lipid signaling and the modulation of surface charge during phagocytosis

Tony Yeung

Sergio Grinstein

Authors’ address

Tony Yeung, Sergio Grinstein

Cell Biology Program, Hospital for Sick Children,

Toronto, Ontario, Canada.

Correspondence to:

Sergio Grinstein

Cell Biology Program

Hospital for Sick Children

555 University Avenue

Toronto, Ontario, Canada M5G 1X8

Tel.: (416)813-5727

Fax: (416)813-5028

E-mail: [email protected]

Immunological Reviews 2007

Vol. 219: 17–36

Printed in Singapore. All rights reserved

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Munksgaard

Immunological Reviews0105-2896

Lipid signaling and themodulation of

surface charge during phagocytosis

Summary: Phagocytosis is an important component of innate andadaptive immunity. The formation of phagosomes and the subsequentmaturation that capacitates them for pathogen elimination and antigenpresentation are complex processes that involve signal transduction,cytoskeletal reorganization, and membrane remodeling. Lipids areincreasingly appreciated to play a crucial role in these events. Sphingoli-pids, cholesterol, and glycerophospholipids, notably the phosphoinosi-tides, are required for the segregation of signaling microdomains and forthe generation of second messengers. They are also instrumental in theremodeling of the actin cytoskeleton and in directing membrane traffic.They accomplish these feats by congregating into liquid-ordered domains,by generating active metabolites that activate receptors, and by recruitingand anchoring specific protein ligands to the membrane, often alteringtheir conformation and catalytic activity. A less appreciated role of acidicphospholipids is their contribution to the negative surface charge of theinner leaflet of the plasmalemma. The unique negativity of the inner aspectof the plasma membrane serves to attract and anchor key signaling andeffector molecules that are required to initiate phagosome formation.Conversely, the loss of charge that accompanies phospholipid metabolismas phagosomes seal facilitates the dissociation of proteins and thetermination of signaling and cytoskeleton assembly. In this manner, lipidsprovide a binary electrostatic switch to control phagocytosis.

Keywords: phagocytosis, phospholipid, phosphoinositide, surface charge, phagosomematuration

Introduction

Phagocytosis is a critical event in the elimination of invading

microorganisms and other foreign particles and in the sub-

sequent presentation of antigens for the development of

acquired immunity. The process can be conceptually divided

into two major steps: phagosome formation and maturation.

The former refers to the engulfment process, whereby the target

particles are initially recognized and subsequently internalized

by the phagocytic cell and enclosed within a membrane-bound

vacuole or phagosome. Maturation refers to the gradual

conversion of the phagosome from a comparatively inert

vacuole to an effective microbicidal and degradative compart-

ment capable of antigen presentation. Note that the nascent

17

phagosome is derived originally from the plasma membrane

and contains, in addition to its prey, a sampling of the

extracellular fluid. Over the course of the next hour, the

phagosome transforms to eventually resemble a lysosome; its

lumen becomes markedly acidic and rich in hydrolases and in

a variety of anti-microbial agents. Mature phagosomes also

acquire the ability to present antigens (1, 2).

Phagocytosis has been studied extensively because of its

paramount importance to the innate immune response and to

its interface with acquired immunity. Both phagosome

formation and maturation are complex sequences of events

requiring signaling, transduction of information, and engage-

ment of the cytoskeleton and of membrane fusion and fission

machinery. A myriad of different proteins are anticipated to be

required for such a sophisticated process, and their identifica-

tion is well underway, thanks to the advent of proteomic

technology and the availability of short-interfering RNA

(siRNA) libraries. Several hundred proteins have been identified

on (one stage of) the phagosomal membrane (3), and many

others have been linked physically, functionally, or genetically

to the phagocytic process (4–6).

By comparison, the role of lipids in phagosome formation

and maturation has been neglected. Our understanding of the

lipids present in the phagosomal membrane and of their role in

signaling and cytoskeletal and membrane remodeling is rather

primitive, lagging sorely behind the proteomic studies. The

intent of this review is to briefly summarize the state of our

knowledge of lipid involvement in phagocytosis while

highlighting the large gaps in understanding. In the spirit of

this series of reviews, recent work from our own laboratory is

emphasized and the role of lipids in dictating the surface charge

of biological membranes featured prominently. It is also

noteworthy that although phagocytosis can be triggered by

a wide variety of receptors that most likely differ in the mode

and eventual outcome of their signaling, most of our

knowledge to date stems from studies of Fcg receptors and,

less frequently, of complement receptors. The vast majority of

the results discussed subsequently were obtained using cells

activated by the former pathway. Although they provide a useful

guideline, data obtained from Fcg-receptor-mediated phago-

cytosis may not be applicable to other forms of phagocytosis.

Glycerophospholipids

Phosphatidylcholine and phosphatidic acid

Phosphatidylcholine (PC) is the most abundant lipid in

mammalian cells, comprising approximately 45–55% of the

total lipids (7). It can be synthesized from choline through the

Kennedy pathway and also in the liver by methylation of the

head group of phosphatidylethanolamine (PE) (8). PC is

prominent in the plasma membrane, where it is distributed

nearly equally on both leaflets, contributing about one-third of

the total lipid on either side (9).

PC can be hydrolyzed by phospholipase D (PLD) to generate

the metabolically active phosphatidic acid (PA). Two isoforms

of PLD have been identified: PLD1 and PLD2. In macrophages,

PLD1 is localized primarily in the late endosomal and lyso-

somal compartments, whereas PLD2 is found on the plasma

membrane (10). Themolecular determinants of this differential

targeting are not entirely defined, but mutations that rendered

PLD1 catalytically inactive or prevented its palmitoylation

resulted in its redistribution to the cytosol (11). Both iso-

forms of PLD require phosphatidylinositol 4,5-bisphosphate

[PI(4,5)P2] for optimal activity, but they differ in their

requirement for other cofactors. At least under some circum-

stances, PLD1 depends on small guanosine triphosphatases

(GTPases) like Rho, Ral, and Arf and/or on protein kinase

C (PKC) to become significantly active, whereas PLD2

is measurably active even in the absence of protein cofactors

(10, 11).

The involvement of PLD in phagocytosis has been studied in

some detail. Kusner et al. (12) showed by biochemical means

that macrophages produce PA on exposure to the quintessential

Fcg receptor target, immunoglobulin G (IgG)-opsonized

erythrocytes. More recently, local production of PA was

observed both at the phagocytic cup and on the nascent

phagosomes using a PA-specific fluorescent probe (10). PLD2

itself was observed at the phagocytic cup and on the extending

pseudopods, whereas PLD1-containing membrane vesicles

were recruited to the vicinity of the forming phagosome

(10), suggesting that these enzymes are the source of the PA

(Figs 1 and 2). The involvement of PLD was confirmed by

stimulating the cells in the presence of 0.5% ethanol. Under

these conditions, instead of PA, the cells generated phos-

phatidylethanol, a specific product of PLD-catalyzed trans-

phosphatidylation (12).

Dominant-negative alleles and RNA interference have been

used to assess the function of PLD in phagocytosis. Catalytically

inactive mutants of both PLD1 and PLD2 caused partial

inhibition of phagosome formation, and similar results were

obtained when the corresponding genes were silenced with

short hairpin RNA (10, 13). These findings strongly suggest

that both isoforms are involved in the early steps of

phagocytosis, namely in particle engulfment. Much less is

known about their role in later stages of the process. PLD2 is

promptly lost from the phagosome on sealing and is therefore

Yeung & Grinstein � Phospholipids and phagocytosis

18 Immunological Reviews 219/2007

unlikely to be of consequence (10). In contrast, vesicles bearing

PLD1 continue to be delivered to the (proximity of) early

phagosomes, suggesting that this isoform may contribute to

maturation.

In all likelihood, the effects of PLD on phagocytosis are

mediated by the localized accumulation of PA, which in resting

cells is a minor but significant species (about 1–2% of the total

lipids) (14). In addition to PLD-mediated hydrolysis of PC, PA

can be formed de novo through the acylation of glycerol-3-

phosphate, by phosphorylation of diacylglycerol (DAG) by

DAG kinase, or by acylation of lysophosphatidic acid (LPA) by

LPA acyltransferase (15–17). Although specific mechanisms

remain to be identified, PA could conceivably participate in

phagocytosis by direct and indirect means. Because the cross-

sectional area of its negatively charged head group is very small

compared with that of its acyl chains, PA is a cone-shaped (type

II) lipid and can therefore induce negative (concave) curvature

on membranes (18). Because of this bilayer-curving property,

PA has been suggested to promote membrane fission. In this

context, the LPA acyltransferase CtBP/BARS is thought to cause

fission of Golgi tubules by generating PA from LPA and acyl-

CoA (19). Similarly, the invagination and detachment of

synaptic vesicles from the plasma membrane may be promoted

by PA formed by endophilin I, which displays LPA acyltransfer-

ase activity (20). In an analogous manner, PA could facilitate

phagosome sealing and fission from the membrane, pre-

sumably by accumulating at the highly curved tips of advancing

pseudopods. Vesicular budding during phagosome maturation

may also depend on PA accumulation. These hypothetical

notions remain to be tested experimentally.

In addition to this direct role on membrane curvature, PA

could also contribute indirectly to phagocytosis by locally

activating signaling molecules. PA has been shown to stimulate

phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) and to

favor recruitment of sphingosine kinase (SK)1 to themembrane

Fig. 2. Time course of lipid metabolism during phagocytosis.Generation of PI(4,5)P2 de novo occurs early during phagocytic cupformation, followed shortly after by its hydrolysis and the formation ofDAG, which persists on the sealed phagosomal membrane for manyseconds to a few minutes. PI(3,4,5)P3 is formed soon after receptorengagement and is still observable after fusion of the pseudopods,lasting approximately 1 min after sealing. PA is observed at thephagosome membrane during the uptake process and is thereafter likelyconverted to other lipids such as DAG.

Fig. 1. Localized lipid metabolism duringphagocytosis. Fcg receptor ligation results inthe activation of PI4P5K (PIPK) and de novosynthesis of PI(4,5)P2. As the macrophageproceeds to internalize the opsonized particle,PI(4,5)P2 is hydrolyzed by PLCg to generateIP3 and DAG, first at the base of the phagocyticcup and then along the pseudopods. The lossof PI(4,5)P2 can also be attributed to itsconversion to PI(3,4,5)P3 by the class I PI3Kp110 subunit, recruited to the formingphagosome by the p85 regulatory subunit.Changes in glycerophospholipids also occurconcurrently with phosphoinositide metabo-lism. PC is hydrolyzed by PLD to form PA,a cone-shaped lipid that promotes membranefission events. The level of PS may also bemodulated by metabolism, membrane dilu-tion, or transbilayer redistribution throughadenosine triphosphate-dependent transport-ers such as ABC1.


Immunological Reviews 219/2007 19

(11, 21). Both of these enzymes and their products are thought

to be important in phagocytosis, as discussed in more detail

subsequently. Last, PA can contribute to phagocytosis when

converted to DAG, another metabolically active lipid, by PA

phosphohydrolase (22).

Phosphatidylserine

Phosphatidylserine (PS) contributes 2–10% of the total cellular

lipids, depending on the cell type (14). It is well established,

however, that PS is preferentially enriched in the plasma

membrane, particularly in its inner leaflet, where it constitutes

15% (14, 23). PS is synthesized in a specialized region of the

endoplasmic reticulum (ER), known as the mitochondria-

associated membranes, by two enzymes: phosphatidylserine

synthase 1 (PSS1) and phosphatidylserine synthase 2 (PSS2).

PSS1 catalyzes a base-exchange reaction that replaces the

choline head group of PC with serine to yield PS (24). By

contrast, PSS2 uses PE as a substrate and exchanges the

ethanolamine head group for serine (14). This reaction is

counterbalanced by the conversion of PS to PE bymitochondrial

decarboxylases (14).

The distribution of PS in the various cellular compartments

can in principle be studied by different means. Annexin V binds

with high affinity to PS and has been used extensively to

monitor its appearance on the outer leaflet of the plasma

membrane, for example when cells are undergoing apoptosis.

However, the interaction of annexin with PS requires the

presence of millimolar calcium, which precludes its use as

a probe of intracellular PS. An alternative approach is the use of

fluorescently labeled PS analogs. One such probe, NBD [6-(7-

nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-PS, has been

used extensively. It can be readily loaded into mammalian cells,

where it distributes in a variety of endomembrane compart-

ments. It is not entirely clear, however, whether this result is

a faithful representation of the distribution of native PS in the

cell. The NBDmoiety attached to one of the fatty acyl chains has

been suggested to loop back to the membrane–water interface,

distorting the normal structure of the lipid and likely altering its

partition properties (25, 26). Just as worrisome, metabolism of

the probe by lipases or decarboxylases can result in the

progressive appearance of fluorescent species that are not

distinguishable from the original NBD-PS. This finding may

explain why shortly after loading, NBD-PS stained primarily the

ER and the Golgi apparatus, whereas at longer times (>2 h),

NBD staining was observed in the mitochondria (27).

PS is important to the biology of both phagocytes and their

prey. In apoptotic cells, which are cleared by phagocytosis, PS is

externalized to the outer leaflet of the plasma membrane by

a calcium-induced scrambling mechanism (28). The external-

ized PS serves as a major recognition signal for phagocytosis by

engaging one or more PS receptors on macrophages. Curiously,

it has been reported that the macrophages themselves also

externalize PS during the internalization of apoptotic cells (29)

(Fig. 1). The adenosine triphosphate-binding cassette trans-

porter 1 (ABC1) is thought to be important for the transbilayer

redistribution of PS during phagocytosis (30); macrophages

deficient in ABC1 or treated with the ABC1 blocker glyburide

failed to externalize PS (29, 30) (Fig. 1). They were also unable

to ingest apoptotic cells, implying that mobilization of PS is an

essential step in the phagocytic sequence. Where and how PS

functions to promote particle engulfment is obscure at present.

The distribution of PS between the two leaflets of the plasma

membrane has also been studied during Fcg-receptor-mediated

phagocytosis. In this system, the phagocytic cupwas not stained

by annexin V (31). Externalization may not occur following

engagement of Fcg receptors; but it is also possible that PS

externalized locally at the cup diffuses laterally, becoming dilute

and undetectable (Fig. 1). Lipid rearrangement may have also

occurred during the fixation that preceded labeling in these

studies.

Because suitable probes to monitor the distribution and

dynamics of PS inside cells are not currently available, much less

is known about intracellular PS in phagocytosis. However,

possible roles can be anticipated based on the known PS

dependence of enzymes that become activated during phag-

osome formation. Together with DAG, which is released by

phospholipase C (PLC) (see subsequently), PS likely contributes

to the recruitment of PKC isoforms to the membrane. In

addition to the C1 domains that recognize DAG, classical and

novel isoforms of PKC contain a C2 domain that associates with

anionic phospholipids, notably PS (32). Macrophages express

PKCa, bI, d, e, and z (33), and PKC isoforms were shown to be

involved in phagosome formation (33, 34) and nicotinamide

adenine dinucleotide phosphate (NADPH) oxidase activation

(35, 36). PKC and possibly PSmay also play a role at later stages,

during phagosome maturation.

Although numerous examples exist showing the role of PKCs

in phagocytic signaling, there are various other signaling

molecules containing C2 domains that may play a role in

phagocytosis in a PS-dependent manner. Such candidates

include PLC, cytosolic phospholipase A2 (cPLA2), phosphati-

dylinositol 3-kinase (PI3K), synaptotagmin, and PTEN (phos-

phatase and tensin homolog deleted on chromosome 10) (32).

In addition, PS likely contributes to the negative surface charge

of the inner leaflet of the plasma membrane and thereby may

help recruit signaling molecules such as K-Ras, Rac1, and c-Src,



which contain polycationic motifs (37). The relationship

between anionic lipids, surface charge, and cell signaling is

discussed in more detail subsequently.

LPA and lysobisphosphatidic acid

LPA is abundant in serum but is a relatively minor constituent of

cells. It is best known for its effects on cell proliferation,

migration, and survival. Extracellular LPA binds to cognate cell

surface receptors that are coupled to heterotrimeric G-proteins

that signal through the Ras/mitogen-associated protein kinase

(MAPK) pathway and also through Rho family GTPases that

control cytoskeletal structure. Monocytic cells can be activ-

ated by LPA through G-protein-coupled receptors (GPCRs),

resulting in an increase in intracellular calcium level (38). In

addition, LPA treatment has been shown to enhance the anti-

mycobacterial activity of macrophages through a PLD-mediated

mechanism (39).

LPA is generated in the ER from glycerol-3-phosphate and

acyl-CoA (15). Much less is known about the function of

intracellular LPA, at least in part because it is short lived,

becoming rapidly acylated to form PA. Unlike PA, which is

a conical or type II lipid, LPA is a type 1 lipid shaped like an

inverted cone that induces a positive (convex) curvature to

membranes. As such, it could promote budding of vesicles from

the phagosomal vacuole. Vesiculation would be directed

outward, if LPA accumulates in the cytosolic monolayer, or

inward [as in the case of multivesicular bodies (MVBs)], if

accumulation occurs on the luminal membrane.

Lysobisphosphatidic acid (LBPA), also known as bis(mo-

noacylglycero)phosphate, is a minor lipid (<1% of total lipids)

formed from the degradation of phosphatidylglycerol and

cardiolipin (40, 41). LBPA is found mostly in late endosomes,

where it accounts for about 15% of the total lipids (42). Late

endosomes are typically multivesicular structures, and immu-

nofluorescence studies found LBPA to be particularly concen-

trated in the luminal vesicles of these complex organelles.

Indeed, in vitro experiments suggest that LBPA may be

instrumental in the inward budding that leads to the formation

of such internal vesicles. Matsuo et al. (42) found that pure lipid

liposomes spontaneously became multivesicular when a pH

gradient mimicking that found across the membrane of late

endosomes was imposed, but only when LBPA was present. It is

conceivable that protonation of luminal LBPA facilitates its

transbilayer redistribution (flipping) to the outer leaflet, where

it would reacquire charge and be retained following deproto-

nation. The progressive asymmetric accumulation of LBPA on

the outer (cystolic) leaflet could deform the bilayer, forcing

inward vesiculation.

Although LBPA induces the formation of multivesicular

liposomes in pure lipid model systems, proteins alsomost likely

contribute to MVB formation in the cell. A protein that interacts

selectively with LBPA, called Alix (interacting partner of ALG-2

and the ESCRT proteins), was identified by Matsuo et al. (42).

Alix is likely to be involved in MVB formation because

downregulation of its expression using siRNA reduced the

number of multivesicular and multilamellar late endosomes.

How this is accomplished is less clear because, paradoxically,

addition of Alix inhibited the formation of multivesicular

liposomes in vitro driven by LBPA and pH (42).

Not all the vesicles that bud into the lumen of MVB are

degraded in the lysosomes. Some fuse back with the limiting

membrane, ostensibly to recycle resident endosomal proteins. It

is interesting that downregulation of Alix prevented not only

the inward budding that gives rise to vesicles but also possibly

the back-fusion of preexisting luminal vesicles (42), suggesting

a wider role in vesicular traffic. LBPA may also be involved in

this back-fusion because occlusion of its head group with

antibodies resulted in the development of cholesterol-rich,

multilamellar endosomes reminiscent of those seen in patients

with Niemann-Pick type C disease(43).

Both LBPA (44, 45) and Alix (3) have been detected on the

phagosomalmembrane (Fig. 3). In fact, LBPA is perhaps the best

if not the sole marker that unambiguously identifies the ‘late

phagosome’ stage that precedes phagolysosome formation

(Fig. 4). Remarkably, there is almost no information addressing

the multivesicular nature of phagosomes, and the possibility of

inward budding at the late stages of maturation has been

explored in only one report. Lee et al. (45) detected delivery of

a cytosolic epitope from the limiting membrane of phagosomes

to their acidic interior and were able to visualize intra-

phagosomal vesicles by electron microscopy (Fig. 3). Whether

LBPA and Alix underlie these events has not been investigated.

Arachidonic acid

Arachidonic acid is generated from the hydrolysis of glycer-

ophospholipids at the sn-2 position by phospholipase A2 (PLA2)

(46). The PLA2 superfamily currently consists of 15 groups and

subgroups and includes five distinct types of enzymes, namely

the secreted, cytosolic, calcium-independent, and lysosomal

PLA2 isoforms and the platelet-activating factor acetylhydrolases

(47). The cPLA2, which may be the most important isoform in

phagocytosis, contains a calcium-dependent C2 domain that

binds to PC-richmembranes (48, 49). Its activity is regulated by

phosphorylation at serine residues by kinases including MAPK,

calcium/calmodulin-dependent protein kinase II, and MAPK-

interacting kinase I (48). Not only is calcium elevated but also



(at least some of) these kinases are stimulated early in

phagocytosis, suggesting that cPLA2 may become activated.

Indeed, direct measurements have confirmed that arachidonate

is generated on engagement of phagocytic receptors (50, 51).

Arachidonic acid is important in various physiological and

pathological processes, including inflammation and asthma,

because it is metabolized to leukotrienes and prostaglandins,

which are potent proinflammatory mediators (46). However,

arachidonate itself is very important in the early stages of

phagocyte activation. Formation of arachidonate at the plasma

membrane was postulated to play a role in both the trans-

location and the activation of subunits of the NADPH oxidase.

Arachidonate is a potent activator of the oxidase in vitro, and

downregulation of cPLA2 using anti-sense technology inhibited

superoxide production (52). Zhao et al. (53) later attributed this

finding to the failure of the p47 and p67 subunits of the oxidase

to translocate from the cytosol to the membrane. However,

Shmelzer et al. (54) suggested that the sequence of events is the

opposite, that is the assembled oxidase recruits cPLA2 to the

membrane, which in turn generates arachidonate necessary for

allosteric activation. The inability of cPLA2 to translocate in cells

deficient in the gp91 subunit of the NADPH oxidase supports

this model (54). Arachidonate was proposed to bind to the

oxidase complex in the vicinity of the flavocytochrome b,

increasing its affinity for oxygen (55, 56).

Despite these seemingly compelling data, macrophages from

cPLA2�/� mice did not manifest any defects in superoxide

production when stimulated by soluble or particulate stimuli

(57). The reasons for these conflicting results are not clear; but

redundant or compensatory effects from other PLA2 isoforms

may have confounded the observations (56).

The hydrolysis reaction that produces arachidonic acid

simultaneously generates a lysophospholipid that, because of

its altered shape, can distort the membrane bilayer. Lysophos-

pholipids are inverted cone-shaped (type I) lipids that impose

membrane curvature in the convex direction and could thereby

promote fusion events. Indeed, exogenous addition of PLA2in vitro promotes fusion of liposomes, and in mast cells, PLA2activity promotes fusion of secretory granules with the

plasma membrane (58). It remains to be examined whether

Fig. 3. Lipid metabolism during phagosome mat-uration. PI(3)P is formed immediately after particleinternalization and lingers on the early phagosomalmembrane for approximately 10 min. It is generatedby the recruitment and activation of the class III PI3KVps34, which associates with a myristoylated p150subunit for membrane anchoring. As maturationprogresses, membrane fusion and fission events occurat the phagosome, resulting in both the delivery andthe selective removal of signaling and other mole-cules. The Fcg receptor, which becomes ubiquity-lated, is recognized by Hrs, which has a ubiquitin-interacting motif and also interacts with the ESCRTcomplex to facilitate inward membrane budding. Asthe phagosome fuses with the late endosomes, LBPAis acquired and possibly distributed on both thecytosolic and the luminal leaflets of the phagosomemembrane. Alix, through its interaction with LBPA,may promote multivesiculation of the phagosome,generating LBPA-positive internal vesicles. As thephagosome transitions into the late phagosome stage,PI(3)P can potentially be converted by PIKfyve toform PI(3,5)P2 or by the 3#-phosphatase myotubu-larin to form PI, although both of these proposedmechanisms remain to be shown experimentally.

Fig. 4. Time course of lipid metabolism during phagosomematuration. PI(3)P is synthesized immediately after particle internal-ization and persists on the phagosomal membrane for approximately10 min. It can be dephosphorylated to PI or potentially be converted toPI(3,5)P2 during the later stage of maturation and subsequently bedephosphorylated to PI(5)P by the 3#-phosphatase myotubularin. LBPAis also observed at the late phagosome stage and presumably disappearswhen phagolysosomes are formed.



arachidonic acid generated at the time of phagocytosis plays any

role in fusion and membrane remodeling.

Cholesterol

Much of the cellular cholesterol is from an exogenous origin.

Dietary cholesterol in the esterified form is taken up by cells

through the receptor-mediated internalization of lipoprotein

complexes like the low-density lipoprotein particles. The

receptors deliver the lipoproteins to the endocytic pathway,

where the cholesterol esters are hydrolyzed (59). The

cholesterol freed within the endolysosomal compartments is

then redistributed to the plasma membrane and to compart-

ments of the secretory pathway, particularly to the ER where it

can be reesterified and packaged for storage in lipid droplets

(59). The Niemann-Pick type C1 and C2 proteins are involved

in this redistribution step. From the plasma membrane,

cholesterol can be extruded from the cell by the ABCA1

transporter to bind to the lipid-poor apoA-I and generate high-

density lipoprotein (60).

Cholesterol can also be synthesized de novo from acetate in

a series of approximately 30 steps, most of which occur in the

ER. During this process, several important biomolecules are also

generated, including precursors for isopentenyl transfer RNA,

dolichol, farnesyl and geranylgeranyl moieties, and ubiquinone

(59). Althoughmost of the enzymatic reactions take place in the

ER, the majority of unesterified cholesterol is delivered to the

plasma membrane by a combination of both vesicular and non-

vesicular mechanisms.

Cholesterol is a planar, rigid molecule that inserts into

bilayers, where it interacts preferentially with sphingolipids

(59). It can pack between the long, saturated acyl chain and the

sphingosine backbone of sphingolipids, stabilized by van der

Waals interactions, whereas hydrogen bonding maintains the

interaction between the lipid head groups (59). The specialized

microdomains that are formed as a result of this interaction and

their possible role in phagosome formation are discussed in

detail subsequently.

In addition to the plasmamembrane, unesterified cholesterol

is also abundant in organelles of the endocytic pathway and is

present in phagosomes (59, 61, 62). What role, if any,

cholesterol plays in phagosome maturation remains to be

studied. One possible mode of action involves the targeting and

retention of Rab-family GTPases, which are geranylgeranylated

(63). In this regard, cholesterol accumulation in the endocytic

pathway induced by the drugU18666A increases the amount of

membrane-associated Rab7 and Rab9 (64, 65). Excess cho-

lesterol is thought to reduce the ability of Rab guanidine

nucleotide dissociation inhibitor (GDI) to extract from the

membrane the guanosine diphosphate (GDP)-bound form of

these Rab GTPases (64, 65). From these pharmacological

observations, it is tempting to speculate that the physiological

levels of cholesterol also influence the partition of Rab isoforms

in phagosomes and other endocytic organelles.

Sphingolipids

Sphingolipids comprise a large class of about 400 different

compounds (66). The typical sphingolipid contains a long

chain (18–20) carbon backbone known as the sphingoid base

that varies in length, hydroxylation, saturation, and branching

(67). Sphingosine and sphinganine are among the most

common sphingoid bases, to which long, saturated fatty acids

can be attached through an amide linkage to form ceramides

(67). Sphingolipids containing the ceramide backbone can be

further modified to sphingomyelin (SM) by the addition of

a phosphocholine head group or to glycosphingolipids by the

stepwise addition of various sugars (67).

Sphingoid bases are formed initially from L-serine and

palmitoyl CoA on the cytosolic side of the ER, where they also

become acylated subsequently to form ceramides (67).

Ceramides are then delivered by both vesicular and non-

vesicular mechanisms to the Golgi apparatus for glycosylation

by glucosyltransferases to form glucoyslceramides (67, 68).

Glucosylceramides then translocate to the lumen of the Golgi to

be further modified by luminal enzymes to yield lactosylcer-

amide and more complex glycosphingolipids (67). The newly

formed glycosphingolipids are delivered to the plasma

membrane and eventually internalized through the endocytic

pathway to the lumen of the lysosomes for degradation (67).

SM is generated in the Golgi by the transfer of phosphocho-

line from PC to ceramide (69). It contributes 5–10% of the total

lipids in the cell (14). Because it is synthesized in the lumen of

the secretory pathway, 80–90% of the plasmalemmal SM is

localized on the outer leaflet (14, 70). SM acts as a reservoir of

lipid signalingmolecules as it can be converted back to ceramide

by both neutral and acidic sphingomyelinases (69). Of note, the

activity of an acidic sphingomyelinase is enhanced when

phagocytic receptors are activated (70, 71).

Sphingolipid- and cholesterol-enriched lipid microdomains

(rafts)

Glycosphingolipids, SM, and their precursor, ceramide, contain

two long, saturated, hydrophobic chains that pack tightly,

conferring rigidity to the lipid bilayer. The straight chains and

the head group spacing of complex sphingolipids also favor



intercalation of unesterified cholesterol, which further increases

the lipid-packing density. These strong interactions promote

the segregation of cholesterol and sphingolipid-rich liquid-

ordered microdomains on the plasma membrane, often called

‘rafts’. Although lipid microdomains are clearly discernible in

model systems, their size, composition, and even their existence

in biological membranes remain subjects of controversy.

Cellular lipid rafts are sometimes equated to the fraction of

detergent-resistant membrane (insoluble in 1% cold Triton-X-

100); but this definition has shortcomings and is not universally

accepted. Indeed, extraction with Triton-X-100 was itself

reported to force the coalescence of otherwise separate

microdomains (72). A more accurate description was derived

from less invasive biophysical methods that suggested, unlike

the originally proposed long-lived and micron-sized moving

platforms, that plasmalemmal rafts are probably nanometer-

sized, transient domains that can interact with transmembrane

proteins and the cytoskeleton (73, 74).

These cholesterol- and sphingolipid-richmicrodomains have

been hypothesized to compartmentalize the plasma membrane

into distinct regions that can preferentially recruit signaling

proteins during receptor stimulation. During phagocytosis, Fcgreceptors that are cross-linked by polyvalent ligands are thought

to be recruited to (or perhaps recruit to their vicinity) lipid rafts.

The significance of this interaction lies in the observation that

Src family kinases like Lyn are normally resident in the rafts.

These kinases are responsible for the phosphorylation of the

immunoreceptor tyrosine-based activation motif (ITAM),

which is a key initiating event in the signal transduction cascade

that leads to particle engulfment (75). In accordance with this

idea, clusters of 200–300 nm diameter where Fcg receptors

colocalized with Lyn were detected by immunoelectron

microscopy after but not before the receptors were cross-

linked (76).

In biological membranes, particularly in the plasmalemma,

cholesterol is believed to be essential for the formation and

maintenance of liquid-ordered microdomains (rafts). For this

reason, the functional requirement for rafts has often been

evaluated by extraction of cholesterol from the membrane,

using agents like b-methyl-cyclodextrin, or its sequestration

within the membrane, using nystatin or filipin (77, 78). Using

this approach, Kwiatkowska et al. (75) and Kwiatkowska and

Sobota (79) found that disruption of lipid rafts by cholesterol

removal prevented the association of Lyn with Fcg receptors,

thereby inhibiting receptor tyrosine phosphorylation and

activation. Jointly, these observations would appear to establish

cholesterol and liquid-ordered microdomains as key compo-

nents of the phagocytic response.

However, extensive cholesterol removal with agents such as

b-methyl-cyclodextrin or the insertion into the bilayer of

cholesterol scavengers like nystatin can have unintended,

potentially deleterious effects on cells. Excessive extraction of

cholesterol was reported to disrupt the organization of

PI(4,5)P2, causing remodeling of the actin cytoskeleton (80).

Treatment with b-methyl-cyclodextrin has also been suggested

to deplete intracellular calcium stores and to depolarize the

plasma membrane (81). Therefore, more sophisticated meth-

ods were needed to overcome the shortcomings of these

traditional pharmacological techniques.

An alternative approach is the identification and manipula-

tion of the molecular determinants of receptor association with

the rafts. Recently, Barnes et al. (82) showed that palmitoylation

of the FcgIIA receptor on cysteine 208, located within

a juxtamembrane region of the cytoplasmic tail, is required

for association with lipid rafts. Remarkably, mutations of this

residue that precluded palmitoylation only moderately reduced

and delayed tyrosine phosphorylation of the receptor, despite

its failure to associate with lipid rafts (82). Others have reported

that alanine 224 is also essential for the association of FcgIIAreceptors with rafts (83). As in the previous study, mutation of

this alanine prevented the interaction with rafts; yet, tyrosine

phosphorylation and,more importantly, phagocytosis proceeded

normally (83). These studies suggest rather convincingly that

lipid rafts are not essential for FcgIIA-receptor-mediated

phagocytosis.

Other members of the Fcg family, however, may be more

dependent on their interaction with lipid-ordered micro-

domains. It is generally acknowledged that IgG-coated particles

activate not only stimulatory receptors like FcgI and FcgIIA but

also inhibitory ones like FcgIIB and that the fine balance

between these receptors is critical for regulated phagocytosis. In

the case of the FcgIIB receptor, an isoleucine residue at position232, which is mutated in a cohort of patients with systemic

lupus, was found to be necessary for its association with rafts

(84). FcgIIB receptors that underwent substitution of this

isoleucine for threonine were excluded from the rafts they

normally share with FcgI receptors after stimulation (84). Cells

bearing this mutation exhibited greater phagocytic capacity and

expressed higher levels of surface major histocompatibility

complex class II than wildtype cells (84). These results suggest

that the failure to associate with lipid rafts disrupts normal

FcgIIB receptor function, resulting in unhindered, exaggerated

FcgI and FcgIIA receptor activity.

Rafts may also be involved in phagosome maturation.

Although the existence of liquid-ordered microdomains in

phagosomes has never been established, Dermine et al. (85)



showed that the raft-associated protein flotillin-1 accumulates

in the phagosomal membrane during maturation. In addition,

proteomic analysis identified subunits of the vacuolar adeno-

sine triphosphatase and heterotrimeric G-proteins in the

detergent-resistant fraction of the phagosomal membrane,

suggesting that lipid rafts may contribute to the recruitment or

activation of themolecularmachineries required formaturation

(85). In fact, it was proposed that lipid rafts play a role in

mediating fusion of the phagosome with late endosomes. This

suggestion was derived from analysis of the maturation of

phagosomes following internalization of Leishmania, which lack

flotillin-1 and fail to fuse with late endosomes (85). It remains

to be established if the absence of flotillin-1 correlates with the

paucity of rafts and whether these alterations are the cause or

merely a consequence of the maturation arrest.

Signaling through sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P), the product of sphingosine

phosphorylation by SK, is well known as an extracellular ligand

that is recognized by a family of five GPCRs (S1PR1–5) (86).

Interest in S1P stems from the findings that it exerts anti-

apoptotic effects in cells and can regulate cell movement,

angiogenesis, and vascular maturation (87). In agreement with

these observations, inhibition of SK activity or S1P inhibitory

antibodies reduce tumor burden in mice (88, 89). Importantly,

S1P can be converted back to sphingosine and ceramide, which

are thought to be anti-tumorigenic and are associated with

growth arrest and apoptosis. The term ‘sphingolipid rheostat’

has been coined to describe the dynamic balance of these

interconvertible species that is vital for the regulation of cell

growth and apoptosis (86).

It is also well established that binding of S1P to its receptors

on the cell surface raises the cytosolic calcium level, which may

be important for phagosome maturation (86). Interestingly, it

has been suggested that S1P may also act intracellularly as

a second messenger that binds to calcium-permeable channels

in the ER and thereby directly mediating calcium mobilization

(90–92).

S1P was shown to be produced in human macrophages,

which express SK1 but not SK2 (93). In macrophages, SK1 was

found to be recruited to sites of phagocytosis during the

internalization of latex particles or serum-opsonized Staphylo-

coccus aureus (93). A sizable increase in SK activity was detected in

membranes of cells isolated after induction of phagocytosis

(93). The mechanisms of SK1 translocation and activation

during receptor-mediated phagocytosis are currently not

known. However, in vitro experiments showed that SK1 binds

anionic phospholipids, particularly PS, with nanomolar affinity

(94). As discussed in more detail subsequently, the plasma

membrane is particularly enriched in anionic lipids, making it

a likely target for SK recruitment. The activity of SK1 can be

stimulated by tyrosine kinases and serine/threonine kinases

(86, 94). Both of these types of kinases are stimulated during

the early stages of phagocytosis and may very well account for

the activation of SK1. In addition, SK1 contains a calmodulin-

binding site that, when occupied, promotes membrane trans-

location of the kinase (86). Because cytosolic calcium becomes

elevated during the initial stages of phagocytosis, complexation

of calmodulin may contribute to the recruitment of SK1.

Although some disagreement persists, several authors have

reported that cytosolic calcium changes are required for

completion of phagosome formation and/or maturation (95–

97). Given the ability of S1P to release endomembrane calcium

stores, whether directly or by activating surface receptors,

Kusner and colleagues (97) wondered whether S1P is involved

in elevating cytosolic [Ca2þ] during phagocytosis. They found

that treatment of the cells with dihydrosphingosine, a compet-

itive inhibitor of SK1, abolished the rise in intracellular [Ca2þ]

associated with particle internalization (97). More importantly,

the inhibition of SK1 and the suppression of calcium fluxes

significantly reduced the acquisition of lysosomal markers by

the phagosomes and blocked its acidification (97). Remarkably,

they also noted that when live Mycobacterium tuberculosis was the

phagocytic prey, SK1 was not recruited to the site of

phagocytosis nor was its activity stimulated (93, 97). Based

on these and other observations, Kusner and his colleagues (93,

97) proposed the interesting hypothesis that virulent mycobac-

teria may use inactivation of SK1 as a means to subvert the

normal maturation process, enabling their prolonged intracel-

lular survival. It is not entirely clear whether elevated [Ca2þ] is

both a prerequisite for SK1 recruitment, in a calmodulin-

dependent manner, and a consequence of its production of S1P.

Similarly, the mechanism used by M. tuberculosis to prevent SK1

activation remains obscure.

Like sphingosine, ceramide can also be phosphorylated,

yielding ceramide-1-phosphate. Ceramide kinase is responsible

for this reaction. Increased levels of ceramide-1-phosphatewere

reported to enhance particle engulfment (98), but it is not clear

whether this pathway is a normal contributor to the phagocytic

response.

Phosphoinositides

Phosphatidylinositol (PI) is synthesized from cytidine diphos-

phate diacylglycerol (CDP–DAG) and inositol by PI synthase in

the ER and also possibly on the plasma membrane (22). PI



comprises about 8% of the total lipid content of cells and

approximately 10% of the lipid on the inner leaflet of the plasma

membrane (9, 99). It serves as the substrate for the biosynthesis

of three different species phosphorylated at position 3, 4, or 5 of

the inositol ring. The resulting monophosphoinositides,

phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylino-

sitol 4-phosphate [PI(4)P], and phosphatidylinositol 5-phos-

phate [PI(5)P], respectively, can in turn be phosphorylated

further to form phosphatidylinositol 3,4-bisphosphate

[PI(3,4)P2], phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2],

PI(4,5)P2, and phosphatidylinositol 3,4,5-trisphosphate

[PI(3,4,5)P3]. PI(4)P and PI(4,5,)P2 are the major phos-

phoinositides in cells, constituting about 0.5% of the total

lipid, whereas the D3-phosphorylated phosphoinositides and

PI(5)P combined contribute to less than 0.025% of the total

lipids (less than 0.25% of the phosphoinositides) (100,

101). To the best of our knowledge, all phosphoinositides

are localized on the cytosolic leaflet of the plasma membrane

and of intracellular organelles.

Phosphatidylinositol 4-phosphate

PI(4)P is found mainly in the Golgi and the plasma membrane

(102, 103). Two classes of PI4 kinases (type II and type III)

synthesize this inositide. As expected, PI4 kinases are present in

the Golgi and the plasma membrane, where their product is

found; but they are also detectable in the ER (104–106). At the

membrane, PI(4)P is most likely important as the source of

PI(4,5)P2, which is without question a key molecule in

phagosome formation. PI(4)P could itself serve to recruit

signaling proteins to the phagocytic cup, as it is thought to do in

the Golgi in the context of recruitment of activator protein-1

(107), although this possibility has not been explored.

We investigated whether PI(4)P is present in phagosomes

using a tandem construct of the pleckstrin homology (PH)

domain of the yeast oxysterol-binding protein (Osh2p) that

reportedly binds to this phosphoinositide (102). Compared

with the concentration detected in the plasma membrane, the

level of PI(4)P in the phagosome was found to be considerably

lower (31). However, these results must be viewed with

caution because the specificity of the Osh2p-PH tandem

construct toward PI(4)P is far from perfect. The probe also

interacts with PI(4,5)P2, which, as described subsequently, is

known with more certainty to be depleted from phagosomes,

compared with the plasmalemma. Nevertheless, because both

PI-3 kinases and PI4P-5 kinase are known to be involved in

phagocytosis, it is conceivable that PI(4)P may serve as

a substrate for the de novo synthesis of PI(3,4)P2 and PI(4,5)P2(108, 109).

Phosphatidylinositol 4,5-bisphosphate

PI(4,5)P2 is largely localized on the inner leaflet of the plasma

membrane, contributing about 1–2% of the lipid on this side of

the bilayer (110, 111). PI(4,5)P2 is well known for its role in

actin remodeling. It influences filament formation and

extension by binding to a variety of actin-capping, severing,

and monomer-binding proteins (100). The distribution and

metabolism of PI(4,5)P2 can be monitored with the PH domain

of PLCd (111). Using this approach, PI(4,5)P2 was found to

undergo a modest and transient accumulation at the forming

phagosome (108) (Fig. 1). Prior to phagocytosis, the enzyme

that synthesizes PI(4,5)P2, PI4P5K, is found at the plasma

membrane, ostensibly bound to its substrate, PI(4)P, through

basic residues in its activation loop (112). The kinase is

therefore also present in the early phagocytic cup (Fig. 1). The

activity of the enzyme at rest is believed to be low; but PI4P5K

isoforms are stimulated by a variety of factors, several of which

are involved in phagocytosis. RhoA and Rac1 can physically

associate with PI4P5K and stimulate its activity (101). In

addition, PA, the product of PLD activity, is also an agonist of

PI4P5K activity (101) (Fig. 1). Because these stimulants

accumulate in the vicinity of activated receptors, it is quite

likely that although present throughout themembrane, PI4P5Ks

become selectively activated in the early phagosomal cup,

explaining the observed accumulation of PI(4,5)P2.

Transfection of inactive mutants of PI4P5K prevented the

accumulation of the PH-PLCd probe at the base of forming

phagosomes (113). More importantly, interference with

PI4P5K activity inhibited phagocytosis as a consequence of

impaired actin polymerization and also possibly by arresting

other signaling pathways (113). PI(4,5)P2 can be hydrolyzed to

generate the second messengers inositol-1,4,5-triphosphate

(IP3) and DAG, whichmobilize calcium stores and activate PKC,

respectively (Fig. 1). Hydrolysis of PI(4,5)P2 was observed

shortly after the initial phase of accumulation (Fig. 2).

Dissociation of the PH-PLCd probe, indicative of elimination

of PI(4,5)P2, was observed even before closure was completed,

first around the base of the phagocytic cup and then throughout

the pseudopods. Disappearance of PI(4,5)P2 likely results, in

part, from PLCg-mediated degradation (Fig. 1). PLCg1 is

recruited to the phagocytic cup through its SH2 domain, and

disruption of PLC function by the inhibitor U73122 prevents

the loss of PI(4,5)P2 (96, 108).

The elimination of PI(4,5)P2 at the time of sealing appears to

be required for successful completion of phagocytosis (Fig. 2).

Treatment with U73122 obliterated particle ingestion (96,

114). Although PLCg2, a highly expressed isoform in

hematopoietic cells, was also observed at the phagocytic cup



(108), macrophages lacking this enzyme perform phagocytosis

normally (115). As in other systems, isoform redundancy may

explain these observations. One of the main functions of PLC-

mediated PI(4,5)P2 hydrolysis is to direct the depolymerization

of the actin meshwork originally accumulated at the forming

phagosome. Indeed, inhibition of PLC activity resulted in

persistent actin accumulation and the inability to complete the

particle internalization process (114).

Another consequence of PI(4,5)P2 hydrolysis is the gener-

ation of IP3 and DAG. The role of IP3 in releasing calcium from

the lumen of the ER is well entrenched, and the occurrence of

calcium transients during phagocytosis has been documented

extensively and is not discussed further. DAG, the other product

of the hydrolysis reaction, can be visualized by the C1 domain of

PKCd, which in resting cells accumulates in a juxtanuclear

organelle, likely the Golgi complex (116). Interestingly, in

macrophages undergoing phagocytosis, the C1-PKCd probe

was recruited to the forming phagosomal membrane and

lingered there for only about 1 min after sealing (108) (Figs 1

and 2). As anticipated, the generation of DAG on the phagosomal

membrane was inhibited by the PLC inhibitor U73122,

implying generation from hydrolysis of PI(4,5)P2 (108).

DAG could conceivably have direct and indirect effects on

phagocytosis. Because of its very small polar head group, DAG is

a type II, cone-shape lipid that can induce negative (concave)

curvature on lipid bilayers, promoting fluidization or disorder

(117). For these reasons, DAGmay facilitatemembrane bulging

and fission. However, the biological effects of DAG are more

likely to be indirect, mediated in large part by its ability to

recruit proteins with C1 domains. These include various

isoforms of PKD, chimaerins, RasGRP, and DAG kinases.

Virtually, nothing is known about these proteins with regards

to phagocytosis. In contrast, a great deal of effort has been

devoted to study another group of C1-domain-bearing

proteins, namely the classical and novel isoforms of PKC. Of

these, most is known about PKCe, thanks to the work of

Lennartz and her group (34). PKCe, through its regulatory

region that includes the C1 domain, is recruited to the forming

phagosome. Competitive displacement of the full-length kinase

using a truncated, kinase-deficient mutant, reduced particle

uptake by approximately 50%, indicating an essential func-

tional role for PKCe. It is noteworthy that a related isoform,

PKCd, that contains an analogous C1 domain failed to be

recruited under identical circumstances, which suggests the

existence of other isoform-specific binding determinants (34).

In contrast, PKCdwas prominent on the membrane of Chlamydia

inclusion vacuoles (118), which bear some similarities to

phagosomes. The target(s) of PKC isoforms and the manner by

which they influence the formation and/or the maturation of

phagosomes are not clearly defined and merit future study.

Phosphatidylinositol 3,4,5-trisphosphate

PI(3,4,5)P3 is a rare phosphoinositide produced by the

phosphorylation of PI(4,5)P2 by class I PI3K. The PI(3,4,5)P3content of cells, which is minimal at rest, increases noticeably

on stimulation by growth factors, chemoattractants, and other

agonists, including ligands of phagocytic receptors. The

appearance, distribution, and fate of PI(3,4,5)P3 can be

monitored in live cells using fluorescently tagged protein

domains that specifically bind to its head group. One such

chimeric construct, consisting of the PH domain of Akt fused to

green fluorescence protein has been used to record the de novo

synthesis of PI(3,4,5)P3 during phagosome formation (109).

The specificity of this construct is imperfect as it also attaches to

PI(3,4)P2 that can be formed by dephosphorylation of

PI(3,4,5)P3 at position 5 or by phosphorylation of PI(4)P on

its position 3, a reaction favored by class II PI3K. Other PH

domains, such as those of Grp1 or Bruton’s tyrosine kinase, are

more specific for PI(3,4,5)P3 but less sensitive than that of Akt.

Use of such fluorescent probes showed that PI(3,4,5)P3becomes markedly accumulated at the cup shortly after

engagement of phagocytic receptors and persists for about

1 min in the newly formed sealed phagosome (109) (Figs 1 and

2). Experiments using cells derived from knockout mice

indicated that class I PI3K is required for this accumulation.

The catalytic p110 subunit of PI3K is associated with a p85

regulatory subunit, which is responsible for targeting of the

complex to the forming phagosome through the interaction of

its SH2 domain with phosphotyrosine residues on activated Syk

(109, 119) (Fig. 1).

Inhibitors of PI3K have been used to evaluate whether

PI(3,4,5)P3 formation is important for phagosome formation

and/or maturation. Interestingly, inhibitors such as wortman-

nin or LY294002 inhibited only the internalization of large

particles (�3 mm in diameter) but affected smaller particles

minimally (120, 121). Moreover, even in the case of large

particles, where phagocytosis was impaired, inhibition of PI3K

did not prevent actin polymerization (120). This result suggests

that class I PI3K likely contributes to membrane remodeling

processes that support the extension of the pseudopods. Indeed,

electron micrographs clearly showed that in wortmannin-

treated cells, incipient pseudopods did form but were unable to

extend sufficiently to surround the prey particle (122). That

delivery of endomembrane vesicles is required for completion

of phagocytosis of large particles was confirmed by electro-

physiological capacitance measurements that showed a net



increase in the area of the plasma membrane during

phagocytosis, as opposed to the decrease that would be

anticipated from simple invagination and vacuolar fission

(123). In addition to its possible role in membrane delivery to

sites of phagocytosis, PI(3,4,5)P3 was also found to be

necessary for the recruitment of myosin X to the phagocytic

cup. Myosin X, a motor protein, is necessary for cell spreading

on IgG-coated substrates and is likely to propel pseudopod

extension and/or close the mouth of open phagosomes in

a purse-string fashion to seal them (124).

The termination of PI(3,4,5)P3-mediated signaling is

important for the timely completion of engulfment. Elimina-

tion of PI(3,4,5)P3 formed on the phagosomal membrane has

been shown to be dependent on the activity of the SH2-

domain-containing inositol 5#-phosphatase (SHIP) (125, 126).

SHIP isoforms have been shown to associate with both the

ITAM and the immunoreceptor tyrosine–based inhibition

motif (ITIM) region of Fcg receptors and are therefore prime

candidates for the dephosphorylation of PI(3,4,5)P3 imme-

diately after phagocytosis is completed. Silencing the gene

encoding SHIP resulted in enhanced Rac activation during Fcgreceptor clustering (127), suggesting that PI(3,4,5)P3 is

required for the maintenance of Rac activation and actin

assembly. However, inhibition of PI3K by LY294002 resulted

in prolonged Rac activation at the phagosomal membrane,

indicating that PI(3,4,5)P3 may also be required for activation

of a Rac-GTPase-activating protein (GAP) that deactivates Rac1

(unpublished results). Interestingly, Beemiller et al. (128)

reported a similar PI3K-dependent inactivation of Arf6 during

phagocytosis, suggesting that PI(3,4,5)P3 may play a more

general role in the termination of GTPase signaling.

Phosphatidylinositol 3-phosphate

PI(3)P is commonly found on the outer (cytosolic) leaflet of

early endosomes and in the internal vesicles of multivesicular

endosomes. This rare phosphoinositide can be generated

through phosphorylation of PI by the class III PI3K, Vps34; by

dephosphorylation of PI(3,5)P2 by 5 phosphatases; or by

sequential dephosphorylation of PI(3,4,5)P3 by 4 and 5

phosphatases (129, 130) (Fig. 3). PI(3)P can be visualized

using probes consisting of two tandem FYVE (Fab1/YOTB/

Vac1/EEA1)domains or the phox (PX) domain tagged with

a fluorophore, by a strategy like that described previously for

other phosphoinositides. In macrophages, PI(3)P was observed

on early endosomes and also on early phagosomes (44, 109).

Microinjection of inhibitory antibodies indicated that phag-

osomal PI(3)P originated from the phosphorylation of PI by

Vps34 (2).

PI(3)P was not readily detectable at the phagosomal cup but

became very apparent about 1 min after the phagosome sealed

(Figs 3 and 4). This observation suggests that PI(3)P is not

important in phagosome formation but may instead play a role

during maturation. Accordingly, Vps34-inhibitory antibodies

were without effect on particle internalization. In contrast,

inhibition of PI(3)P production, whether using antibodies or by

addition of wortmannin immediately after particle engulfment,

prevented fusion of the phagosome with late endosomes/

lysosomes (44, 109). The block of phagosome maturation may

be as a result of the reduced accumulation of the FYVE-domain-

containing protein early endosomal autoantigen 1 (EEA1). In

addition to a FYVE domain, EEA1 possesses two Rab5 binding

domains that are thought to mediate homotypic fusion of early

endosomes (131). Recruitment of Rab5 to endosomes and

phagosomes occurs independently of PI(3)P, and in fact,

elimination of PI(3)P, which inhibits the recruitment of EEA1,

enhanced and prolonged the association of Rab5 with

phagosomes (132).

In addition to its effects on EEA1 and Rab5, wortmannin

treatment significantly reduced the acquisition of the hepato-

cyte growth factor-regulated tyrosine kinase substrate (Hrs) by

the phagosome (133). Hrs is known to interact with the ESCRT

complex to generate MVBs and is also involved in phagosome

maturation (Fig. 3). siRNA directed against Hrs inhibited the

acquisition of LBPA by phagosomes and prevented their luminal

acidification (133). Combined together, these results provide

strong evidence of a role for PI(3)P in phagosome maturation

through the recruitment of various effectors that recognize the

head group of this inositide. How these interactions orchestrate

the fusion of the phagosome with late endosomes and

lysosomes remains to be clarified.

Because of its central role in the early stages of maturation,

PI(3)P is targeted by some pathogens in an attempt to subvert

phagolysosome formation and avoid being killed by macro-

phages. One such pathogen is M. tuberculosis. Vergne et al.

(134) reported that M. tuberculosis secrete a phosphoinositide

3-phosphatase (SapM) on internalization by macrophages.

These investigators found that SapMwas capable of reducing the

levels of PI(3)P on the phagosomal membrane, thereby

contributing to the maturation arrest (134). It is not obvious

how the phosphatase, which is presumably secreted into the

lumen of the phagosome, reaches its cytosolic face where

PI(3)P resides. Nevertheless, it is clear that elimination of the

phosphoinositide could readily explain the inability of

phagosomes to fuse with lysosomes.

In addition to its role in mediating phagosome maturation,

PI(3)P has been shown recently to be critical in the activation of



the NADPH oxidase assembled on the phagosome. Two groups

reported simultaneously that unlike the respiratory burst

elicited at the surface membrane by soluble agonists, the

activation triggered by particulate stimuli required the presence

of the p40 subunit of the NADPH oxidase (135, 136). The p40

subunit is characterized by the presence of a PX domain that

binds with high affinity and selectivity to PI(3)P. Although the

precise mode of action of p40 is not yet clear, it is likely that its

recruitment to the phagosomal membrane is required to

maintain all the necessary subunits in the active configuration

on the phagosomal membrane. After being recruited to the

phagosome by PI(3)P, p40 may fulfill the role normally played

by components like Rac, which are unlikely to remain

associated with the vacuole after sealing (see section on surface

charge subsequently).

Is lipid diffusion limited by a barrier during phagocytosis?

A striking feature of the particle ingestion process is the highly

restricted distribution of some of the products of phosphoi-

nositide metabolism; several lipid species are seen exclusively

at the cup, whereas others are selectively eliminated from this

area, despite its apparent physical continuity with the

remainder of the plasma membrane. Thus, when visualized

using the PH domain of Akt, PI(3,4,5)P3 and/or PI(3,4)P2form in the patch of membrane subtending the engaged

particle, yet are never seen to extend to the immediately

neighboring, unengaged plasma membrane (137) (Fig. 1).

Similarly, the reduction of PI(4,5)P2 seen to occur at the

phagocytic cup was not replenished by lateral diffusion of

undegraded lipid from the rest of the plasma membrane

(108) (Fig. 1). These standing gradients often last for

minutes. Several mechanisms could potentially explain these

observations. One possibility is that the lipids generated

locally by enzymes associated with the phagocytic receptor

complex are continuously and rapidly degraded by phospha-

tases or lipases as they reach the outer edge of the cup. Such

a ‘focal source-peripheral degradation’ model would require

preferential distribution of the degradative enzymes at the rim

of the cup or at least their presence in the unengaged

membrane. In the case of PI(3,4,5)P3, hydrolysis is

principally mediated by SHIP and PTEN; yet, neither one of

these was accumulated at the edge or outside the cup. In fact,

although PTEN appeared to have a diffuse cytosolic distribu-

tion, SHIP was concentrated at the cup itself (137). Hence, no

direct evidence exists to date to support the focal source-

peripheral degradation model. Alternatively, the lipids could

be immobilized through their association with components

of the Fcg receptor signaling complex, as they coalesce into

microdomains on clustering. Using lipid-anchored fluores-

cent proteins as probes to assess lateral mobility in the plane of

the membrane, it was found that diffusion was significantly

reduced at the phagocytic cup compared with the bulk plasma

membrane, and interestingly, the effect was particularly

noticeable for the inner and not the outer leaflet of the

membrane (138). These observations are consistent with

direct binding of some lipids to the receptor complex; but

other explanations are possible. The lipids could be trapped

within the phagocytic cup by a diffusional barrier, perhaps

analogous to the tight junctions of polarized epithelial cells,

which could serve as a fence to constrain lipid exchange across

the boundary of the cup. In this case, we envisage the barrier

to form at the edge of the advancing pseudopods and to

consist of integral membrane components maintained in an

orderly structure through interactions with the underlying

cytoskeleton. Clearly, much additional study is necessary to

understand the mechanism responsible for the formation of

standing lipid gradients in phagocytic cells.

Surface charge, phospholipids, and phagocytosis

The plasma membrane is rich in anionic phospholipids (15–

20 mol%), the majority of which are preferentially distributed

to the inner leaflet (99, 139). This distinctive composition

confers a uniquely negative surface charge to the cytosolic

aspect of the cell membrane. Although monovalent anionic

phospholipids, primarily PS and PI, contribute most of this

surface charge, the polyvalent anionic phosphoinositides,

PI(4)P, PI(4,5)P2, and possibly PI(3,4,5)P3, may also play an

important role, inasmuch as they may be laterally sequestered

into microdomains by polycationic peptides (37, 140). The

accumulation of negative charges on the inner leaflet creates an

electric field equivalent to 105 V/cm that strongly attracts

cationic molecules, including inorganic ions and peptides or

proteins with clusters of cationic residues (141, 142) (Fig. 5).

This electrostatic attraction is effective within the Debye length

(estimated to approximate 1 nm) and, as described by the

Gouy–Chapman theory, is directly proportional to the surface

charge density and inversely proportional to the ionic strength

(141) (Fig. 5). The surface potential is different from the

transmembrane potential, which is largely an electrodiffusional

(Nernst) voltage generated by the differential permeability of

the membrane to inorganic ions. Transmembrane proteins,

such as ion channels, are sensitive to the combined surface and

Nernst potentials, whereas peripheral proteins are only

subjected to the surface potential on their side of the bilayer

(Fig. 5).



The negative surface charge on the inner leaflet of the plasma

membrane is important for the targeting of many intracellular

signaling proteins. Peripheral proteins including the myristoy-

lated alanine-rich C-kinase substrate (MARCKS), c-Src, K-Ras,

and Rac1 contain a cationic motif that is necessary for their

electrostatic attachment to the inner leaflet of the plasmalemma

(143–147). Mutations that reduce or eliminate the charge of

such cationic regions cause dissociation of the proteins from the

inner aspect of the membrane (144–146, 148). It is interesting

that the electrostatic interaction with the membrane depends

neither on the structure of the cationic motif nor on the

particular type of anionic phospholipids. Thus, mutations that

alter the original sequence yet preserve the net cationic charge

of the region have no effect on the localization of the protein

(144–146, 148, 149). However, the cationic motif by itself

appears to be insufficient for polypeptides to localize to the

plasma membrane. It has become apparent that cationic motifs

of membrane-targeted proteins are usually located close to an

acylated or a prenylated moiety (e.g. a myristoyl or a farnesyl

tail) that is important in membrane targeting. Successful

anchorage therefore seems to require two independent

components: an electrostatic interaction and partitioning of

a hydrophobic tail into the bilayer. This ‘coincidence detector’

mechanism ensures that proteins intended to bind to the inner

aspect of the plasma membrane are not mistargeted to other

(non-membranous) polyanionic structures, like DNA.

Because both components of the coincidence detector are

required for successful anchorage, changes in the electrostatic

interaction can alter membrane localization. Modulation of the

net charge of the polypeptide or changes in the surface charge of

the membrane could release bound polypeptides, thereby

operating as binary electrostatic switches, an idea originally

proposed by McLaughlin and Aderem (143). In this manner,

electrostatics could provide a new dimension to the regulation

of a large number of GTPases and of transmembrane proteins

with juxtamembrane cationic domains (150–153).

There are several imaginable ways to toggle the electrostatic

switch. One is to alter the net charge of the bound protein

through phosphorylation of residues in the immediate vicinity

of the cationic motif. In this regard, it has been shown that

phosphorylation of serine and/or threonine residues within the

cationic domain of K-Ras, MARCKs, and the MAPK cascade

scaffold protein Ste5 cause their dissociation from the

membrane (153–155). A secondmechanism involves regulated

ligation of a polyanionic protein to (or near) the polycationic

motif, altering the net charge of the complex. The calcium-

dependent binding of calmodulin, which is polyanionic, has

been documented to promote detachment of K-Ras, MARCKs,

and a juxtamembrane peptide of the epidermal growth factor

receptor from the membrane (155–157). Alternatively, instead

of modifying the protein, the electrostatic change may take

place at the membrane. Metabolism of anionic phospholipids

on the inner leaflet could similarly cause dissociation of cationic

proteins from the plasma membrane. This possibility had not

been contemplated until recently, partly because of the paucity

of information about the surface charge of the inner aspect of

the plasma membrane and of endomembranes in general.

Because extensive phospholipid remodeling occurs during

phagocytosis, as described previously, we decided to explore

whether particle ingestion is accompanied by changes in the

surface charge on the phagosomal membrane. Fast-response

dyes like aminonaphthylethenylpyridinium had been used for

measurements of the outer surface potential of the plasma

membrane. However, this type of probe cannot be selectively

delivered to desired intracellular targets and exhibit very small

fluorescence increments in response to potential changes

(�10% change in fluorescence per 100 mV) and are therefore

Fig. 5. Surface potential attracts cationic molecules to the inner

leaflet of the plasma membrane. Accumulation of anionic phospho-lipids on the inner leaflet of the plasma membrane leads to generationof a negative surface potential (fsurface) within the Debye layer(approximately 1 nm). The surface potential is directly proportional tothe surface charge density and inversely proportional to the ionicstrength of the cytosol. This potential attracts cations and macro-molecules with polybasic motifs. A smaller surface potential is alsothought to exist on the outer surface because of the accumulation ofnegatively charged glycolipids and proteins. Superimposed on thesesurface potentials is an electrodiffusional (Nernst) potential caused byan imbalance in the transmembrane ionic composition and thedifferential ionic conductance of the membrane. Although peripheralproteins are affected by the surface potential on the inner leaflet, theyare not affected by the transmembrane (Nernst) potential. Trans-membrane proteins are sensitive to the combined effects of surface andtransmembrane potential.



not applicable to the study of other surface potentials (158).

This limitation prompted us to develop probes to measure the

charge of the inner surface of the plasmamembrane. Our design

was based on the known properties of K-Ras, which

accumulates on the inner leaflet of the plasmalemma by virtue

of its C-terminal farnesylation and neighboring polycationic

motif. The isolated C-terminal hypervariable region of the

protein behaves similarly. Because posttranslational modifica-

tion such as phosphorylation and ubiquitylation could

potentially alter the membrane localization of the C-terminal

tail of K-Ras, all lysine residues were mutated to arginine, and

the intervening serine/threonine residues that are prime targets

of phosphorylation were replaced with alanine. Covalent

attachment of fluorescent moieties enabled us to monitor the

distribution of the probes bymicroscopy or spectrofluorimetry.

The resulting cationic probe, designated R-pre for arginine (R)

and prenylation, was capable of sensing surface charge changes

both in vitro and inside live cells (31). Two other coincidence

detector probes, designed based on the same principles as R-

pre, behaved similarly.

We proceeded to apply these probes to the measurement of

surface charge during the course of phagocytosis in live

macrophages. Drastic and very acute changes in the distribution

of R-pre were seen to occur during the course of phagocytosis,

and similar observations were made using the two other surface

charge probes. The redistribution of the probes was indicative

of a sudden drop in the negativity of the inner leaflet of the

phagosomal membrane, whereas the unengaged (bulk) plasma

membrane remained unaffected (31). The change in chargewas

correlated with the metabolism of phosphoinositides, specif-

ically with the sharp drop in the content of PI(4,5,)P2 (108).

We also obtained preliminary indications that the PS content of

the phagosomal membrane may have decreased; but caution

must be exercised when interpreting these results, which were

obtained using probes that required fixation, permeabilization,

and, in one case, elevation of calcium, manipulations that may

have altered lipid architecture.

Regardless of whether PS contributes to the drop in surface

charge, these surprising findings imply that receptor-mediated

stimulation of lipid metabolism can alter the net anionic charge

of the membrane. Such alterations can have important

consequences on the localization of surface-charge-sensitive

signaling proteins, which in turn can affect their activity. The

loss of negativity observed during the course of phagosome

formation is expected to cause the redistribution of K-Ras, the

original farnesylated and polycationic protein after which the

surface charge probes were patterned, and other GTPases that

are anchored by similar motifs. In accordance with this

prediction, full-length K-Ras detached quantitatively from the

membrane of the nascent phagosome, whereas the closely

related H-Ras did not (31). The latter is anchored to the

membrane hydrophobically through a dual acylation motif and

hence is insensitive to the charge of the inner leaflet.

Perhaps more importantly, Rac1 was also found to be

released from the phagosomal cup as the lipids remodeled and

the charge fell. Rac1, which initiates actin assembly that drives

pseudopod projection during phagocytosis, also displays a C-

terminal polycationic motif. However, unlike Ras isoforms,

inactive (GDP bound) Rac1 exists in a soluble form, forming

a complexwith GDI proteins (Fig. 6i). Only the active guanosine

triphosphate (GTP)-bound form is thought to be membrane

associated. Partition to and from the membrane is convention-

ally thought to be dictated by guanine nucleotide exchange

factors (GEFs) and GAPs, respectively (Fig. 6iii,v). Accordingly,

constitutively active mutants of Rac1 that are permanently

associated with GTP fail to associate with GDI and reside at the

membrane. Of all the cellular membranes, active Rac1 chooses

to partition almost exclusively at the plasma membrane, which

we attribute to the preferential interaction between its

polycationic tail and the uniquely negative inner leaflet of the

plasmalemma. Importantly, even the constitutively active

mutant form of Rac1 was released from the membrane during

phagocytosis, in parallel with the probes of surface charge (31)

(Fig. 6iv). Because this mutant is permanently associated with

GTP, detachment from the membrane cannot be attributed to

either GAP stimulation or termination of GEF activity. These

results raised the possibility that changes in the lipid

composition of the target membrane may be a major

determinant of the localization and therefore of the activity of

not only Rac1 but also other GTPases with polycationic

targeting motifs. This new layer of control would operate

independently of the state of nucleotide binding of the GTPase.

Charge-dependent control of GTPase activity may occur not

only at the level of release from the membrane but may be

critical also for their recruitment and activation. Indeed, it was

shown recently that Rho-family proteins in their GDP-bound

form can be released from their association with GDIs and bind

to membranes (159, 160) (Fig. 6ii). Using an in vitro assay,

Ugolev et al. (160) showed that Rac1-GDP detached from the

Rac1–RhoGDI complex and partitioned into anionic but not

neutral liposomes. This charge-driven transfer reaction may in

fact be required for the GEFs to access the GTPase (Fig. 6ii,iii).

Crystallographic analysis has shown that GDIs bind not only to

the geranylgernayl tail of the Rho proteins but also to residues in

the switch I and switch II domains, regions of the GTPase that

need to be accessed by the GEFs to exert nucleotide exchange



(161). Detachment of the GDP-bound form of the Rho proteins

from the GDI may therefore be conducive if not an essential

preliminary step for their interaction with GEFs (Fig. 6ii,iii). In

support of this concept, the GDP-to-GTP exchange mediated by

the GEF Tiam1 occurs 10 times more rapidly when its target,

Rac1, is bound to liposomes than when it is complexed with

RhoGDI (159). Therefore, for GTPases with a cationic targeting

motif, the negative surface charge of the membrane may

function as the elusive GDI-displacement factor (161) (Fig. 6ii).

The phagosomal membrane undergoes additional extensive

remodeling during the course of maturation to phagolyso-

somes. Phospholipids such as PI(3)P and LBPA, which are not

present in the plasmalemma or nascent phagosome, appear

transiently in maturing phagosomes. PI(3)P may in turn be

converted to PI(3,5)P2 by PIKfyve; but this has not been shown

directly (Figs 3 and 4). For the most part, however, the precise

composition and sidedness of the lipids of maturing phag-

osomes have not been studied, and there is little evidence

therefore to predict the surface charge of the different stages of

the phagosome. Use of probes like R-premay provide ameasure

of the negativity of these membranes andwill suggest what type

of proteins can interact with them electrostatically.

Concluding remarks and future directions

The lipid distribution of the plasma membrane is highly

asymmetrical, and this asymmetry impacts on the initiation and

development of the phagocytic response. In addition to the

specific role each lipid species plays in recognizing and

recruiting ligands, they act jointly by generating a sizable

surface charge that attracts polycationic ligands electrostatically,

with less structural selectivity. The extent to which each lipid

species contributes to this charge is the subject of debate, with

some groups favoring the polyphosphoinositides (151),

whereas others believe that the very abundant PS and PI must

contribute importantly, despite being monovalent. Although

Fig. 6. Surface charge as a binary regulator of Rho GTPases. (i) Rhofamily GTPases in the GDP-bound state are retained by GDIs in thecytosol. (ii) Rho proteins are occasionally released from the Rho–GDIcomplex and tend to partition onto the plasma membrane as a result ofthe electrostatic interaction between their C-terminal polybasic motifand the negative surface charge on the inner leaflet of theplasmalemma. In this way, surface charge acts as the putative GDF thatpromotes the dissociation of Rho proteins from GDIs and facilitatestheir association with membranes. (iii) Membrane-anchored Rhoproteins in the GDP state can be activated by exchange factors (GEFs),

converting into GTP-bound species that associate with and activate theirrespective effectors. (iv) Membrane and lipid remodeling events duringthe internalization process alter drastically the surface charge on thephagosome, leading to the net loss of negative charge, which in turninduces dissociation of Rho proteins from the phagosome, regardless oftheir nucleotide-binding status. As such, surface charge functions as anelectrostatic switch or binary regulator of Rho GTPases or otherpolycationic ligands. (v) Activated (GTP bound) Rho proteins may alsobe deactivated by GAPs, thereby reentering an equilibrium that favorstheir retention in the cytosol by GDIs.



considerably less abundant, the polyphosphoinositides may

weigh disproportionately if they congregate in islands, a phe-

nomenon that has been suggested to occur in the presence of

polyvalent cationic ligands (37, 140).

Althoughmuch has been learned about lipid distribution and

dynamics in recent years, enormous gaps exist in our

understanding. The development of non-invasive probes in

conjunction with ever more sensitive and quantitative imaging

methods has afforded a new window into the biochemistry and

physiology. However, the availability of probes is still limited,

and some crucial lipids have not been investigated at all. Notable

among these is PS, a major component that serves both the

apoptotic targets and the phagocytic cells to complete the

ingestion process. Ongoing work from several groups,

including our own, may soon yield useful probes to better

monitor and understand the biological life of PS.

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