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PHYSIOLOGY 22: 366–372, 2007; 10.1152/physiol.00028.2007
Fusion, Fission, and Secretion
During Phagocytosis
Jason G.
Kassidy K. Huynh,1*
Jennifer L. Stow,2
and Sergio Grinstein1
Kay,2*
1Division
Phagocytosis is essential for the elimination of pathogens and for clearance of apoptotic bodies. The ingestion process entails extensive remodeling of the cellular membranes, particularly when large and/or multiple particles are engulfed. The mem-
of Cell Biology, The Hospital for Sick Children,
Toronto, Canada; and
2Institute for Molecular Bioscience,
University of Queensland, Brisbane, Queensland, Australia
[email protected]
*Contributed equally to the preparation of this review.
brane fusion and fission events that accompany phagocytosis are described. The
coordinated sequence of membrane trafficking events required for phagocytosis
involves multiple organelles and also serves other cellular functions, such as
cytokine secretion.
366
internalized during engulfment is replaced by membranes derived from an intracellular reservoir.
The fusion of endomembrane vesicles with the plasma membrane is, of course, not unprecedented.
Although regulated exocytosis is most often regarded
as a means of delivering soluble cargo to the extracellular milieu, a variety of instances have been documented where insertion of intrinsic membrane components and the accompanying extra surface area are
the primary objectives. Osteoclasts, specialized cells
designed for bone remodelling, gain their ability to
form large, acidic resorptive lacunae by the targeted
exocytosis of late endosomes bearing V-ATPases to
areas of the plasma membrane in contact with the
bone matrix (38). Also, in cells that are not professional phagocytes, exocytosis is known to be stimulated by
invading pathogens. Trypanosoma cruzi, an obligate
intracellular parasite, gains entry into host cells by a
process that requires fusion of lysosomes with the
plasma membrane at the site of contact (2). Finally, all
cells have the need to patch their plasma membrane,
using endomembranes, during abscission in cytokinesis (29). The objectives of this review is to briefly summarize the evidence supporting the occurrence of
focal exocytosis during phagocytosis, to discuss the
potential endomembrane compartments involved,
and to describe a recently appreciated aspect of the
phenomenon, namely the opportunity this affords to
simultaneously secrete cytokines that accompany exocytosis during the inflammatory response.
Evidence for Focal Exocytosis
During Phagocytosis
The notion that the plasma membrane needs to be
restored following phagocytosis was first introduced
35 years ago by Werb and Cohn (46). These authors
noted that surface-specific markers were depleted
from phagocytes following particle uptake and were
subsequently replenished. The replenishment
occurred during a comparatively long period of time,
and its sensitivity to protein and RNA synthesis
1548-9213/05 8.00 ©2007 Int. Union Physiol. Sci./Am. Physiol. Soc.
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Cells of the innate immune system remove invading
microorganisms and other foreign particles by phagocytosis. Phagocytosis is also used for the ongoing
clearance of apoptotic bodies from senescent or
remodeling tissues. The engulfment of apoptotic or
potentially noxious particles by phagocytes is a summation of carefully orchestrated events beginning
with receptor engagement, pseudopod extension,
“zippering” of the membrane around the target, and
finally scission of the nascent vacuole or phagosome
from the plasmalemma. As illustrated in FIGURE 1,
phagocytosis involves a large expanse of membrane
to encircle the prey. Because the patch of the plasma
membrane that engages the particle is ultimately
internalized, the surface area of the cells has the
potential to decrease during phagocytosis, especially
when multiple and/or large particles such as apoptotic bodies are engulfed by a single cell (FIGURE 2).
Indeed, phagocytes have an astounding appetite,
being capable of internalizing so many particles that
the area of the vacuoles formed can be comparable to
that of the cell’s own surface! Yet, video microscopy
reveals that the cell shape and size are remarkably
conserved. It has been suggested that, because the
surface of phagocytes is corrugated, “unwrinkling”
may suffice to maintain the cell size when a sizable
area is internalized (23). This would enable the
phagocyte to retain its volume while decreasing its
area by smoothing it. However, at least in the case of
macrophages, quantitative spectroscopic and electrophysiological measurements have demonstrated that
the surface area of the cell in fact increases following
phagocytosis (22, 26). Elastic lateral stretching of the
bilayer could conceivably account for the increased
surface. However, both synthetic pure-lipid bilayers
and biological membranes can tolerate only a maximum of 4% stretch before their structural integrity is
compromised (24). This small contribution cannot
account for the changes observed experimentally
during phagocytosis of even a few mid-size particles
(e.g., FIGURE 2B). The inescapable conclusion is,
therefore, that the expanse of the surface membrane
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uration. These elegant studies revealed that stepwise
increases in capacitance, indicative of exocytic events,
occurred when phagocytosis was induced.
Jointly, these studies provided convincing indications that endomembranes are stimulated to fuse with
the surface membrane during the course of particle
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inhibitors served to implicate biosynthetic
pathways (46).
More recently, the advent of modern biophysical
methods enabled investigators to re-analyze the phenomenon with greater temporal resolution.
Greenberg and colleagues (12) used the dye FM1-43, a
solvochromic membrane indicator, to estimate the
surface area of macrophages undertaking “frustrated
phagocytosis,” an abortive form of phagocytosis where
overambitious macrophages try to engulf a flat surface
coated with an opsonic ligand. Remarkably, an
increase of over 20% of the original surface area of the
cells was recorded in these studies. The change was
blocked by inhibitors of phosphatidylinositol 3-kinase,
an enzyme critical to endocytic membrane traffic, suggesting involvement of endomembrane fusion events.
Of note, the increased binding of FM1-43 is not a
peculiarity of the frustrated phagocytosis model, since
it was subsequently replicated in cells that had performed bona fide phagocytosis of yeast particles that
were fully ingested (22).
The surface area of phagocytes was studied with
even finer temporal resolution by Palfrey and colleagues (36), who used an electrophysiological
approach. These investigators monitored the electrical
capacitance, a reliable index of the plasmalemmal
area, of macrophages patched in the whole cell config-
FIGURE 1. Membrane engagement during phagocytosis
Scanning electron micrograph showing phagocytosis of beads. An
expanse of membrane protrudes from the cell surface, forming the
phagocytic cup, which initially surrounds and ultimately engulfs particles.
FIGURE 2. Surface area changes during phagocytosis
A: comparison of the size of phagocytes and their prey (latex beads). To simplify the calculations of surface area,
macrophages were assumed to be spherical and smooth, i.e., without any corrugations. R and r denote the radius of
the macrophage and latex beads, respectively. The fractional surface area internalized (FI) is dependent on the number
of latex beads taken up and is denoted by n. B–C: RAW 264.7 macrophages were exposed to IgG-opsonized latex
beads of 3.87-m (B) or 8.1-m (C) diameter, and images were acquired at regular intervals. The FI was calculated at
the end of the experiment and found to be 0.18 and 0.16 for B and C, respectively. Internalized beads are indicated by
an asterisk. Size bar = 10 m.
PHYSIOLOGY • Volume 22 • December 2007 • www.physiologyonline.org
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ingestion. In accordance with this conclusion, interference with the fusogenic machinery of the cells was
found to impair phagocytosis. Cleavage of defined
SNARE proteins by injection of tetanus neurotoxin
reduced the phagocytic efficiency, and comparable
results were also obtained when a dominant negative
form of NSF (N-ethylmaleimide-sensitive factor) was
expressed (9).
The Source of Endomembranes
FIGURE 3. Recruitment of VAMP3 to the
phagocytic cup
F-actin (red) and nuclei (blue) were stained in
macrophages expressing GFP-VAMP3 (green) before
confocal imaging and three-dimensional reconstruction.
Actin-rich protrusions constituting the phagocytic cup
(arrows) colocalize with VAMP3 (yellow) at the base of
the cup, signifying delivery of recycling endosomes to
assist in pseudopod extension.
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What is the origin of the endomembranes that fuse
with the surface membrane to compensate for the area
internalized during phagocytosis? An unnervingly
large number of different intracellular compartments
have been implicated in the process, including elements from the endocytic and secretory pathways
(FIGURE 4). This surprising multiplicity of candidates
may be attributable to the variety of experimental systems utilized but may also be an indication that several sources need to be tapped to offset the large area
internalized.
Several subcompartments of the endocytic pathway
feature most prominently among the sources of
endomembrane. This consensus was reached on the
basis of the appearance of organelle-specific markers at
sites of ingestion before or at the time of phagosome
sealing. These markers feature prominently Rab
GTPases, which direct the traffic of vesicles, and SNARE
proteins, which mediate their fusion. Because phagocytosis was found earlier to be sensitive to tetanus and
botulinum B toxins, the involvement of the R-SNAREs
that are sensitive to these proteases was suspected (22).
Accordingly, GFP-tagged VAMP3 was found to be
recruited to nascent phagocytic cups in CHO cells that
were rendered phagocytic by transfection with Fc
receptors (5). Such engineered phagocytes were used to
circumvent the difficult task of transfecting myeloid
cells. This technical limitation was subsequently overcome, and two other groups independently confirmed
that, in macrophages, VAMP3 is also recruited to forming phagosomes (7, 34) (see also FIGURE 3). This
SNARE is found almost exclusively in recycling endosomes, and the importance of this compartment for the
phagocytosis of some particles (but not others) was
documented using VAMP3 knockout mice. Bone marrow-derived macrophages obtained from VAMP3-null
animals have a reduced ability to ingest zymosan particles, whereas they take up complement or
IgG-opsonized particles normally (1).
That recycling endosomes are indeed critical to the
early stages of phagocytosis is also suggested by the
effects of mutant forms of Rab11. Like VAMP3, Rab11
is a normal resident of recycling endosomes (45),
where it is thought to control traffic toward the surface.
Predictably, Cox et al. (11) detected Rab11 on nascent
phagosomes. Moreover they also found that mutant
alleles that prevent the normal cycling of Rab11
between its GDP- and GTP-bound states reduced the
ability of macrophages to engulf particles.
The traffic of recycling vesicles to the membrane is
also controlled by Arf6 (14, 45), which prompted two
different studies analyzing the role of this GTPase in
phagocytosis. In one, mutants of Arf6 that alter its
cycling were reported to depress the phagocytic efficiency (48). A subsequent study by Niedergang et al.
(35) not only confirmed these findings but in addition
demonstrated that inhibitory forms of Arf6 prevent the
focal delivery of VAMP3 to the phagocytic cup and
block pseudopod extension.
Other endosomal membranes are also recruited to
the phagocytic cup. Evidence exists for the recruitment of both late endosomes and lysosomes to sites of
phagocytosis. VAMP7, a SNARE that localizes to late
endosomes and lysosomes (27), was also detected in
nascent phagosomes, where it appears shortly after
the delivery of VAMP3 (7). The appearance of VAMP7
is functionally significant, since its depletion reduced
not only the exocytosis of late endocytic vesicles but
also the extension of pseudopods and, as a consequence, the phagocytic efficiency.
The fusion of VAMP7-containing vesicles is regulated
by synaptotagmin VII, a ubiquitously expressed calcium-sensing protein that controls the secretion of lysosomes (32). Recent observations in mice lacking synaptotagmin VII support of a role for late endocytic/
lysosomal vesicles in the formation of phagosomes.
Macrophages from such animals displayed an impaired
ability to engulf particles, a defect that was modest
when few small particles were ingested but became
more apparent as the phagocytic load increased (13). In
view of these observations, it is tempting to propose that
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large particles. Second, by delivering foreign particles
to the ER, this mode of phagocytosis could favor antigen cross-presentation (the presentation on class I
histocompatibility complexes of antigens internalized
by endocytosis). However, because antigens require
proteasomal degradation before class I presentation,
direct exposure to the ER lumen would not simplify
the loading procedure; the antigens would purportedly be retrotranslocated across the ER membrane to the
cytosol, where proteasomal degradation would ostensibly occur, followed by re-uptake of the resulting
peptides into the ER by the TAP transporter.
However attractive, the notion that the phagosome
is composed largely of ER-derived membranes is
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the delivery of endocytic membranes is graded and
occurs on demand, with recycling endosomes delivered
when only a smaller area of the plasmalemma is internalized and later compartments being summoned only
when compensation for a larger area is required.
In addition to membranes derived from the endosomal pathway, some specialized secretory organelles
present in phagocytes can contribute to phagosome formation. Neutrophils contain a number of specialized
secretory granules and vesicles that are seemingly
unique to this cell type. Three types of secretory granules
are formed and stored during neutrophil ontogeny: the
primary (or azurophilic), secondary (or specific), and
tertiary (or gelatinase) granules. All of these granule
types, plus small secretory vesicles, can be mobilized, to
varying extents, during neutrophil activation (47). Two of
these, the primary and secondary granules, have been
shown to fuse with the plasma membrane during phagocytosis (42). The primary granules, which are lysosomal
in nature, are secreted focally at the phagocytic cup and
likely supply membrane to the forming phagosome (41).
The Endoplasmic Reticulum
Controversy
The data reviewed thus far clearly point to a significant
contribution of the plasma membrane and endosomes to the nascent phagosome membrane. In
recent years, the endoplasmic reticulum (ER) was
invoked as an alternative source of internal membranes to forming and early phagosomes. Based on
proteomic analyses, in combination with electron
microscopy and glucose-6-phosphatase cytochemistry, Gagnon et al. (19) proposed that during particle
engulfment the ER fuses with the plasma membrane at
the base of the phagocytic cup. The establishment of
continuity between these two membranes was proposed to establish a pathway whereby the target particle could “slide” into the lumen of the ER. Scission of
the ER and resealing of the plasmalemma was envisaged to complete the phagocytic event.
Independent evidence in support of this model was
provided by targeting the SNARE proteins of the ER. In
one report, trapping antibodies to ERS24/Sec22b
inside macrophages depressed the efficiency of phagocytosis (6). A related study re-evaluated the role of
ERS24/Sec22b and additionally analyzed two other
SNARE proteins of the ER: syntaxin 18 and D12.
Interference with the function of syntaxin 18 and D12
produced a modest reduction in the phagocytic activity, whereas impairment of ERS24/Sec22b had no
discernible effect (25). The source of the apparent discrepancy between these studies is not readily apparent.
A contribution of the ER to phagosome formation
and maturation is attractive in several respects. First,
as the largest single intracellular compartment, the ER
can potentially provide enormous amounts of membranes to satisfy the need for entrapment of multiple
FIGURE 4. Sources of membrane delivered to the
phagocytic cup
A: early and recycling endosomes are the primary source of membrane
for the enlargement of the phagocytic cup. VAMP3 and Rab11-containing early/recycling endosomes are translocated to the cup, carrying
secretory cargo such as TNF-<alpha. Other molecules such as ARF6,
dynamin, and amphiphysin are reportedly involved in this process. B:
VAMP7 translocation to nascent phagosome heralds the involvement of
late endosomes, which fuse under the regulation of synaptotagmin VII
and syntaxin 7. C: in neutrophils, primary granules (identifiable by the
presence of CD63) and secondary granules (bearing CD66b) fuse with
the plasma membrane during phagocytosis in a calcium-dependent
manner. CD63 has been detected at the phagosomal cup. D: the endoplasmic reticulum has also been suggested to participate in phagocytosis, although this source of membrane has been contested.
PHYSIOLOGY • Volume 22 • December 2007 • www.physiologyonline.org
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“An obvious possibility is that dynamin and
amphiphysin are required for fission of vesicles from
the endosomal compartment...”
Research in our laboratory sought to reconcile the
classical model of phagocytosis with the paradigm of
ER-mediated entry. To this end, we compared the contribution of the ER and of endosomes to the formation
of nascent phagosomes by a variety of quantitative
methods, including biochemical, immunological,
fluorescence imaging, and electron microscopy techniques. Not one of these approaches supported the
fusion of the ER with plasma membrane during
phagocytosis or a contribution of the ER to phagosome maturation (43). Instead, the limiting
membrane of phagosomes was confirmed to derive
largely from the plasma membrane, becoming subsequently modified through a series of fusion reactions
with subcompartments of the endocytic pathway. In
view of the existing evidence, we conclude that the
contribution of the ER to the phagosomal membrane
is at best small and favor the canonical concept that
phagocytosis and maturation involve primarily the
plasma membrane and endocytic organelles.
Exocytosis and the Secretion
of Cytokines During
Phagosome Formation
Using the endocytic pathway, particularly the recycling endosomes, as a source of membrane for phagosome formation has an added bonus feature.
Recycling endosomes are a key station for the delivery
of pro-inflammatory cytokines in the secretory pathway, en route to the cell surface after being synthesized in the ER and transported through the Golgi
complex (31, 34). When VAMP3-positive membrane
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PHYSIOLOGY • Volume 22 • December 2007 • www.physiologyonline.org
from recycling endosomes is being delivered to the
surface for phagocytic cup formation, it simultaneously carries cargo such as the cytokine TNF-. VAMP3containing vesicles are delivered to the phagocytic cup
where the cognate SNAREs (syntaxin 4 and SNAP-23)
promote fusion. VAMP3-positive recycling compartment en route to the plasma membrane, where the
soluble form of the cytokine is released by proteolysis.
During phagosome formation, the stimulated exocytosis of recycling endosomes delivers VAMP3-containing vesicles to sites where the cognate SNAREs (syntaxin 4 and SNAP-23) promote fusion. The cytokine
precursor is also inserted into the phagocytic cup,
where the specific protease TACE cleaves and releases
soluble TNF- (34). This focal secretion process is
selective, however, in that IL-6, another pro-inflammatory cytokine that also traffics via recycling endosomes, is not focally delivered to the phagocytic cup
(31); indeed, neither are transferrin receptors, the
quintessential markers of recycling endosomes, which
are not delivered preferentially to the phagocytic cup
(31) but are instead recruited to the phagosome shortly after sealing (15, 16, 39). These events imply that
sorting within the recycling endosomes ensures that
membrane and cargo can be selectively deployed for
phagocytosis and concomitant secretion without
compromising other endosomal functions. Although
the mechanisms for this sorting are not yet understood, cholesterol-rich lipid rafts in phagocytic membranes may be involved (30).
Fission
To complete the internalization of particles, the nascent phagosome must undergo fission from the plasma membrane to form an autonomous internal compartment. For many forms of endocytosis, including
receptor-mediated endocytosis, rapid recycling of
neurotransmitter vesicles, and internalization of caveolae, the protein dynamin is required for scission to
occur (for review, see Ref. 33). In the case of phagocytosis, a role for dynamin has also been postulated. An
initial study found that expression of dominant-negative mutants dynamin-2, the ubiquitously expressed
dynamin isoform, greatly reduced the efficiency of
phagocytosis (21). Moreover, the dynamin-recruiting
protein amphiphysin was also found to be required for
optimal phagocytosis (20). Yet, contrary to expectation, these studies found that phagocytosis was not
arrested just before fission. Instead, when dynamin-2
was inhibited, the extension of membrane around the
forming phagosome was precluded, halting phagocytosis at an earlier stage.
Two subsequent studies have attempted to clarify
this apparent paradox. In a nutshell, what these studies concluded was that the main site of dynamin-2
action may not be at the plasma membrane but on
internal membranes. Cells transfected with a
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inconsistent with a plethora of earlier biochemical and
immunostaining data and is seemingly incompatible
with at least some of the established physical attributes of phagosomes. In dendritic cells and
macrophages, the lumen of the nascent phagosome
becomes acidic shortly after sealing. This acidification
has been attributed to the inward pumping of protons
by V-ATPases, which are thought to be acquired
through fusion with endosomes. The acidification
becomes more accentuated as phagosomes age, and
this correlates with the graded acidification of the
compartments of the endocytic pathway that fuse
sequentially with maturing phagosomes. It is difficult
to envisage how acidification would develop in a
phagosome composed largely of ER, which is believed
to be devoid of V-ATPases and is inherently permeable
to protons (37).
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machinery that is engaged for engulfment (8). For all
of these reasons, not all phagosomes are created
equal, and extrapolation can be very misleading.
Surely, new and unexpected features will be unveiled
when systems other than Fc receptor-mediated
phagocytosis are studied in detail.
Original work in the authors’ laboratories is supported
by the Canadian Institutes of Health Research (CIHR), the
Canadian Cystic Fibrosis Foundation (CCFF), the Heart
and Stroke Foundation of Ontario, the National Health
and Medical Research Council of Australia, and the
National Institutes of Health. K. K. Huynh is the recipient
of a CIHR Graduate Studentship. S. Grinstein is the current holder of the Pitblado Chair in Cell Biology.
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