Phagosome maturation is therefore of
particles and initiate their standing how apoptotic cells are phagocytosed and pro great importance to normal homeostasis of the immune
phagocytosis. cessed is a fundamentally important biological problem. system, to the response to bacterial pathogens and in
Apoptotic cell turnover begins with the induction of influencing the immune response to our normal bacte
an apoptotic programme or other cellular changes that rial flora. Moreover, as certain pathogens seem to use the
mark the cell for removal6,7. The subsequent recognition phagocytic machinery to enter and/or live within
of altered features by phagocytes leads to their highly the host, understanding the various steps of cargo
efficient and immunologically silent removal8. Apoptotic handling in the context of phagocytosis could be key in
cells can be taken up either by neighbouring cells or by therapeutic design.
Beirne B. Carter Center for professional phagocytes, such as macrophages9–11 and We focus on how apoptotic cellcontaining phago
Immunology Research and
dendritic cells12,13. The contribution of each type of somes mature within the phagocyte, and on the recent
the Department of
Microbiology, phagocyte to clearance in vivo has not been extensively developments in our understanding of the genetic,
University of Virginia, addressed. molecular and functional aspects of phagosome matura
Charlottesville, Virginia Phagosome maturation can be viewed as the end of tion in various contexts. Extensive studies on receptor
22902, USA. the phagocytic process, during which internalized apop mediated endocytosis over the past few decades have
e-mails:
kinchen@virginia.edu;
totic cells or bacteria are efficiently degraded. After a allowed the development of a general paradigm for this
ravi@virginia.edu particle is internalized, it ‘matures’ through a series of process: after the binding of a soluble ligand (such as a
doi:10.1038/nrm2515 increasingly acidified membranebound structures growth factor) to a receptor, the membrane invaginates
and the vesicle that contains the receptor and the ligand activation of multiple types of receptors that are distinct
or cargo from the plasma membrane is excised20–22 from those involved in receptormediated endocytosis
(FIG. 1a). Timelapse studies have shown that this vesicle or phagocytosis of bacteria8,32 (BOX 1). Therefore, the
moves within the cell through a series of stages (dur paradigm that phagocytosed particles mature through
ing which they acquire rAb5 and rAb7 (ReF. 23)) that progressively acidic structures seems to be true 33
lead to progressive acidification and eventually to fusion (FIG. 1b). However, how to define which molecules regu
with lysosomes24,25. During these acidification steps, the late phagosome maturation, and our knowledge of how
cargo dissociates from the receptor, which might be signalling events overlap or differ from endocytosis and
recycled back to the membrane and the cargo might other phagocytic events, have become active areas of
be trafficked for degradation or use within the cell26,27. investigation.
Although this paradigm has been useful for our current
knowledge of vesicular transport within cells, membrane Insights from proteomics approaches
biogenesis and fusion events, the phagocytic process using targets such as whole bacteria or latex beads,
poses several unique challenges. early biochemical and proteomics studies attempted
The first challenge stems from the need of a plasma to identify proteins that are involved in phagosome
membrane to temporarily extend over the entire surface maturation33. Although this resulted in the identifica
area of a neighbouring apoptotic cell during engulf tion of a vast number of candidate proteins that are
ment; in many cases, the neighbouring cell is as large or localized to phagosomes, in general these studies did
larger than the phagocyte14. This engulfment is accom not address the functional requirements for these pro
Fc receptors plished by the mobilization of intracellular vesicles to teins in phagosome maturation. However, an excellent
A group of proteins that bind the plasma membrane; these vesicles also potentially descriptive analysis of the different stages of phagosome
to immunoglobulin (Ig)G- transport signalling proteins to the site of internaliza maturation was obtained from these studies, which also
opsonized particles, leading to
tion14,28,29. second, the contents of the phagocytic cargo led to the development of a basic descriptive model
the activation of phagocytosis.
brought into the cell (for example, bacteria or apoptotic for protein recruitment and release from the nascent
Rab GTPases cells) need to be ‘unpacked’ and processed in a way phagosome33–35. various proteins must be delivered to
A family of proteins that cycle that allows for either an immunological response (in the phagosome, including vacuolar (v)type ATPases
between GDP-bound inactive the case of microorganisms) or immunological toler (which acidify the phagosome), acidic proteases (such
and GTP-bound active
forms and have a role in
ance (to selfantigens derived from apoptotic cells; as cathepsins) and major histocompatibility complex
targeting transport to BOX 2)30,31. Third, the engulfment of larger particles, (MHC) class II molecules (for the presentation of
membrane-bound organelles. such as apoptotic cells, often involves the simultaneous phagosomederived antigens on the cell surface).
a b Apoptotic cell
Dynamin
Dynamin
Receptor-bound Clathrin
Phagocytic
cargo receptor
Adaptins
Recycling
endosome
Clathrin
removal
RAB5 recruitment
RAB5
recruitment
RAB7 recruitment
Concentration
stage
RAB7
recruitment
LAMP1 recruitment
Recycling Loss of
endosome RAB5
Lysosome
Fusion to
lysosomes
Lysosome Golgi Golgi
Figure 1 | endocytosis as a paradigm for phagocytosis. Phagocytosis and receptor-mediated endocytosis are similar
processes that also have distinct features. a | During receptor-mediated endocytosis, the activated receptor sinks into
the cell, in this case into a clathrin-coated pit21. Clathrin is removed from the vesicle after dynamin-mediated scission
from the membrane20; receptors are then trafficked to the recycling endosome, whereNature Reviews
they are | Molecular
eventually sortedCell
backBiology
to
the cell surface22. Activated endocytic receptors are also removed from phagosomes and presumably are also targeted
to recycling endosomes (see part b). Endocytosed vesicles acquire the GTPase RAB5, which is activated by guanine
nucleotide-exchange factors (GEFs; such as RABEX5 and GAPEX5)150. Active RAB5 allows homotypic fusion with other
RAB5-positive structures23,57, as well as heterotypic fusion with RAB4-positive recycling endosomes70. RAB5 is then
exchanged for RAB7 through the HOPS (homotypic fusion and vacuole protein sorting) complex23, which contains the
RAB7 GEF vacuolar sorting protein-39 (VPS39), resulting in the recruitment of the RAB7 effector RAB7-interacting
lysosomal protein (RILP) and, ultimately, fusion with acidic lysosomes151 and acquisition of the lysosomal markers
lysosome-associated membrane protein-1 (LAMP1) and LAMP2. b | By contrast, during phagocytosis the plasma
membrane is extended around the apoptotic cell, forming the phagosome8,14. Phagosomes also mature through
RAB5-positive and RAB7-positive stages, which result in fusion with lysosome structures28,33,41,52,57,58; however, phagosomes
seem to use a distinct mechanism for GTPase regulation. In the nematode, known RAB-5 GEFs are not required for
phagosome maturation, and the HOPS complex is not required for the recruitment of RAB-7 to the phagosome. This might
reflect the observations that phagosomes rarely fuse together and tend to mature individually. Other events have been
described43, such as the trafficking of proteins from the Golgi to the phagosome, although whether these occur during
endocytosis remains to be determined. A number of GTPases are associated with the phagosome during phagosome
maturation (BOX 3), but the roles of many of these on the endosome have not been described.
The acid test and its consequences. Acidification of the the phagocytosed particle can begin to be degraded36. In
phagosome is perhaps the most common readout used addition to degrading proteins into peptides for loading
for addressing protein function in phagosome maturation. onto class II and class I MHC molecules, cathepsin D also
Recycling endosome
A membrane-bound organelle
Acidification is essential during phagosome maturation: it has a role in the activation of MHC class II proteins, a role
for the recycling of receptors is only when the phagosome hits a sufficiently low pH that that is essential for peptide loading37,38. Thus, acidification
following ligand dissociation. the cathepsin family of acidic proteases become active and is a marker of a ‘functional’ phagosome.
Box 2 | The consequences of apoptotic cell removal budding from the Golgi, is also required for phagosome
maturation49, although a role for CoPI in vesicle budding
There are several consequences associated with the internalization of apoptotic cell from the phagosome (or other organelles) is possible.
corpses. These consequences fall into three general classes that might also be affected studies in zebrafish have suggested that vtype ATPases
by signalling from the phagosome.
might have roles in phagosome maturation in addition to
First, the removal of apoptotic cells has been linked to the enhanced secretion of
phagosome acidification: the vtype ATPase A1 subunit
‘pro‑healing’ cytokines (such as transforming growth factor‑β10 and interleukin‑10
(ReF. 139)) that serve to reduce inflammation in the extracellular surroundings and to
is required for apoptotic cellcontaining phagosome
promote wound healing. The signalling pathways that alter cytokine secretion are not fusion with lysosomes, although the exact stage at which
well characterized, but many phagocytic receptors (for example, stabilin‑2 (ReF. 140) phagosomes are arrested is unknown50. A screen for
and CD36 (ReF. 139)) have been implicated. Intriguingly, endothelial cells have been genes that are required in the early stages of phagosome
reported to induce pro‑inflammatory signalling (increased secretion of tumour‑ maturation identified C. elegans vha‑16, which encodes
necrosis factor‑α) following recognition of apoptotic cells141; this signalling has the D subunit of the v0 ATPase41. A direct role for these
implications for atherosclerotic disease. ATPases in vesicle fusion seems unlikely, but it is possible
Second, phagocytes must also dispose of internalized components of the apoptotic that a low pH might induce conformational changes in
cell. Few studies have tracked the fate of apoptotic cell components, but two recent
proteins that are required for phagosome maturation.
studies have addressed signalling pathways that lead to cholesterol efflux from the cell
Alternatively, acidification might result in cleavage of a
and suggest that internalization of apoptotic cells (or targets) activates the nuclear
receptor LXR. LXR activation results in enhanced transcription of the transporter target protein by activated cathepsins, which would then
protein ABCA1 (ReFs 103,142). induce maturation. These are mere speculations, however,
Third, protein components of the cell are degraded and cross‑presented by major and the mechanism by which vtype ATPases regulate
histocompatibility complex (MHC) class I molecules11,143 and tend to be excluded from maturation remains uncertain.
class II presentation97. Processing of apoptotic cell‑associated antigens seems to be
especially important, and has been linked to the maintenance of self‑tolerance12,19,144. Proteomics and contamination. one must sound a note of
Potential pathways for DNA degradation have been described in the nematode145–147 caution when analysing proteomics data. A recent study
and mouse models17; defects in this process, as with defects in cell corpse removal, has suggested that proteomics purification schemes suf
have been linked to autoimmune disease, emphasizing that efficient phagosome
fer from relatively high contamination from other cellular
maturation is required for health of the organism.
membranes, which could be misleading51. Although trans
ient protein localization to the phagosome might provide
an argument for a role in this process, further studies are
Acidification of the phagosome occurs in two stages. required to validate such candidates, either by rnA inter
First, a poorly understood ‘early’ acidification step results ference (rnAi) or dominantnegative approaches. In this
in a relatively small drop in pH39. second, vtype ATPases respect, recent studies in model organisms, such as the
—ATPhydrolysisdriven proton pumps40 — are sub nematode C. elegans, have for the first time identified a
sequently trafficked to the phagosome at relatively late functional pathway for phagosome maturation41,52,53.
stages. In the nematode Caenorhabditis elegans, phago
some acidification begins quite early: rAb5positive Defining signalling pathways
phagosomes stain weakly with acridine orange, a marker Whereas studies in cultured cells have been instrumental
for acidic organelles41. However, these phagosomes do in identifying the protein components of the phagosome,
not stain as strongly as latestage apoptotic cells, a pattern an understanding of a comprehensive pathway for how
that is consistent with multiple modes of acidification that phagosome maturation is regulated has been lacking.
depend on the stage of maturation. recent genetic studies, combined with cell biological
approaches in model organisms, have begun to define
Vacuolar ATPases. The vtype ATPase is a multisubunit the basic pathway for the acidification of apoptotic
complex composed of 14 subunits in two separate cellcontaining phagosomes.
structures: the v1 complex mediates ATP hydrolysis, C. elegans has been a powerful genetic tool for the
whereas the v0 complex transports H+ across the endo identification of genes that are involved in programmed
somal membrane40. vtype ATPases are trafficked to the cell death; and genes identified in this model have also
phagosome and acidify its contents, but where do these been shown to have a role in mammalian phagocytic
ATPases (and other proteins trafficked to the phagosome) uptake (BOX 1). However, the events leading to cell corpse
come from? degradation following phagocytosis have been addressed
In Dictyostelium discoideum, yeastcontaining phago only recently. one of the major impediments to these
somes acquire vtype ATPases by fusion with vtype studies has been the requirement of many endocytic
ATPasecontaining endosomes42. In multicellular organ proteins for cell viability; these challenges necessitate
isms, the trafficking of vtype ATPases is more complex, the identification of rare temperaturesensitive muta
because rather than delivery by endosomal fusion, these tions41 or timeconsuming clonal screens28,54. rnAi has
proteins seem to be trafficked from the transGolgi to the bypassed this issue as it is remarkably efficient in the
phagosome along with cathepsins43. Proteomics studies nematode41, although complementary studies using
Programmed cell death have identified a large number of components that local mutant alleles are preferable when such mutants are
The process by which a healthy ize to phagosomes33–35,44–48. However, the mechanism by available. rnAibased approaches, combined with
cell is induced to die,
undergoes apoptosis and is
which many of these components arrive at the phago genetic and fluorescent markers that define specific
then cleared and degraded by somes has yet to be addressed. Intriguingly, coatomer steps in phagosome maturation, allow unbiased screens
a phagocyte. protein complexI (CoPI), which is required for vesicle to identify regulators at various steps in phagosome
Table 1 | Genes and proteins required for phagosome maturation during apoptotic cell removal
categories gene or mammalian Function loss- or reduction-of-function refs
protein or nematode phenotypes
name orthologue
Caenorhabditis elegans
Phagocytic ced-1 LRP, MEGF10 Recognition of the apoptotic cell and Delayed phagocytosis and accumulation of 53,104,
genes intracellular signalling RAB-7 and FYVE marker 134,136,
ced-6 GULP, human 137,153
CED6
ced-7 ABCA1, ABCA7
ced-5 DOCK180 GEF for CED-10 (also known as RAC1) Delayed phagocytosis and accumulation of 53,131,
FYVE marker 154,155
GTPases rab-2 RAB2 ER-to-Golgi transport and potentially Delayed (or possibly abnormal) maturation 52,54
ER- or Golgi-to-phagosome transport
rab-5 RAB5 Early endosome maturation Arrested phagosomes, block in acidification 41
rab-7 RAB7 Late endosome maturation Arrested phagosomes at the RAB-5-positive 41,53
stage
dyn-1 Dynamin Recruitment of RAB-5 Enlarged phagosomes, pre-RAB-5 arrest 41
RAB-5 vps-34 VPS34 PI3K Blocked in RAB-5 recruitment, no 41
effectors acidification
RAB-7 vps-11 VPS11 RING-motif-containing protein Arrested phagosomes at RAB-7-positive 41
effectors stage
vps-16 VPS16 Unknown
vps-18 VPS18 SNARE binding?
vps-33 VPS33 Sec1 homologue, SNARE binding?
vps-39 VPS39 GEF for RAB-7
Unassigned vps-41 VPS41 Adaptor protein, vacuolar biogenesis No gross effect on recruitment of RAB-5 or 41
genes RAB-7 to phagosomes
vps-45 VPS45 Sec1 homologue, vacuolar biogenesis
Mammals
RAB5 Dynamin DYN-1 Required for RAB5 recruitment Arrested nascent phagosome, block in 28,41
recruitment acidification
GTPases RAB5 RAB-5 Required for maturation from nascent Block in acidification 41,61
phagosome to RAB7-positive stage
RAB7 RAB-7 Maturation from the RAB5-positive to Unknown 89
LAMP1-positive phagosome
RAB5 GEF GAPEX5 RME-6 RAB5 activation Blocked RAB5 activation during phagosome 61
maturation
Unknown RhoA RHO-1 Regulate early steps of phagosome Blocked RAB7 recruitment, phagosome 89
role maturation acidification and protease delivery
ERM proteins ERM-1
Recent studies in the nematode Caenorhabditis elegans and mammalian systems have helped identify proteins that are involved in various steps of phagosome
maturation during apoptotic cell removal. The genes in the nematode were identified in genetic screens, whereas those in the mammalian systems were identified
using a candidate approach that targets individual molecules. ER, endoplasmic reticulum; ERM, ezrin–radixin–moesin; GEF, guanine nucleotide-exchange factor;
LAMP1, lysosome-associated membrane protein-1; PI3K, phosphoinositide 3-kinase; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein
receptor; VPS, vacuolar sorting protein.
maturation (TABLe 1). several groups have made great and allowing it to bind membranous organelles. rab
strides towards developing the nematode as an in vivo proteins can then bind to a guanine nucleotideexchange
model for phagosome maturation41,52,53. factor (GEF), resulting in the exchange of GDP for GTP55.
The now active rabGTP can bind to effector proteins57,
Phagosome maturation: Rab GTPases and protein resulting in the recruitment of machinery for further steps
trafficking. rab GTPases have been implicated in con of maturation.
trolling transport between membranebound organelles. recent genetic studies have shown that rAb5
These proteins function as ‘molecular switches’: in the (ReF. 41) and rAb7 (ReFs 41,53) are required for the
Prenylated inactive conformation, the rab protein is GDP bound removal of apoptotic cells in the nematode, with both
The process by which a and resides in the cytoplasm in complex with a rab GDP GTPases marking distinct (but partially overlapping)
hydrocarbon moiety is
attached to a conserved CAAX
dissociation inhibitor (rabGDI), which keeps the protein stages of maturation41. similar to our current knowledge
motif in the C terminus of inactive55,56. The rabGDI releases the rab GTPase follow of maturation of internalized particles in mammalian
GTPases. ing a signalling event, thereby exposing the prenylated tail systems15,58, rAb5 functions upstream of rAb7, as
phagosomes are arrested at the rAb5positive stage GEF might have this role. regulation of rAb5 is thus
in rab‑7deficient worms. based on the role of rAb5 distinct from other endocytic events (such as the inter
and rAb7 on endosomes, rab GTPases seem to func nalization of proteins from the extracellular space) that
tion as part of a fusion pore that determines the types jointly require rME6 and rAbX5, suggesting that the
of interactions that vesicles have with other membrane regulation of phagosome maturation differs from that of
bound organelles59. In this respect, rAb5 and rAb7 receptormediated endocytosis.
potentially serve as nodes for recycling proteins back to Intriguingly, a recent study in which a fluorescence
the plasma membrane and as entry points for vesicles resonance energy transfer (FrET) probe was used to
to deposit lysosomal proteases, with ‘effector’ proteins monitor rAb5 activation in swiss 3T3 cells suggested
binding to the activated rab protein to mediate this that the GEF GAPEX5 might have a role in regulating
function (see below). GTP loading of rAb5 during removal of apoptotic
cells61. one caveat to this is that the experimental system
A novel recruitment strategy for RAB5. one of the is somewhat artificial, requiring overexpression of
earliest known maturation events in a number of sys integrin proteins (and opsonization with the integrin
tems is the recruitment of rAb5 to phagosomes41,58,60,61, ligand MFGE8) rather than the monitoring of basal
although a detailed mechanism for how this is accom engulfment. The importance of GAPEX5 in the context
plished during phagosome maturation is lacking. of endogenous cell corpse removal (which does not
recent studies in the nematode41 have implied an require the opsonin MFGE8 in most cases65) therefore
unexpected role for dynamin and the rAb5 effector remains to be determined.
vacuolar sorting protein34 (vPs34) in rAb5 recruit
ment to nascent phagosomes (FIG. 2), which suggests Phosphoinositides and RAB5 effectors. The enrichment
that phagosomal recruitment of GTPases is a complex, of PtdIns3P on the surface of the phagosome can be
regulated process41,62. regarded as a sign of phagosome maturation from the
The GTPase DYn1, the nematode orthologue of rAb5positive to the rAb7positive stage in both mam
dynamin, was identified in forward and reverse genetic malian and nematode models (see below). The recruit
screens for genes that are involved in programmed cell ment of rAb5 effectors (such as rabenosyn5 (ReF. 66))
death28,41. Although initial reports linked DYn1 to the by interaction with GTPbound rAb5 (in conjunction
internalization of apoptotic cells (by having a role in with PtdIns3P57) results in the subsequent recruitment
focal exocytosis28), this view has now been revised41,53. of rAb7 and continued maturation of the phagosome
The current view, based on studies in mammalian (see below). There are many known rAb5 effectors in
cells and nematodes 41,62, is that DYn1 (and mam cultured cells that serve diverse functions57. Effectors
malian dynamin) has a role not in exocytosis or the typically mediate homotypic fusion events during endo
internalization of cell corpses, but is instead essential cytosis — for example, among rAb5positive vesicles,
for phagosome maturation, namely the recruitment of as in the case of early endosome antigen1 (EEA1)59,67,68
rAb5 to the nascent phagosome. Phagosomes in dyn‑1 — or they mediate heterotypic fusion between structures
lossoffunction mutant worms appear arrested, without coated with different rab proteins (potentially between
recycling of the phagocytic receptor CED1 back to the rAb5 and rAb7staining structures, as in the case of
plasma membrane41. Further genetic and cell biological rabenosyn5 (ReFs 66,69), or between rAb5 and rAb4
analysis identified the phosphoinositide 3kinase (PI3K) coated structures, as in the case of rabaptin5 (ReF. 70)).
vPs34 (an orthologue of human PIK3C3 or vPs34) Typically, rAb5 effectors share a similar architecture;
Dynamin
as another protein that regulates rAb5 recruitment41. they contain a FYVe domain for binding PtdIns3P that is
A large GTPase that is involved biochemical analysis revealed a novel protein complex in generated on the phagosome by vPs34 (ReF. 57).
in membrane scission, actin which DYn1 mediates the recruitment of GDPbound In the nematode, vPs34 is required for the accu
cytoskeletal dynamics and rAb5 through interaction with vPs34, which serves mulation of PtdIns3P on the phagosome41, and green
phagosome maturation.
as a bridging protein. To complicate matters, rAb5 also fluorescent protein (GFP)tagged constructs contain
Focal exocytosis seems to regulate vPs34 activity63 (this is also shown ing the FYvE domain from EEA1 (a component of the
small vesicles are targeted to during removal of immunoglobulin (Ig)Gopsonized red rAb5 fusion pore) or HGrs1 (which is required for
the plasma membrane during blood cells in mammals58,63), and knockdown of rAb5 formation of multivesicular endosomes71) have been
phagocytosis. In turn, this is blocks the accumulation of PtdIns3P on the phagosome successfully used to monitor this process41,53,54. vPs34 is
thought to produce a local
increase in the volume of
(see below). currently the only rAb5 effector to be linked to phago
plasma membrane, thereby Although this novel complex explains how rAb5 some maturation in the nematode, and worms that are
allowing the cell to extend its is recruited to apoptotic cellcontaining phagosomes, it deficient in eea‑1 or hgrs‑1 show no defect in cell corpse
membrane around large does not suggest a mode of regulation, as rAb5 must removal41, although EEA1 is required for maturation
particles.
still be converted to the GTPbound form for phago of Mycobacterium speciescontaining phagosomes in
FYVE domain somes to mature41,64. There are at least three canonical mammalian macrophages72. Knockdown of another
A zinc-finger-containing protein rAb5 GEFs in the nematode genome, all of which are known rAb5 effector, F22G12.4 (the nematode ortho
motif that binds to the vPs9domaincontaining proteins41: rME6, rAbX5 logue of rabankyrin), which is required for maturation
membrane lipid phosphatidyl- and TAG333 (orthologues of mammalian GAPEX5, of pinocytosed particles73, suggested that this protein
inositol-3-phosphate. This
results in the targeting of the
rAbEX5 and rIn1, respectively). However, not one of is dispensable for maturation41. Moreover, a targeted
FYVe-containing protein to these proteins is required (either singly or redundantly) screen of all FYvEdomaincontaining proteins in the
the membrane. for the removal of apoptotic cells, suggesting that a novel genome failed to identify other candidate effectors.
>ciZgcVa^oVi^dc
6edeidi^XXZaa
9ncVb^c9NC"&
KEH()
G67*Ä<9E
G67*Ä<IE
G67,Ä<9E
EiY>ch(E
GVW<9>
:[[ZXidg4 E]V\dhdbZ
G67*
gZXgj^ibZci
G67*
VXi^kVi^dc
G67,gZXgj^ibZci
dgZmX]Vc\Z
:[[ZXidg
gZXgj^ibZci
Figure 2 | activation of rab5 during phagosome maturation. During internalization of apoptotic cells, dynamin and
vacuolar sorting protein-34 (VPS34) are recruited to the phagosome by an unknown mechanism. VPS34 can interact with
inactive (GDP bound) or active (GTP bound) RAB5; the recruitment of dynamin to theCVijgZGZk^ZlhqBdaZXjaVg8Zaa7^dad\n
forming phagosome results in the
recruitment of VPS34 and RAB5–GDP to the phagosome41. Dynamin (and DYN-1 in nematodes) has a role in the
maintenance of the actin cytoskeleton; this role also has implications for phagocytosis41,152. Once on the phagosome, an
unidentified guanine nucleotide-exchange factor (GEF) probably activates RAB5 (converting it to the GTP-bound state),
thereby activating VPS34 (ReF. 61), a kinase that generates phosphatidylinositol-3-phosphate (PtdIns3P) on the
phagosome. It is noteworthy that GAPEX5 has been identified as a possible GEF for RAB5 in one mammalian cell context61.
Effector proteins bind to active RAB5 in the context of PtdIns3P on the phagosome57; the assembly of unidentified
effectors on the phagosome results in the exchange of RAB5 for RAB7 and leads to the continued maturation of the
phagosome. RabGDI, Rab GDP-dissociation inhibitor.
Phox homology (PX) domain other phospholipidbinding proteins, such as those In summary, it seems that the function and regulation
A lipid- and protein-interaction containing the phox homology (PX) domain, have been of rAb5 during phagosome maturation is distinct from
domain that consists of described to function in endocytic events and can bind other endocytic events. Intriguingly, studies in mammalian
100–130 amino acids and is to PtdIns3P. Targeted screening of these factors identi macrophages have suggested that Mycobacterium species
defined by sequences that are
found in two components of
fied two previously uncharacterized genes with weak target EEA1 as a means of arresting phagosome matura
the phagocyte NADPH oxidase defects in cell corpse removal, but could not place these tion72, which suggests that the requirements for some
(phox) complex. genes within the pathway for phagosome maturation41. proteins might vary depending on the phagosome cargo.
Efficient targeting of vesicles to the phagosome (and thereby potentially altering signalling and disrupting
appropriate acidification) also has important implica maturation. However, our knowledge of how early
tions for antigen presentation. Dendritic cells isolated steps in phagocytosis dictate later events of phagosome
from rAb27Adeficient mice show defects in antigen maturation is minimal.
presentation related to overacidification of the phago
some87. Another factor, noX2, is an nADPH oxidase Do phagocytic receptors control phagosome maturation?
that generates reactive oxygen species, contributing to The contribution of proteins that are involved in apoptotic
the alkalinization of the phagosome88. Fusion of latex cell phagocytosis to phagosome maturation was recently
beadcontaining phagosomes with noX2positive addressed. In engulfmentdeficient worms (for example,
structures slows acidification, allowing the controlled a ced‑1 or ced‑10 mutant), the efficiency of phago
generation of antigenic peptides and loading onto MHC cytosis is greatly decreased, although many cells are
molecules. Loss of rAb27A, which is required for this eventually engulfed53,98. using timelapse microscopy of
fusion event, leads to overacidified latex beadcontaining embryogenesis as a model, it was shown that CED1,
phagosomes, thereby greatly reducing efficiency of a transmembrane protein that is thought to function as a
antigen presentation 87. This work emphasizes the phagocytic receptor (BOX 1), was required for the recruit
complex sorting requirements of phagosomes and the ment of a FYvEdomain marker and of rAb7 to the
importance of rab proteins to phagosome maturation phagosome53. This work also suggested that another key
(BOX 3). other studies have suggested that this method of engulfment protein, CED5 (DoCK180 in mammals; a
delayed acidification might also be relevant in apoptotic racGEF that functions in an alternate engulfment path
cell removal, as apoptotic cellcontaining phagosomes way downstream of an unknown receptor), also showed
are acidified more slowly in dendritic cells compared reduced phagosomal PtdIns3P and a mild delay in
with macrophages89. rAb7 recruitment28. studies of apoptotic cell removal
in mammalian macrophages have shown that signalling
LAMP proteins and lysosome fusion. LAMP1 and LAMP2 from rhoA (which negatively regulates apoptotic cell
(and the nematode orthologues LMP1 and LMP2) removal99) and ErM (ezrin–radixin–moesin) proteins
are glycoproteins that are specifically localized to acidic also have a role in the timely recruitment of rAb7 to
lysosome structures90, although the precise molecular the phagosome89, suggesting that there might be feed
functions of these proteins are unknown. LAMP1 and back between phagosome maturation and apoptotic cell
LAMP2 (and nematode LMP1) are recruited to the uptake. Indeed, studies using nondigestible latex beads
phagosome; intriguingly, in Lmp1–/– Lmp2–/–‑mutant have shown that the inability to degrade a target can
mice, phagosomes that contain IgGopsonized beads result in decreased uptake100.
appear arrested at the rAb5positive, PtdIns3Ppositive Many of the studies of this process focus on localiza
stage91, which suggests that these two proteins have roles tion of rAb7 or other lysosomal markers (rather than
in the recruitment of rAb7 to the phagosome. rAb7 on earlier markers such as rAb5 or vPs34). studies on
and its effector rILP can be found on LAMPpositive endocytosis have revealed that internalized receptors
structures91, although a molecular mechanism for LAMP can modulate rAb5 activity on vesicles93,94, and one
function during phagosome maturation remains to be tantalizing possibility is that a receptor (or coreceptor)
determined. would recruit rAb5 or a rAb5regulatory machinery
(such as a GEF or GTPaseactivating protein (GAP)) to
Phagocytic receptors and maturation the phagosome. It is intriguing to note that the punc
Conceptually, recognition of the target during phago tate localization of dynamin on the phagosome appears
cytosis is a desirable signalling mechanism for distin similar to the localization pattern of phosphatidylserine
guishing cargo. Therefore, the possibility that phagocytic (Ps) or LrP1 on the phagocytic cup41 (Ps is exposed
receptors could have a key role in determining how a by the apoptotic cell during apoptosis and LrP1 is a
given target is processed for degradation is enticing. phagocytic receptor that is required for apoptotic cell
In endocytosis, ligandbound receptors (for example, removal). These localization patterns suggest that the
epidermal growth factor receptor) have been shown to recruitment of proteins involved in phagosome matur
control rAb5 recruitment, although the mechanisms ation might indeed be mediated by receptors. studies
by which this occurs are sketchy92–94, and the nerve in mammalian systems have also suggested that GuLP,
growth factor receptor actively signals from the endo a mammalian orthologue of the adaptor protein CED6
some to activate the ras–MAPK signalling pathway95. (which binds to the phagocytic receptor CED1 (LrP1
signalling during internalization might be particularly in mammals; see BOX 1)), can interact with the endocytic
important for generating either a tolerogenic (apoptotic machinery102,103. Whether this is a mechanism for recy
cells) or an immunogenic (bacteria) immune response. cling CED1 to the cell surface or a mechanism to recruit
recent studies in both nematode53 and mouse models96,97 maturation proteins remains to be addressed.
have led to the proposal of a ‘phagosome autonomous’ one caveat with these studies is that they do not
model, in which signalling proteins that are enriched address how delays in phagocytosis might affect phago
on the phagosome during internalization determine how some maturation. For example, one recent study has
the phagosome will mature. This becomes especially shown that microtubules are required for delivery of
relevant in the case of internalized pathogens, some GAPEX5 (a GEF for rAb5) to the phagosome61. In
of which can inject factors outside of the phagosome, this respect, a delay in microtubule attachment to the
phagosome could affect the timing of maturation with the phagocyte and/or phagosome maturation dictate
out a ‘direct’ role in signalling. Addressing this issue is how the antigens derived from the cargo would be pro
likely to be difficult and might require the identification cessed. This influence on phagosome maturation could
of mutant proteins that can affect phagosome maturation have important implications for immune responses to
but leave phagocytosis intact. In the case of CED1 (LrP1 antigens that are derived from the target.
in mammals), one study has analysed functional domains similar to mammals, the fly has developed primi
of the protein that are required for cell corpse removal104. tive innate and cellular immune systems to target and
Further clarification of whether higher numbers of remove both foreign and self particles109. A number of
apoptotic cells in this case represent defects in uptake genes that encode phagocytic receptors have been iden
versus maturation would strengthen the hypothesis tified in the fly, including croquemort (for the removal
that these proteins direct phagosome maturation. of apoptotic cells and Staphylococcus aureus)110,111, draper
(apoptotic cells and injured axons)112,113, eater (S. aureus,
Mammalian TLRs and phagosome maturation. Toll-like Escherichia coli and Serratia marcescans)114, nimrod C1
receptors (TLrs) have many functions and can recog (S. aureus)115 and peste (Mycobacterium fortuitum)116.
nize multiple bacterial antigens to mediate their effects. The corresponding receptors have yet to be linked to
During phagocytosis, TLrs serve as coreceptors for phagosome maturation in the fly, but orthologues of the
bacteria (in conjunction with a primary receptor such Croquemort protein in mammals (CD36) have been
as CD14 or CD36)105; typically, TLrs do not function as linked to TLr2 and TLr6 signalling, which suggests
phagocytic receptors, but instead induce macrophage that these receptors might also modulate phagosome
activation in response to bacterial antigens, such as maturation in the fly. In addition, TLrs have been impli
lipopolysaccharide (LPs), resulting in the induction of cated in the innate immune response to bacteria in the
an inflammatory response106. Most importantly in this nematode: tol‑1‑deficient worms show significant inva
context, TLr2 and TLr4 seem to control phagosome sion of Salmonella species into the pharynx117. Whether
maturation and steer the bacterial cargo into the ‘fast this is true ‘invasion’ or due to deficiencies in phagosome
lane’ for both antigen presentation and the induction maturation and bacterial degradation remains to be
of an inflammatory immune response96. When bacteria determined.
and apoptotic cells (which do not engage TLrs) are co
incubated with phagocytes, the different targets are not Overlap between signalling pathways. The outcomes of
present within the same phagosome, and each target recognizing bacteria (immunogenic response) and apop
matures at a different rate. It is important to note that totic cells (tolerogenic response) are different; the differ
fasttracking a phagosome might require multifactorial ential activation of signalling during both phagocytosis
signalling events, as studies have shown that the addi and phagosome maturation might be the molecular basis
tion of TLr agonists alone is insufficient to enhance the of this difference. Little is known about how apoptotic
maturation of IgGopsonized beads and other partic cells generate a tolerogenic state, but much work has gone
les107, although this needs to be addressed in greater into the identification of signalling pathways that regu
detail in vivo. late the inflammatory response to bacteria (reviewed in
one of the consequences at the end of phagosome ReFs 105,118). The activation of MHC class II molecules
maturation is the generation of peptide antigens that are on bacterial phagosomes (but not phagosomes that con
subsequently loaded onto MHC molecules and targeted tain apoptotic cells) supports distinct signalling during
to the cell surface38,108. The differential maturation of each process97. The molecular basis for this difference is
phagosomes seems to be an important regulatory mecha difficult to understand because MHC class II molecules
nism that excludes apoptotic cell antigens from class II are present on phagosomes that contain apoptotic cells97,
presentation (and avoids the subsequent stimulation of a but apoptotic cellderived antigens are not loaded; this
selfreactive response). MHC class II molecules are typi suggests a negative regulatory event. other signalling
cally activated by a proteolyic event on the phagosome events on the phagosome seem to be conserved: the
(as a result of cathepsin activation37); engagement of TLr GTPases rAb5 and rAb7 are present on both bacteria
signalling and fasttrack maturation results in steps that and apoptotic cellcontaining phagosomes; further, both
favour MHC class II loading, leading to phagocyte activa types of phagosomes accumulate and lose PtdIns3P dur
tion and induction of an immune response. Apoptotic ing maturation and require vPs34 (ReFs 8,9,15,41,58).
cellcontaining phagosomes do not possess peptide How these rab proteins are regulated during bacterial
loaded MHC class II, and apoptotic cell antigens are not phagocytosis has yet to be addressed.
presented on the cell surface on class II molecules97.
normally, peptides derived from antigens that enter Escaping a timely death
an antigenpresenting cell (for example, macrophages The immune system is incredibly efficient at seeking
or dendritic cells) are presented on class II MHC mole out and destroying potential pathogens. However, cer
cules. However, it is well documented that antigens tain pathogens have developed mechanisms to escape
derived from apoptotic cells are presented by a pro degradation, persisting (and often replicating) within
Toll-like receptor cess referred to as antigen crosspresentation on class I phagosomes by coopting the phagosomematuration
A transmembrane protein that
recognizes bacterial pathogens
MHC molecules, which generally present Erderived or machinery (TABLe 2) . Legionella pneumophila, for
and induces an inflammatory endogenously produced proteins11. This again suggests example, changes the composition of the phagosomal
response. that early steps in the recognition of targets entering membrane so that it contains many features of the Er,
Table 2 | Bacteria have evolved mechanisms to modify the phagosome Where do we go from here?
The recent identification of genes and proteins involved
bacteria mode of action refs
in phagosome maturation is of key importance to basic
Brucella abortus Converts the phagosome into an 156,157 science and for the understanding of bacterial patho
autophagosome or ER-like vesicle genesis and drug design. unbiased genetic screening
Chlamydia spp. Form inclusions that are enriched in 158,159 approaches have identified >60 proteins that are poten
sphingomyelin tially involved in phagosome maturation in C. elegans.
Coxiella burnetii Converts phagosome into a hybrid 160,161 Further characterization of proteins identified by genetic
autophagosome–RAB7-positive and proteomics approaches and testing them in mam
phagosome malian systems is likely to shed light on the control of
Cryptococcus neoformans Phagosome is extruded from living cells by 162,163 phagosome maturation.
an unknown mechanism Most studies have focused on the acidification of
Helicobacter pylori Sequesters RAB7 to block delivery of 164,165 the phagosomes as the primary readout. However,
proteases there are a number of other steps in phagosome matu
Legionella pneumophila Converts phagosome into rough ER 120–122 ration, such as the accumulation of specific proteins
Leishmania spp. Inhibits phagosome maturation 166,167 on the phagosome (for example, ATPases involved in
phagosome acidification, myosin motors and acidic
Mycobacterium spp. Block at the RAB5-positive stage 168 proteases89,125,126) or other steps in processing (BOX 2),
Salmonella spp. Blocks maturation at the RAB5-positive 166,169 that could provide new insights. Indeed, a recent study
phagosome, potentially by influencing in zebrafish in which v0 ATPase function was moni
RAB7 function
tored revealed a novel role for the protein in phagosome
Toxoplasma spp. Phagosome has characteristics of host 164,165 fusion50, although the molecular details (or the stage at
membrane (non-receptor-mediated
which the phagosome is arrested) remain to be defined.
uptake)
Moreover, whereas significant attention has been paid
ER, endoplasmic reticulum.
to the rab family of GTPases in phagosome matura
tion (and justifiably so, given the importance of rab
allowing it to persist and replicate within the cell119. To GTPases in vesicular traffic in other systems; BOX 3), the
achieve this, the bacteria have developed proteins that role of the ARF GTPases, another family that is linked to
act specifically on rAb1, although how this leads to vesicular traffic in cells, is less well defined. As one of the
phagosome transformation is not well understood120–122. engulfment proteins, GuLP, has been linked to ArF6
Many other bacteria similarly modify signalling mediated signalling103, this might be worthy of future
to block their degradation within the cell (TABLe 2). investigation. similarly, the role of microtubules, which
Furthermore, the yeast Cryptococcus neoformans, have recently been suggested to have a role in delivery
rather than blocking phagosome maturation, can induce of a rAb5 GEF to the phagosome61 during phagosome
expulsion of the phagosome from the cell. one can maturation, needs more detailed investigation.
speculate that this yeast might convert the phagosome Another important topic is whether phagosome
into an exocytic vesicle, although the mechanism of how maturation and autophagy might share a common
this would be accomplished is unknown. mechanism. both processes share a similar aetiology:
Intriguingly, recent work has shown that one patho the degradation of a target (internalized particles or
gen, Shigella species, seems to use some of the proteins organelles, respectively) within a doublemembraned
required for cell corpse removal, for example ELMo1 vesicle. Markers for autophagy (such as LC3 and beclin)
and DoCK180, for entry into the mammalian cells123 are recruited to mammalian phagosomes127, and loss of
(BOX 1). As phagocytic receptors and coreceptors have autophagy protein5 (ATG5) or ATG7 impairs phago
been linked to maturation events in other contexts, and some acidification. How these proteins are recruited to
ced‑5 is required for aspects of phagosome maturation the phagosome (and whether they overlap with rAb5,
in the nematode53, it is possible that some bacteria might rAb7 or LAMP1) has not been addressed. However,
use the apoptotic celluptake machinery to disguise itself these studies suggest that the view of phagosome
and avoid detection. A recent study showed that vaccinia maturation as the movement from a phagosome into a
virus might disguise itself in a Ps coat to mimic an apop lysosomal structure is simplistic, and signalling might
totic cell124, but the consequence of such Psdependent be more complex than envisioned.
entry to phagosome maturation and presentation of Current knowledge of how bacteria can adapt phago
bacterial or viral epitopes is not yet defined. cytic and phagosomal signalling to their own designs
one of the major problems in designing drugs to treat is sketchy (BOX 3), and further research on this topic is
diseases that are associated with these pathogens is our important for the generation of novel therapeutic tar
poor understanding of how they adapt the phagosome to gets. removal of apoptotic cells is important for innate
ARF GTPases their life cycle. A more thorough evaluation of the func immunity, and defects in apoptotic cell phagocytosis and
(ADP-ribosylation factor). tional role of different proteins on the phagosome, and degradation have been shown to result in autoimmune
A family of small GTPases that an assembly of these proteins into a coherent pathway, disease. Comparative studies on how apoptotic cells
regulate aspects of membrane
trafficking that are related to
is of key importance to the development of therapeutics and bacteria are degraded is therefore of great clinical
the budding of vesicles from that can restore ‘normal’ phagosome maturation, rather importance, and further studies into the basic biology of
membranes. than inhibiting bacterial growth or replication. phagosome maturation are crucial to the identification
of relevant therapeutic targets for persistent bacterial In particular, the rab GTPases (BOX 3) might be the best
diseases. How signalling proteins that are required for targets for study, and the identification of the diverse
phagosome maturation (TABLe 1) can be coopted by functions of these proteins in phagosome maturation is
bacterial pathogens (TABLe 2) is a topic of great interest. of paramount importance to the field.
1. van Lookeren Campagne, M., Wiesmann, C. & Brown, 18. Wallet, M. A. et al. MerTK is required for apoptotic 40. Beyenbach, K. W. & Wieczorek, H. The V-type H+
E. J. Macrophage complement receptors and cell-induced T cell tolerance. J. Exp. Med. 205, ATPase: molecular structure and function,
pathogen clearance. Cell. Microbiol. 9, 2095–2102 219–232 (2008). physiological roles and regulation. J. Exp. Biol. 209,
(2007). 19. Albert, M. L., Jegathesan, M. & Darnell, R. B. 577–589 (2006).
2. Underhill, D. M. & Ozinsky, A. Phagocytosis of Dendritic cell maturation is required for the cross- 41. Kinchen, J. M. et al. A pathway for phagosome
microbes: complexity in action. Annu. Rev. Immunol. tolerization of CD8+ T cells. Nature Immunol. 2, maturation during engulfment of apoptotic cells.
20, 825–852 (2002). 1010–1017 (2001). Nature Cell Biol. 10, 556–566 (2008).
3. Swanson, J. A. & Hoppe, A. D. The coordination of 20. Ungewickell, E. J. & Hinrichsen, L. Endocytosis: Uses C. elegans as a model to screen for regulators
signaling during Fc receptor-mediated phagocytosis. clathrin-mediated membrane budding. Curr. Opin. Cell of phagosome maturation, identifies a genetic
J. Leukoc. Biol. 76, 1093–1103 (2004). Biol. 19, 417–425 (2007). pathway for phagosome maturation and shows
4. Nimmerjahn, F. & Ravetch, J. V. Fcγ receptors: old 21. Young, A. Structural insights into the clathrin coat. evolutionary conservation of this pathway in
friends and new family members. Immunity 24, Semin. Cell Dev. Biol. 18, 448–458 (2007). mammals. This work also identifies an unexpected
19–28 (2006). 22. Hanyaloglu, A. C. & von Zastrow, M. Regulation of role for dynamin in the recruitment of VPS-34 and
5. Metzstein, M. M., Stanfield, G. M. & Horvitz, H. R. GPCRs by endocytic membrane trafficking and its RAB-5 to the phagosome.
Genetics of programmed cell death in C. elegans: past, potential implications. Annu. Rev. Pharmacol. Toxicol. 42. Clarke, M. et al. Dynamics of the vacuolar H+-ATPase
present and future. Trends Genet. 14, 410–416 48, 537–568 (2008). in the contractile vacuole complex and the endosomal
(1998). 23. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab pathway of Dictyostelium cells. J. Cell Sci. 115,
6. Lettre, G. & Hengartner, M. O. Developmental conversion as a mechanism of progression from early 2893–2905 (2002).
apoptosis in C. elegans: a complex CEDnario. Nature to late endosomes. Cell 122, 735–749 (2005). 43. Fratti, R. A., Chua, J., Vergne, I. & Deretic, V.
Rev. Mol. Cell Biol. 7, 97–108 (2006). This ground-breaking study used time-lapse Mycobacterium tuberculosis glycosylated
References 5 and 6 are an excellent introduction to microscopy to track endocytic vesicles as they phosphatidylinositol causes phagosome maturation
the field of apoptosis in the nematode. Reference 5 acquired the GTPases RAB5 and RAB7. This work arrest. Proc. Natl Acad. Sci. USA 100, 5437–5442
is a classic in the field and is an excellent provides support for the direct exchange of RAB5 (2003).
introduction to the genetics of programmed cell for RAB7 on the endosome; studies in the 44. Fratti, R. A., Vergne, I., Chua, J., Skidmore, J. &
death, whereas reference 6 extends and updates nematode have also observed this ‘Rab conversion’ Deretic, V. Regulators of membrane trafficking and
these findings. during phagosome maturation. Mycobacterium tuberculosis phagosome
7. Taylor, R. C., Cullen, S. P. & Martin, S. J. Apoptosis: 24. Russell, M. R., Nickerson, D. P. & Odorizzi, G. maturation block. Electrophoresis 21, 3378–3385
controlled demolition at the cellular level. Nature Rev. Molecular mechanisms of late endosome morphology, (2000).
Mol. Cell Biol. 9, 231–241 (2008). identity and sorting. Curr. Opin. Cell Biol. 18, 45. McCoy, J. J. & Mann, B. J. Proteomic analysis of
8. Ravichandran, K. S. & Lorenz, U. Engulfment of 422–428 (2006). Gal/GalNAc lectin-associated proteins in Entamoeba
apoptotic cells: signals for a good meal. Nature Rev. 25. Luzio, J. P. et al. Lysosome–endosome fusion and histolytica. Exp. Parasitol. 110, 220–225
Immunol. 7, 964–974 (2007). lysosome biogenesis. J. Cell Sci. 113, 1515–1524 (2005).
9. Miyake, Y. et al. Critical role of macrophages in the (2000). 46. Okada, M. et al. Proteomic analysis of phagocytosis in
marginal zone in the suppression of immune 26. van Ijzendoorn, S. C. Recycling endosomes. J. Cell Sci. the enteric protozoan parasite Entamoeba histolytica.
responses to apoptotic cell-associated antigens. 119, 1679–1681 (2006). Eukaryot. Cell 4, 827–831 (2005).
J. Clin. Invest. 117, 2268–2278 (2007). 27. Maxfield, F. R. & McGraw, T. E. Endocytic recycling. 47. Okada, M. et al. Kinetics and strain variation of
10. Fadok, V. A. et al. Macrophages that have ingested Nature Rev. Mol. Cell Biol. 5, 121–132 (2004). phagosome proteins of Entamoeba histolytica by
apoptotic cells in vitro inhibit proinflammatory 28. Yu, X., Odera, S., Chuang, C. H., Lu, N. & Zhou, Z. proteomic analysis. Mol. Biochem. Parasitol. 145,
cytokine production through autocrine/paracrine C. elegans dynamin mediates the signaling of 171–183 (2006).
mechanisms involving TGF-β, PGE2, and PAF. J. Clin. phagocytic receptor CED-1 for the engulfment and 48. Jutras, I. et al. Modulation of the phagosome
Invest. 101, 890–898 (1998). degradation of apoptotic cells. Dev. Cell 10, 743–757 proteome by interferon-γ. Mol. Cell. Proteomics 7,
The first characterization of the anti-inflammatory (2006). 697–715 (2008).
cytokine response that is elicited by apoptotic cell 29. Lee, W. L., Mason, D., Schreiber, A. D. & Grinstein, S. 49. Botelho, R. J., Hackam, D. J., Schreiber, A. D. &
recognition by phagocytes. This response occurs in Quantitative analysis of membrane remodeling at the Grinstein, S. Role of COPI in phagosome maturation.
both professional and non-professional phagocytes. phagocytic cup. Mol. Biol. Cell 18, 2883–2892 J. Biol. Chem. 275, 15717–15727 (2000).
11. Bellone, M. et al. Processing of engulfed apoptotic (2007). 50. Peri, F. & Nusslein-Volhard, C. Live imaging of
bodies yields T cell epitopes. J. Immunol. 159, 30. Albert, M. L. Death-defying immunity: do apoptotic neuronal degradation by microglia reveals a role for
5391–5399 (1997). cells influence antigen processing and presentation? V0-ATPase A1 in phagosomal fusion in vivo. Cell 133,
12. Huang, F. P. et al. A discrete subpopulation of Nature Rev. Immunol. 4, 223–231 (2004). 916–927 (2008).
dendritic cells transports apoptotic intestinal epithelial 31. Blander, J. M. Signalling and phagocytosis in the 51. Touret, N. et al. Quantitative and dynamic assessment
cells to T cell areas of mesenteric lymph nodes. J. Exp. orchestration of host defence. Cell. Microbiol. 9, of the contribution of the ER to phagosome formation.
Med. 191, 435–444 (2000). 290–299 (2007). Cell 123, 157–170 (2005).
13. Albert, M. L. et al. Immature dendritic cells 32. Akira, S. & Takeda, K. Toll-like receptor signalling. 52. Lu, Q. et al. C. elegans Rab GTPase 2 is required for
phagocytose apoptotic cells via αvβ5 and CD36, and Nature Rev. Immunol. 4, 499–511 (2004). the degradation of apoptotic cells. Development 135,
cross-present antigens to cytotoxic T lymphocytes. 33. Desjardins, M., Huber, L. A., Parton, R. G. & Griffiths, G. 1069–1080 (2008).
J. Exp. Med. 188, 1359–1368 (1998). Biogenesis of phagolysosomes proceeds through a 53. Yu, X., Lu, N. & Zhou, Z. Phagocytic receptor CED-1
Defects in apoptotic cell removal have long been sequential series of interactions with the endocytic initiates a signaling pathway for degrading engulfed
associated with autoimmune disease; it was apparatus. J. Cell Biol. 124, 677–688 (1994). apoptotic cells. PLoS Biol. 6, e61 (2008).
previously thought that the release of cellular 34. Garin, J. et al. The phagosome proteome: insight into 54. Mangahas, P. M., Yu, X., Miller, K. G. & Zhou, Z. The
contents led to the generation of an immune phagosome functions. J. Cell Biol. 152, 165–180 small GTPase Rab2 functions in the removal of
response. This study suggests that there are (2001). apoptotic cells in Caenorhabditis elegans. J. Cell Biol.
defects in the establishment or maintenance of 35. Stuart, L. M. et al. A systems biology analysis of the 180, 357–373 (2008).
tolerance rather than in the induction of an immune Drosophila phagosome. Nature 445, 95–101 References 52–54 introduce time-lapse microscopy
response. (2007). to the study of phagosome maturation in the whole
14. Swanson, J. A. Shaping cups into phagosomes and 36. Lennon-Dumenil, A. M. et al. Analysis of protease organism and identify UNC-108/RAB-2
macropinosomes. Nature Rev. Mol. Cell Biol. 9, activity in live antigen-presenting cells shows (references 52 and 54) and phagocytic signalling
639–649 (2008). regulation of the phagosomal proteolytic contents (reference 53) as being important for phagosome
15. Smith, A. C. et al. A network of Rab GTPases controls during dendritic cell activation. J. Exp. Med. 196, maturation.
phagosome maturation and is modulated by 529–540 (2002). 55. Ullrich, O., Horiuchi, H., Bucci, C. & Zerial, M.
Salmonella enterica serovar Typhimurium. J. Cell Biol. 37. Bryant, P. W., Lennon-Dumenil, A. M., Fiebiger, E., Membrane association of Rab5 mediated by GDP-
176, 263–268 (2007). Lagaudriere-Gesbert, C. & Ploegh, H. L. Proteolysis dissociation inhibitor and accompanied by GDP/GTP
Uses dominant-negative approaches to identify the and antigen presentation by MHC class II molecules. exchange. Nature 368, 157–160 (1994).
GTPases that are required for the maturation of Adv. Immunol. 80, 71–114 (2002). 56. Seabra, M. C. & Wasmeier, C. Controlling the location
Salmonella-containing phagosomes. 38. Ramachandra, L., Boom, W. H. & Harding, C. V. Class and activation of Rab GTPases. Curr. Opin. Cell Biol.
16. Beertsen, W. et al. Impaired phagosomal maturation II MHC antigen processing in phagosomes. Methods 16, 451–457 (2004).
in neutrophils leads to periodontitis in lysosomal- Mol. Biol. 445, 353–377 (2008). 57. Grosshans, B. L., Ortiz, D. & Novick, P. Rabs and their
associated membrane protein-2 knockout mice. 39. Hackam, D. J. et al. Regulation of phagosomal effectors: achieving specificity in membrane traffic.
J. Immunol. 180, 475–482 (2008). acidification. Differential targeting of Na+/H+ Proc. Natl Acad. Sci. USA 103, 11821–11827
17. Kawane, K. et al. Chronic polyarthritis caused by exchangers, Na+/K+-ATPases, and vacuolar-type (2006).
mammalian DNA that escapes from degradation in H+-ATPases. J. Biol. Chem. 272, 29810–29820 An excellent review of Rab effectors and their
macrophages. Nature 443, 998–1002 (2006). (1997). functions.
58. Vieira, O. V. et al. Modulation of Rab5 and Rab7 through the late endosomal/lysosomal compartment. 100. Schrijvers, D. M., De Meyer, G. R., Kockx, M. M.,
recruitment to phagosomes by phosphatidylinositol Traffic 9, 678–694 (2008). Herman, A. G. & Martinet, W. Phagocytosis of
3-kinase. Mol. Cell. Biol. 23, 2501–2514 (2003). 80. Sun, J. et al. Mycobacterium bovis BCG disrupts the apoptotic cells by macrophages is impaired in
Shows the requirements of kinase function (as interaction of Rab7 with RILP contributing to atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 25,
inhibited by wortmannin) for fusion of phagosomes inhibition of phagosome maturation. J. Leukoc. Biol. 1256–1261 (2005).
with late endosomes or lysosomes. Intriguingly, 82, 1437–1445 (2007). 101. Gardai, S. J. et al. Cell-surface calreticulin initiates
RAB7 can still be recruited to the phagosome, 81. Tisdale, E. J. A Rab2 mutant with impaired GTPase clearance of viable or apoptotic cells through trans-
supporting a model for ‘Rab conversion’ on the activity stimulates vesicle formation from pre-Golgi activation of LRP on the phagocyte. Cell 123,
phagosome. intermediates. Mol. Biol. Cell 10, 1837–1849 (1999). 321–334 (2005).
59. McBride, H. M. et al. Oligomeric complexes link Rab5 82. Tisdale, E. J. & Balch, W. E. Rab2 is essential for the 102. Martins-Silva, C. et al. A rat homologue of CED-6 is
effectors with NSF and drive membrane fusion via maturation of pre-Golgi intermediates. J. Biol. Chem. expressed in neurons and interacts with clathrin. Brain
interactions between EEA1 and syntaxin 13. 271, 29372–29379 (1996). Res. 1119, 1–12 (2006).
Cell 98, 377–386 (1999). 83. Tisdale, E. J., Bourne, J. R., Khosravi-Far, R., Der, C. J. 103. Ma, Z., Nie, Z., Luo, R., Casanova, J. E. &
60. Roberts, E. A., Chua, J., Kyei, G. B. & Deretic, V. & Balch, W. E. GTP-binding mutants of rab1 and rab2 Ravichandran, K. S. Regulation of Arf6 and ACAP1
Higher order Rab programming in phagolysosome are potent inhibitors of vesicular transport from the signaling by the PTB-domain-containing adaptor
biogenesis. J. Cell Biol. 174, 923–929 (2006). endoplasmic reticulum to the Golgi complex. J. Cell protein GULP. Curr. Biol. 17, 722–727 (2007).
61. Kitano, M., Nakaya, M., Nakamura, T., Nagata, S. & Biol. 119, 749–761 (1992). 104. Zhou, Z., Hartwieg, E. & Horvitz, H. R. CED-1 is a
Matsuda, M. Imaging of Rab5 activity identifies 84. Chun, D. K., McEwen, J. M., Burbea, M. & transmembrane receptor that mediates cell corpse
essential regulators for phagosome maturation. Kaplan, J. M. UNC-108/Rab2 regulates post- engulfment in C. elegans. Cell 104, 43–56 (2001).
Nature 453, 241–245 (2008). endocytic trafficking in C. elegans. Mol. Biol. Cell 19, 105. Underhill, D. M. & Gantner, B. Integration of Toll-like
Uses a Raichu–RAB5 biosensor probe to monitor 2682–2695 (2008). receptor and phagocytic signaling for tailored
RAB5 activation during apoptotic cell removal, and 85. Schuck, S. et al. Rab10 is involved in basolateral immunity. Microbes Infect. 6, 1368–1373 (2004).
suggests that GAPEX5 is a major player in RAB5 transport in polarized Madin–Darby canine kidney 106. Taylor, P. R. et al. Macrophage receptors and immune
activation during lactadherin and MFG-E8- cells. Traffic 8, 47–60 (2007). recognition. Annu. Rev. Immunol. 23, 901–944
mediated uptake. 86. Chen, C. C. et al. RAB-10 is required for endocytic (2005).
62. Huynh, K. K. & Grinstein, S. Phagocytosis: dynamin’s recycling in the Caenorhabditis elegans intestine. Mol. 107. Yates, R. M. & Russell, D. G. Phagosome maturation
dual role in phagosome biogenesis. Curr. Biol. 18, Biol. Cell 17, 1286–1297 (2006). proceeds independently of stimulation of toll-like
R563–R565 (2008). 87. Jancic, C. et al. Rab27a regulates phagosomal pH and receptors 2 and 4. Immunity 23, 409–417
63. Vieira, O. V. et al. Distinct roles of class I and class III NADPH oxidase recruitment to dendritic cell (2005).
phosphatidylinositol 3-kinases in phagosome phagosomes. Nature Cell Biol. 9, 367–378 (2007). Questions the phagosome-autonomous model and
formation and maturation. J. Cell Biol. 155, 19–25 Highlights the importance of trafficking events for instead suggests that maturation does not depend
(2001). processing of internalized cargo: trafficking of on signalling from Toll-like receptors.
64. Bucci, C. et al. The small GTPase rab5 functions as a RAB27A-positive vesicles to the phagosome slows 108. Houde, M. et al. Phagosomes are competent
regulatory factor in the early endocytic pathway. Cell acidification, allowing efficient proteolysis of organelles for antigen cross-presentation. Nature
70, 715–728 (1992). proteins and loading of antigens for presentation. 425, 402–406 (2003).
65. Hanayama, R. et al. Autoimmune disease and 88. Savina, A. et al. NOX2 controls phagosomal pH to 109. Stuart, L. M. & Ezekowitz, R. A. Phagocytosis and
impaired uptake of apoptotic cells in MFG-E8-deficient regulate antigen processing during crosspresentation comparative innate immunity: learning on the fly.
mice. Science 304, 1147–1150 (2004). by dendritic cells. Cell 126, 205–218 (2006). Nature Rev. Immunol. 8, 131–141 (2008).
66. Nielsen, E. et al. Rabenosyn-5, a novel Rab5 effector, 89. Erwig, L. P. et al. Differential regulation of phagosome 110. Franc, N. C., Heitzler, P., Ezekowitz, R. A. & White, K.
is complexed with hVPS45 and recruited to maturation in macrophages and dendritic cells Requirement for croquemort in phagocytosis of
endosomes through a FYVE finger domain. J. Cell Biol. mediated by Rho GTPases and ezrin–radixin–moesin apoptotic cells in Drosophila. Science 284,
151, 601–612 (2000). (ERM) proteins. Proc. Natl Acad. Sci. USA 103, 1991–1994 (1999).
67. Christoforidis, S., McBride, H. M., Burgoyne, R. D. & 12825–12830 (2006). 111. Franc, N. C., Dimarcq, J. L., Lagueux, M., Hoffmann, J.
Zerial, M. The Rab5 effector EEA1 is a core The first characterization of maturation relating to & Ezekowitz, R. A. Croquemort, a novel Drosophila
component of endosome docking. Nature 397, apoptotic cell removal. It also suggests a link hemocyte/macrophage receptor that recognizes
621–625 (1999). between RhoA (the activity of which is apoptotic cells. Immunity 4, 431–443 (1996).
68. Simonsen, A. et al. EEA1 links PI(3)K function to Rab5 downregulated during apoptotic cell engulfment) 112. Awasaki, T. et al. Essential role of the apoptotic cell
regulation of endosome fusion. Nature 394, 494–498 and ERM-family proteins for phagosome engulfment genes draper and ced-6 in programmed
(1998). maturation. axon pruning during Drosophila metamorphosis.
69. Gengyo-Ando, K. et al. The SM protein VPS-45 is 90. Eskelinen, E. L., Tanaka, Y. & Saftig, P. At the acidic Neuron 50, 855–867 (2006).
required for RAB-5-dependent endocytic transport in edge: emerging functions for lysosomal membrane 113. Freeman, M. R., Delrow, J., Kim, J., Johnson, E. &
Caenorhabditis elegans. EMBO Rep. 8, 152–157 proteins. Trends Cell Biol. 13, 137–145 (2003). Doe, C. Q. Unwrapping glial biology: Gcm target genes
(2007). 91. Huynh, K. K. et al. LAMP proteins are required for regulating glial development, diversification, and
70. Vitale, G. et al. Distinct Rab-binding domains mediate fusion of lysosomes with phagosomes. EMBO J. 26, function. Neuron 38, 567–580 (2003).
the interaction of Rabaptin-5 with GTP-bound Rab4 313–324 (2007). 114. Kocks, C. et al. Eater, a transmembrane protein
and Rab5. EMBO J. 17, 1941–1951 (1998). 92. Barbieri, M. A., Fernandez-Pol, S., Hunker, C., mediating phagocytosis of bacterial pathogens in
71. Williams, R. L. & Urbe, S. The emerging shape of the Horazdovsky, B. H. & Stahl, P. D. Role of rab5 in EGF Drosophila. Cell 123, 335–346 (2005).
ESCRT machinery. Nature Rev. Mol. Cell Biol. 8, receptor-mediated signal transduction. Eur. J. Cell 115. Kurucz, E. et al. Nimrod, a putative phagocytosis
355–368 (2007). Biol. 83, 305–314 (2004). receptor with EGF repeats in Drosophila
72. Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S. 93. Barbieri, M. A. et al. Epidermal growth factor and plasmatocytes. Curr. Biol. 17, 649–654 (2007).
& Deretic, V. Role of phosphatidylinositol 3-kinase and membrane trafficking. EGF receptor activation of 116. Philips, J. A., Rubin, E. J. & Perrimon, N. Drosophila
Rab5 effectors in phagosomal biogenesis and endocytosis requires Rab5a. J. Cell Biol. 151, RNAi screen reveals CD36 family member required for
mycobacterial phagosome maturation arrest. J. Cell 539–550 (2000). mycobacterial infection. Science 309, 1251–1253
Biol. 154, 631–644 (2001). 94. Lanzetti, L. et al. The Eps8 protein coordinates EGF (2005).
73. Schnatwinkel, C. et al. The Rab5 effector Rabankyrin-5 receptor signalling through Rac and trafficking 117. Tenor, J. L. & Aballay, A. A conserved Toll-like receptor
regulates and coordinates different endocytic through Rab5. Nature 408, 374–377 (2000). is required for Caenorhabditis elegans innate
mechanisms. PLoS Biol. 2, e261 (2004). 95. Howe, C. L., Valletta, J. S., Rusnak, A. S. & Mobley, immunity. EMBO Rep. 9, 103–109 (2008).
74. Storrie, B. & Desjardins, M. The biogenesis of W. C. NGF signaling from clathrin-coated vesicles: 118. O’Neill, L. A. When signaling pathways collide: positive
lysosomes: is it a kiss and run, continuous fusion and evidence that signaling endosomes serve as a platform and negative regulation of toll-like receptor signal
fission process? Bioessays 18, 895–903 (1996). for the Ras–MAPK pathway. Neuron 32, 801–814 transduction. Immunity 29, 12–20 (2008).
75. Rothman, J. H. & Stevens, T. H. Protein sorting in (2001). 119. Kagan, J. C., Stein, M. P., Pypaert, M. & Roy, C. R.
yeast: mutants defective in vacuole biogenesis 96. Blander, J. M. & Medzhitov, R. Regulation of Legionella subvert the functions of Rab1 and Sec22b
mislocalize vacuolar proteins into the late secretory phagosome maturation by signals from toll-like to create a replicative organelle. J. Exp. Med. 199,
pathway. Cell 47, 1041–1051 (1986). receptors. Science 304, 1014–1018 (2004). 1201–1211 (2004).
76. Peplowska, K., Markgraf, D. F., Ostrowicz, C. W., 97. Blander, J. M. & Medzhitov, R. Toll-dependent 120. Machner, M. P. & Isberg, R. R. Targeting of host Rab
Bange, G. & Ungermann, C. The CORVET tethering selection of microbial antigens for presentation by GTPase function by the intravacuolar pathogen
complex interacts with the yeast Rab5 homolog Vps21 dendritic cells. Nature 440, 808–812 (2006). Legionella pneumophila. Dev. Cell 11, 47–56
and is involved in endo-lysosomal biogenesis. Dev. Cell References 96 and 97 present evidence for a (2006).
12, 739–750 (2007). phagosome-autonomous model for maturation, in 121. Machner, M. P. & Isberg, R. R. A bifunctional bacterial
77. Seals, D. F., Eitzen, G., Margolis, N., Wickner, W. T. & which receptor ligation during internalization protein links GDI displacement to Rab1 activation.
Price, A. A Ypt/Rab effector complex containing the determines the fate of the cargo and whether Science 318, 974–977 (2007).
Sec1 homolog Vps33p is required for homotypic antigens will be presented on MHC class II 122. Ingmundson, A., Delprato, A., Lambright, D. G. & Roy,
vacuole fusion. Proc. Natl Acad. Sci. USA 97, molecules at the cell surface. C. R. Legionella pneumophila proteins that regulate
9402–9407 (2000). 98. Kinchen, J. M. et al. Two pathways converge at Rab1 membrane cycling. Nature 450, 365–369
78. Peterson, M. R., Burd, C. G. & Emr, S. D. Vac1p CED-10 to mediate actin rearrangement and corpse (2007).
coordinates Rab and phosphatidylinositol 3-kinase removal in C. elegans. Nature 434, 93–99 123. Handa, Y. et al. Shigella IpgB1 promotes bacterial
signaling in Vps45p-dependent vesicle (2005). entry through the ELMO–Dock180 machinery. Nature
docking/fusion at the endosome. Curr. Biol. 9, 99. Tosello-Trampont, A. C., Nakada-Tsukui, K. & Cell Biol. 9, 121–128 (2007).
159–162 (1999). Ravichandran, K. S. Engulfment of apoptotic cells is 124. Mercer, J. & Helenius, A. Vaccinia virus uses
79. Starr, T., Ng, T. W., Wehrly, T. D., Knodler, L. A. & Celli, J. negatively regulated by Rho-mediated signaling. macropinocytosis and apoptotic mimicry to enter host
Brucella intracellular replication requires trafficking J. Biol. Chem. 278, 49911–49919 (2003). cells. Science 320, 531–535 (2008).
References 123 and 124 suggest that pathogens The cytokine response of phagocytes to apoptotic 158. Scidmore, M. A., Fischer, E. R. & Hackstadt, T.
can co-opt phagocytic receptors for apoptotic cells cells is typically tolerogenic. However, this paper Sphingolipids and glycoproteins are differentially
to gain entry into the cell. Whether such a mode of suggests that, in certain circumstances, the trafficked to the Chlamydia trachomatis inclusion.
entry also alters phagosome maturation remains to presence of apoptotic cells can elicit an J. Cell Biol. 134, 363–374 (1996).
be determined. inflammatory response. 159. Heinzen, R. A., Scidmore, M. A., Rockey, D. D. &
125. Santic, M., Asare, R., Skrobonja, I., Jones, S. & Abu 142. Gerbod-Giannone, M. C. et al. TNFα induces ABCA1 Hackstadt, T. Differential interaction with endocytic
Kwaik, Y. Acquisition of the vacuolar ATPase proton through NF-κB in macrophages and in phagocytes and exocytic pathways distinguish parasitophorous
pump and phagosome acidification are essential for ingesting apoptotic cells. Proc. Natl Acad. Sci. USA vacuoles of Coxiella burnetii and Chlamydia
escape of Francisella tularensis into the 103, 3112–3117 (2006). trachomatis. Infect. Immun. 64, 796–809 (1996).
macrophage cytosol. Infect. Immun. 76, 143. Blachere, N. E., Darnell, R. B. & Albert, M. L. 160. Romano, P. S., Gutierrez, M. G., Beron, W.,
2671–2677 (2008). Apoptotic cells deliver processed antigen to dendritic Rabinovitch, M. & Colombo, M. I. The autophagic
126. Araki, N. Role of microtubules and myosins in Fc γ cells for cross-presentation. PLoS Biol. 3, e185 pathway is actively modulated by phase II Coxiella
receptor-mediated phagocytosis. Front. Biosci. 11, (2005). burnetii to efficiently replicate in the host cell. Cell.
1479–1490 (2006). 144. Perruche, S. et al. CD3-specific antibody-induced Microbiol. 9, 891–909 (2007).
127. Sanjuan, M. A. et al. Toll-like receptor signalling in immune tolerance involves transforming growth 161. Beron, W., Gutierrez, M. G., Rabinovitch, M. &
macrophages links the autophagy pathway to factor-β from phagocytes digesting apoptotic T cells. Colombo, M. I. Coxiella burnetii localizes in a Rab7-
phagocytosis. Nature 450, 1253–1257 Nature Med. 14, 528–535 (2008). labeled compartment with autophagic characteristics.
(2007). 145. Wu, Y. C., Stanfield, G. M. & Horvitz, H. R. NUC-1, Infect. Immun. 70, 5816–5821 (2002).
128. Gardai, S. J., Bratton, D. L., Ogden, C. A. & Henson, a Caenorhabditis elegans DNase II homolog, 162. Ma, H., Croudace, J. E., Lammas, D. A. & May, R. C.
P. M. Recognition ligands on apoptotic cells: a functions in an intermediate step of DNA degradation Expulsion of live pathogenic yeast by macrophages.
perspective. J. Leukoc. Biol. 79, 896–903 (2006). during apoptosis. Genes Dev. 14, 536–548 Curr. Biol. 16, 2156–2160 (2006).
129. Devitt, A. et al. Persistence of apoptotic cells without (2000). 163. Alvarez, M. & Casadevall, A. Phagosome extrusion
autoimmune disease or inflammation in CD14–/– mice. 146. Parrish, J. Z., Yang, C., Shen, B. & Xue, D. CRN-1, a and host-cell survival after Cryptococcus neoformans
J. Cell Biol. 167, 1161–1170 (2004). Caenorhabditis elegans FEN-1 homologue, phagocytosis by macrophages. Curr. Biol. 16,
130. Reddien, P. W. & Horvitz, H. R. CED-2/CrkII and cooperates with CPS-6/EndoG to promote apoptotic 2161–2165 (2006).
CED-10/Rac control phagocytosis and cell migration in DNA degradation. EMBO J. 22, 3451–3460 164. Terebiznik, M. R. et al. Helicobacter pylori VacA toxin
Caenorhabditis elegans. Nature Cell Biol. 2, 131–136 (2003). promotes bacterial intracellular survival in gastric
(2000). 147. Parrish, J. Z. & Xue, D. Functional genomic analysis of epithelial cells. Infect. Immun. 74, 6599–6614 (2006).
131. Gumienny, T. L. et al. CED-12/ELMO, a novel member apoptotic DNA degradation in C. elegans. Mol. Cell 165. Allen, L. A. Intracellular niches for extracellular
of the CrkII/Dock180/Rac pathway, is required for 11, 987–996 (2003). bacteria: lessons from Helicobacter pylori. J. Leukoc.
phagocytosis and cell migration. Cell 107, 27–41 References 145–147 report the characterization Biol. 66, 753–756 (1999).
(2001). of DNAses that have roles during the degradation 166. Scianimanico, S. et al. Impaired recruitment of the
132. Park, D. et al. BAI1 is an engulfment receptor for of apoptotic cells in the nematode. small GTPase rab7 correlates with the inhibition of
apoptotic cells upstream of the ELMO/Dock180/Rac 148. Gagnon, E. et al. Endoplasmic reticulum-mediated phagosome maturation by Leishmania donovani
module. Nature 450, 430–434 (2007). phagocytosis is a mechanism of entry into promastigotes. Cell. Microbiol. 1, 19–32 (1999).
133. Albert, M. L., Kim, J. I. & Birge, R. B. αvβ5 integrin macrophages. Cell 110, 119–131 (2002). 167. Dermine, J. F., Goyette, G., Houde, M., Turco, S. J. &
recruits the CrkII–Dock180–rac1 complex for 149. Kyei, G. B. et al. Rab14 is critical for maintenance of Desjardins, M. Leishmania donovani lipophosphoglycan
phagocytosis of apoptotic cells. Nature Cell Biol. 2, Mycobacterium tuberculosis phagosome maturation disrupts phagosome microdomains in J774
899–905 (2000). arrest. EMBO J. 25, 5250–5259 (2006). macrophages. Cell. Microbiol. 7, 1263–1270 (2005).
134. Su, H. P. et al. Interaction of CED-6/GULP, an adapter 150. Delprato, A. & Lambright, D. G. Structural basis for 168. Chua, J., Vergne, I., Master, S. & Deretic, V. A tale of
protein involved in engulfment of apoptotic cells with Rab GTPase activation by VPS9 domain exchange two lipids: Mycobacterium tuberculosis phagosome
CED-1 and CD91/low density lipoprotein receptor- factors. Nature Struct. Mol. Biol. 14, 406–412 maturation arrest. Curr. Opin. Microbiol. 7, 71–77
related protein (LRP). J. Biol. Chem. 277, (2007). (2004).
11772–11779 (2002). 151. Cantalupo, G., Alifano, P., Roberti, V., Bruni, C. B. & 169. Meresse, S., Steele-Mortimer, O., Finlay, B. B. &
135. Su, H. P. et al. Identification and characterization of a Bucci, C. Rab-interacting lysosomal protein (RILP): the Gorvel, J. P. The rab7 GTPase controls the maturation
dimerization domain in CED-6, an adapter protein Rab7 effector required for transport to lysosomes. of Salmonella typhimurium-containing vacuoles in
involved in engulfment of apoptotic cells. J. Biol. EMBO J. 20, 683–693 (2001). HeLa cells. EMBO J. 18, 4394–4403 (1999).
Chem. 275, 9542–9549 (2000). 152. Orth, J. D. & McNiven, M. A. Dynamin at the actin-
136. Liu, Q. A. & Hengartner, M. O. Candidate adaptor membrane interface. Curr. Opin. Cell Biol. 15, 31–39 Acknowledgements
protein CED-6 promotes the engulfment of apoptotic (2003). The authors wish to thank colleagues in the engulfment field
cells in C. elegans. Cell 93, 961–972 (1998). This excellent review describes the role of and members of our laboratory for insightful discussions. This
References 130–136 identify intracellular dynamin-2 in actin-related processes, such as work was supported by grants from the National Institute of
signalling molecules that function during apoptotic maintenance of the actin cytoskeleton. General Medical Sciences/National Institutes of Health and
cell removal in the nematode and mammals, and 153. Wu, Y. C. & Horvitz, H. R. The C. elegans cell corpse the Strategic Program for Asthma Research (K.S.R), and an
also provide insights into their mechanism of engulfment gene ced-7 encodes a protein similar to Arthritis Foundation Postdoctoral Fellowship (J.M.K).
action. ABC transporters. Cell 93, 951–960 (1998).
137. Hamon, Y., Chambenoit, O. & Chimini, G. ABCA1 and 154. Wu, Y. C. & Horvitz, H. R. C. elegans phagocytosis
the engulfment of apoptotic cells. Biochim. Biophys. and cell-migration protein CED-5 is similar to DATABASES
Acta 1585, 64–71 (2002). human DOCK180. Nature 392, 501–504 Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
138. Kinchen, J. M. & Ravichandran, K. S. Journey to the (1998). fcgi?db=gene
grave: signaling events regulating removal of 155. Brugnera, E. et al. Unconventional Rac-GEF activity is croquemort | draper | eater | nimrod C1 | peste | vha-16
apoptotic cells. J. Cell Sci. 120, 2143–2149 mediated through the Dock180–ELMO complex. UniProtKB: http://www.uniprot.org
(2007). Nature Cell Biol. 4, 574–582 (2002). DYN-1 | EEA-1 | GAPEX5| LAMP1 | LAMP2 | LRP1| RAB2 |
139. Voll, R. E. et al. Immunosuppressive effects of 156. Pizarro-Cerda, J. et al. Brucella abortus transits RAB5 | RAB7 | RAB27A | RABEX5 | RILP | RIN1 | TLR2 | TLR4 |
apoptotic cells. Nature 390, 350–351 (1997). through the autophagic pathway and replicates in the VPS-34 | VPS-45
140. Park, S. Y. et al. Rapid cell corpse clearance by endoplasmic reticulum of nonprofessional phagocytes.
stabilin-2, a membrane phosphatidylserine receptor. Infect. Immun. 66, 5711–5724 (1998). FURTHER INFORMATION
Cell Death Differ. 15, 192–201 (2008). 157. Pizarro-Cerda, J., Moreno, E., Sanguedolce, V., Mege, Kodi S. Ravichandran’s homepage: http://www.
141. Kirsch, T. et al. Engulfment of apoptotic cells by J. L. & Gorvel, J. P. Virulent Brucella abortus prevents healthsystem.virginia.edu/internet/cmb/immunology.
microvascular endothelial cells induces lysosome fusion and is distributed within cfm?uva_id=kr4h&hideintro=1
proinflammatory responses. Blood 109, 2854–2862 autophagosome-like compartments. Infect. Immun. all links are active in the online PdF
(2007). 66, 2387–2392 (1998).