Phagocytosis: latex leads the way

Phagocytosis: latex leads the wayMichel Desjardins� and Gareth Griffithsy

Phagocytosis is the process that cells have evolved to internalise

large particles such as mineral debris, which they store, or

apoptotic cells and pathogens, which they have the capacity to

kill and degrade. However, several important pathogens can

suppress these killing functions and survive and multiply within

phagosomes, causing disease. Recent advances in phagosome

biology have been made possible largely by a model system that

uses inert latex beads. The ability to purify latex bead-containing

phagosomes has opened the door to allow comprehensive

biochemical analyses and functional assays to study the

molecular mechanisms governing phagosome function. These

approaches have led to unique insights directly relevant for the

understanding of the biology of intracellular pathogens and the

ways by which they subvert their hosts.

Addresses�Departement de Pathologie et Biologie Cellulaire, Universite de

Montreal, CP 6128, Succ. centre ville, Montreal, Canada H3C 3J7

e-mail: [email protected] Molecular Biology Laboratory, Meyerhofstrasse 1,

D-69117 Heidelberg, Germany

e-mail: [email protected]

Current Opinion in Cell Biology 2003, 15:498–503

This review comes from a themed issue on

Membranes and organelles

Edited by Alice Dautry-Varsat and Alberto Luini

0955-0674/$ – see front matter

� 2003 Elsevier Science Ltd. All rights reserved.

DOI 10.1016/S0955-0674(03)00083-8

AbbreviationsLBP latex-bead(-containing) phagosome

M.tb Mycobacterium tuberculosis

PIP2 phosphatidylinositol 4,5-bisphosphate

PM plasma membrane

IntroductionThe process of phagocytosis is restricted to relatively

large particles; a minimum size of 0.5 mm is often stated

but rarely justified. In fact, we believe the credit for

determining a lower size limit for this process should

go to Roberts and Quastel [1] who introduced an elegant

spectrophotometric method to quantify precisely the mass

of latex internalised by cells following solvent solubilisa-

tion of the latex. These authors, and later Weisman and

Korn [2], showed convincingly that the total mass of inert

latex material taken up by neutrophils or Acanthamoebawas exactly the same when they were fed any size of

particle between a lower limit of 0.13–0.26 mm and an

upper limit of 3.0 mm. This argued that cells appear to

have a rather strict sensor of their feeling of ‘fullness’.

Their observation that the total intake dropped consider-

ably when smaller beads were used allowed them to

conclude, quite reasonably, that these were being inter-

nalised by a less efficient process of endocytosis. Despite

a massive internalisation of membrane surface during

phagocytic uptake, Tsan and Berlin [3] showed, using

the Roberts and Quastel method, that neutrophils and

alveolar macrophages could internalise beads without

losing the ability to transport adenosine and adenine into

the cells, implying that some plasma membrane (PM)

transporters were likely to be excluded from the forming

phagosome. These studies were later to be complemen-

ted by those of Pitt et al. [4], who showed that many

receptors that do become internalised with the phagosome

are recycled from phagosomes within 15–30 min. Thus,

some components are excluded from forming phagosome,

while others enter, some of which are recycled; additional

components are acquired by the fusion with endocytic

organelles [5].

In this review, we focus on recent advances in phagosome

biology made possible by using the latex bead system

introduced by Weisman and Korn, which we re-discov-

ered in the early 1990s [6]. These beads are a versatile

system for both in vitro and in vivo analyses of many

phagosome functions. Moreover, one has the option of

coating these beads with insert proteins, or with specific

ligands that bind selectively to cellular receptors.

Use of latex beads as an in vitrophagosome modelThe high level of complexity linked to the molecular

mechanisms involved in the early steps of phagocytosis

and cell surface remodelling, discussed recently by

Greenberg and Grinstein [7], is only one example of

the intricate pathways that coordinate particle entry

and phagolysosome biogenesis. Although it is important

to address these processes at the cellular and tissue levels,

it seems likely that detailed mechanisms will ultimately

emerge from the use of in vitro systems. In the case of

membrane organelles, an obvious requirement for in vitroassays and biochemical analyses is that the organelles be as

pure as possible. For most intracellular organelles, includ-

ing phagosomes containing microorganisms, it is very

difficult to achieve this goal and a variable, and often

significant, extent of contamination can hardly be avoided.

In the early 1990, we ‘re-discovered’ the method intro-

duced by Wetzel and Korn [8] to isolate latex-bead-

containing phagosomes (LBPs), and used it to analyse

phagosome functions in the J774 mouse macrophage cell

498

Current Opinion in Cell Biology 2003, 15:498–503 www.current-opinion.com

line. Our first protein and lipid analyses, indicating that

phagosome composition was not as simple as expected,

reflected the high level of complexity of the cellular

processes involved in phagolysosome biogenesis [6,9].

The early finding that sets of small GTPases, shown to

be involved in the regulation of membrane fusion [10],

associated sequentially with phagosomes led to the obser-

vation that phagolysosome biogenesis proceeded by a

series of transient interactions between phagosomes,

early endosomes, late endosomes and lysosomes [6,11].

Recent proteomic analysis of LBPs from J774 macro-

phages led to the identification of about 150 proteins

[12]. Although most of these proteins, such as hydrolase,

subunits of the vacuolar proton pump, or small GTPases,

were expected on an organelle involved in the killing and

degradation of microorganisms, other unexpected ones

led to surprises, as we shall now discuss.

Origin of the phagosomal membraneUntil recently, every model describing phagocytosis pre-

sented the PM as the major and only source of membrane

used to form phagosomes. Indeed, because particles were

internalised from outside the cell, the only possible and

logical source of membrane seemed to be the PM. How-

ever, Vicker [13] showed that an important part of the

phagosome membrane was apparently made of newly

synthesised membranes of undefined origin. Capacitance

measurements made during phagocytosis of latex beads

in macrophages led to the proposal that endomembranes

were recruited at the cell surface for phagosome forma-

tion by a process referred to as ‘focal exocytosis’ [14].

Grinstein and colleagues [15,16] have shown recently that

exocytosis of membranes, possibly originating from recy-

cling endosomes, at or near the site of phagocytosis is

required for complete particle internalisation. Consider-

ing the amount of membrane needed for the internalisa-

tion of several parasites or of large particles within a few

minutes, the notion that other organelles, in addition to

recycling endosomes, might be involved in the entry

process was reasonable.

Theidentificationbyproteomicsanalysisofseveralproteins

from the endoplasmic reticulum (ER) in LBPs_suggested

that ER might be involved in the formation of phago-

somes or phagolysosomes [12]. Recently, elegant work by

Gerisch and co-workers [17�] provided evidence that the

ER is functionally involved in the phagocytic uptake

process in Dictyostelium. Using green fluorescent protein

constructs of the ER proteins calreticulin and calnexin,

they observed transient contacts between elements of

the ER and forming phagosomes. Strikingly, phagocy-

tosis was strongly inhibited when they deleted both of

these ER proteins. In macrophages, we demonstrated

using biochemical approaches and immunogold electron

microscopy labelling that ER proteins are genuine com-

ponents of phagosomal membranes [18�]. Furthermore,

we provided evidence that the ER is recruited at the cell

surface, where it appears to fuse directly with the PM

underneath phagocytic cups, to supply membrane for the

formation of nascent phagosomes [18�]. This process,

referred to as ‘ER-mediated phagocytosis’, was not only

used during the internalisation of inert latex beads, but

also for the internalisation of intracellular pathogens

including Salmonella and Leishmania.

Interestingly, although the molecular mechanisms involv-

ed in ER recruitment and ER–PM fusion remain to be

elucidated, reconstituted liposomes displaying Sec22, an

ER SNARE (soluble N-ethylmaleimide-sensitive factor

attachment protein receptor) molecule, could support

fusion with other liposomes displaying the plasma mem-

brane t-SNARE Sso1/Sec9c [19]. The obvious advantage

of using ER as a source of membrane for phagocytosis in

macrophage is its abundance. It is not clear whether ER-

mediated phagocytosis merely provides a reservoir of

membrane, or whether it also contributes directly to the

functional properties of phagosomes, for example by pro-

viding lipids or proteins that can modulate phagosomal

membrane signalling. Some of the possible new phago-

some functions linked to ER-mediated phagocytosis have

been discussed recently [20].

Phagosome–actin interactionsProteomic analysis of purified LBPs also led to the

identification of numerous actin-binding proteins, pre-

sent on phagosomes at various steps of their maturation,

indicating a potential role for actin at all stages of

phagolysosome biogenesis [9,12]. When a phagocytic

particle contacts receptors at the cell surface, among

the first demonstratable events is the transmembrane

signalling leading to a local membrane-induced poly-

merisation of actin on the cytoplasmic surface of forming

phagosomes [7]. This rapid process, accompanied by an

equally rapid depolymerisation of actin [21], has been

seen in many PM signalling systems (see [22]). Although

the significance of these phenomena is still poorly under-

stood, it has recently emerged that the LBP provides an

excellent system to analyse different kinds of membrane

interactions with actin.

In vitro assays to monitor actin binding andnucleation on phagosomesThe ability to isolate highly purified preparations of LBPs

has allowed us to develop in vitro assays to study the

molecular mechanisms governing the interaction of phago-

somes with the actin cytoskeleton. Al Haddad et al. [23�]established a simple in vitro fluorescence-based assay to

monitor the binding of LBP to F-actin. In contrast to actin

assembly, which operates independently of cytosol, the

LBPs do not bind significantly to actin unless exogenous

cytosol is added. This analysis has revealed that at least two

different cytosolic factors are responsible for binding LBP

to F-actin. First, an unknown ATP-independent factor of

about 600 kDa has been identified by gel filtration [23�].

Phagocytosis: latex leads the way Desjardins and Griffiths 499

www.current-opinion.com Current Opinion in Cell Biology 2003, 15:498–503

The second factor is myosin V. The binding of this, like all

myosins, is abrogated in the presence of ATP. The func-

tion of myosin V has also been investigated with respect to

LBPs in macrophages. It appears that when this myosin

links phagosomes to F-actin it retards the rate at which

these organelles move along microtubules from the cell

periphery to the perinuclear region; in cells lacking this

myosin, the LBPs move more quickly to cell centre. As in

other systems, myosin–actin interactions operate in a kind

of antagonistic relationship with the microtubule cytoske-

leton (see [23�] and references therein).

A second, relatively simple fluorescence-based assay was

developed by Defacque et al. [21,24] to monitor the denovo assembly of F-actin by the LBP membrane. Since

neither cytosol nor GTP is needed for this process, the

nucleation and polymerisation of actin appear to be

linked to inherent properties of the phagosomal mem-

brane. In the LBP system, as in all known examples of

membrane-catalysed (end-on) actin assembly, the polar-

ity of the filaments is such that the fast-growing barbed

ends of actin are localised at the membrane, while the

slow-growing pointed end faces away from the membrane

(Figure 1) [25]. This means that the machinery must

nucleate the filament and then continually insert actin

monomers at the assembly site. Recently, Dickinson et al.[26] and Dickinson and Purich [27�] have introduced an

interesting model that takes into consideration how the

filament stays attached to a surface (including a mem-

brane) while still allowing monomer insertion (Figure 1).

These authors propose an ingenious bridge-like clamp

structure that is stably attached to the membrane surface,

allowing actin monomers to insert into its core to nucleate

filaments that grow outwards from the membrane. They

propose that the clamp binds tightly to ATP–monomeric-

actin; but when ATP hydrolysis occurs (at the barbed

end), this changes the conformation of actin, leading to

the loosening of the clamp, which then binds to the next

ATP monomer, closer to the membrane. Although the

molecular composition of this machine is not yet specified

(except that profiling–actin is considered to be an impor-

tant part of it), we consider this idea to be an important

conceptual advance — because this is the first model

explicitly to acknowledge that the machinery needs to

stay membrane-bound during the (still elusive) process of

nucleation/insertion/growth of F-actin.

In the LBP actin-assembly assay, an important role for

ezrin and/or moesin has been demonstrated [21]. These

proteins are known to bind to both actin and phosphati-

dylinositol 4,5-bisphosphate (PIP2) [28] and the PIP2-

binding site of ezrin is necessary for this protein to

function efficiently on the LBP [29�]. We believe that

synthesis and breakdown of PIP2 are important in the

assembly process. Gelsolin and profilin are also impli-

cated ([24]). In addition to the standard process of actin

nucleation as it occurs on the surface of the LBP, a recent

study by Zhang et al. [30] revealed that a small fraction of

LBP (both uncoated and IgG-coated beads) is transiently

able to nucleate actin comets that can propel the LBP at

rather slow speeds (around 0.1 mm/s) in macrophages.

These comets, first seen on the surface of intracellular

Listeria monocytogenes [31], appear to move phagosomes

predominantly from the perinuclear region to the periph-

ery, similarly to an outward actin-based movement of late

endosomes and lysosomes seen previously by Taunton

et al. [32]. What signals the activation of these comets, as

well as their function, remains to be addressed. Given the

fact that in so many systems different states of phago-

somes can exist in the very same cell (e.g. one fraction is

fusogenic while the rest are not), it seems likely that the

signal that switches on these comets will operate locally at

the level of the individual phagosome.

Role of membrane-dependent actinassembly: from latex-bead-containingphagosomes to mycobacterial phagosomesIn vitro analysis in the presence of cytosol has led to the

working hypothesis that the actin that polymerises on the

Figure 1

A

A

B

Current Opinion in Cell Biology

The actin track model postulates that actin filaments assembled on the

surface of a membrane organelle A (e.g. a phagosome) can provide

tracks for a fusion partner organelle B (e.g. a lysosome) to be attractedto A, to facilitate docking before fusion. The polarity of actin (i.e. with

actin barbed ends at the membrane) ensures that any organelle

containing a myosin (except myosins VI or X) will be transported towards

the nucleator. The mechanism by which actin is nucleated and inserted

at the phagosome membrane surface (or indeed any membrane surface)

is quite mysterious, despite identification of some of the components

involved (see text for full details). An attractive theoretical,

mechanochemical model has recently been proposed by Dickinson et al.

(see [26,27�] for full details), which they refer to as the ‘lock, load and

fire’ model.

500 Membranes and organelles


surface of phagosomes and late endocytic organelles (but

not early endosomes, which failed to nucleate actin invitro in our hands) has the right polarity (i.e. with the actin

plus or barbed ends adjacent to the membrane) to provide

tracks for fusion partner organelles to move, using their

bound myosins, towards the nucleating organelle ([33�];Kjeken et al., unpublished data; Figure 1). This ‘actin

track’ hypothesis would also be consistent with the role of

actin in other systems, such as the transport of secretory

vesicles from the yeast mother cell to the daughter bud

(see [34]). Although we do not yet have a realistic model

for how the actin-assembly machinery operates on the

LBP membrane, it recently emerged that a large number

of signalling molecules are involved in the regulation of

this process, even in vitro. We have identified over 30

lipids and protein effectors that, when added to the LBP,

can induce either stimulation or inhibition of actin assem-

bly, in a process that seems to be further modulated by

ATP (E Anes et al., unpublished data).

This finding became crucial when we applied the LBP

technology to the analysis of mycobacterial phagosomes.

It is well established that non-pathogenic mycobacteria,

such as Mycobacterium smegmatis, or killed pathogenic ones

such as M. tuberculosis (M.tb) or M. avium, enter macro-

phage phagosomes, which fully mature. These phago-

somes fuse with the entire endocytic pathway, including

the late endosomes and lysosomes, thereby acquiring

both the acid hydrolases and the full complement of

the proton ATPase that allows the phagosome to acidify

to pH 5 or below [35,36]. As a consequence, these bacteria

are effectively cleared by macrophages [37].

By contrast, the phagosomes containing live pathogens

fail to fuse with late endocytic organelles, are defective in

acidification, and pathogens have a high probability of

survival and growth within phagosomes, facilitating dis-

ease. When we tested isolated phagosomes containing

mycobacteria in the LBP actin-assembly assay, we

observed yet another difference between the non-patho-

gens (or killed pathogens) and the pathogens. The live

M. smegmatis, killed M. avium or M.tb phagosomes

nucleated actin in a manner that was remarkably similar

to the LBP, with a similar response towards a large

number of effectors. By contrast, the phagosomes enclos-

ing pathogens were strongly inhibited in actin assembly,

both in vitro and in vivo, in agreement with Guerin and de

Chastellier [38]. Lipids have recently been identified

that can switch on not only actin, but also the fusion with

lysosomes, leading to a significant increase in phagosome

maturation and pathogen killing (E Anes et al., unpub-

lished data).

From latex to other intracellular pathogensIn addition to their use in analyses of mycobacteria, LBPs

have also provided important insights into the functions

of other pathogens that reside within host cells, and

especially concerning the nature of the alterations occur-

ring on phagosomes during infection. To cite a few

examples, the use of latex beads coated with proteins

from Shigella flexneri or Listeria monocytogenes demonstrated

that a complex comprising IpaB, IpaC and IpaD, or

internalin (Inl) A and B, were sufficient to promote the

respective internalisation of these bacteria [39,40]. More-

over, differences in the proteome of phagosomes formed

by the internalisation of beads through InlA and InlB

were observed [41]. More recently, the observation that

flotillin-1, a protein originally assigned to lipid rafts at the

cell surface [42], was present in latex bead-containing

phagosomes [12] led to the demonstration that lipid rafts

were also present on this organelle [43].

Thus, instead of being made of lipids and proteins

randomly distributed in their membrane, phagosomes

display distinct membrane microdomains where specific

functions are likely to occur. Although the nature of these

functions is still unknown, the fact that the intracellular

parasite Leishmania donovani survives in phagosomes

lacking flotillin-1-enriched microdomains indicates that

these microdomains are likely to play critical roles in the

microbicidal properties of this intracellular pathogen [43].

ConclusionsOur ongoing proteomics analyses of LBPs from J774 cells

indicates that at least 600 different proteins are present

out of an estimated 1000 polypeptides on phagosomes;

this number does not consider the plethora of forms

displaying post-translational modifications. We also

estimate that there are more than 150 integral mem-

brane proteins, including several uncharacterised ones

(M Desjardins, unpublished data). The amount of several

of these proteins on phagosomes is modulated during

phagolysosome biogenesis. Mass spectrometry analyses

also indicate that several dozen distinct lipids are present

on phagosomes (G Griffiths, unpublished data). These

results confirm the tremendous complexity associated with

the functional properties of phagosomes at the molecular

level. Since many of these molecules will be essential for

intraphagosomal pathogens to grow, targeting some of

them might be considered as a therapeutic strategy.

Whereas LBPs are easy to isolate in a pure state, it is much

more difficult to purify pathogen-containing phagosomes.

Towards this goal, further refinements, such as fluores-

cence-assisted organelle sorting [44], can allow the isola-

tion of phagosomes containing fluorescently labelled

pathogens; alternatively, affinity-based approaches, such

as immunoisolation could also be useful. A key goal of

such analyses will be to identify molecular differences

between non-virulent strains of a pathogen that are gen-

erally killed by professional phagocytes, and the virulent

strains that are more likely to survive and grow in these

cells. The use of latex particles as a system to mimic

host–pathogen interaction during infectious diseases will



continue to have an important impact in our understand-

ing of phagocytosis and phagolysosome biogenesis in

health and disease.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest��of outstanding interest

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This is an impressive study using GFP-labelled ER proteins calnexin andcalreticulin, showing that the ER transiently contacts (�30 s) the formingphagosome in Dictyostelium. Although knock out of either protein alonehas little effect, the double knockout effectively blocks phagocytosis. Thisstudy beautifully complements the work of Gagnon et al. (2002) [18�].

18.�

Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH,Steele-Mortimer O, Paiement J, Bergeron JJM, Desjardins M:

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A simple but elegant assay was developed to monitor the cytosol-dependent binding of LBP to F-actin bound to glass. This assay revealedan important role for myosin-V, and other components mentioned in thebody of the review.

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33.�

Jahraus A, Egeberg M, Hinner B, Habermann A, Sackmann E, PralleA, Faulstich H, Rybin A, Defacque H, Griffiths G: ATP-dependentmembrane assembly of F-actin facilitates membrane fusion.Mol Biol Cell 2001, 12:155-170.

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502 Membranes and organelles


measurements showed that at the end of the actin polymerisation process(�30 min), the actin rapidly re-organises into a gel-like state: The parallelstudy by Kjeken et al. (unpublished data; see text) shows that actin bundleformation and the actin-assisted fusion of LBPs and late endocyticorganelles occurs before the gel-like state.

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Phagocytosis: latex leads the way

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