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European Journal of Cell Biology 85 (2006) 1001–1010

0171-9335/$ - se

doi:10.1016/j.ej

�Correspondfax: +1405 271

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www.elsevier.de/ejcb

Phagocyte meets prey: Uptake, internalization, and killing of bacteria by

Dictyostelium amoebae

Margaret Clarke�, Lucinda Maddera

Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, 825 N.E. 13th Street,

Oklahoma City, OK 73104, USA

Abstract

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probeshave allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we havevisualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf abacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome awayfrom the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we havemonitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicatingpermeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during aperiod of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidicendosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysisand acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of thephagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur muchmore rapidly than had been inferred from previous population studies.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Amoeba; Phagocytosis; Actin; Microtubule; Coronin; Arp2/3; Microtubule; Bacteria

Tribute to Gunther Gerisch

I (MC) have had the privilege of collaborative work

with Dr. Gerisch since 1999. I learned from him the

delights and revelations of live-cell microscopy. He shared

freely his unparalleled knowledge of Dictyostelium cell

biology, his wonderful and always-expanding collection of

fluorescent probes, his access to equipment and expertise,

and his rigorous and incisive analysis of data. Time spent

with him has enriched my life not only in science, but also

e front matter r 2006 Elsevier GmbH. All rights reserved.

cb.2006.05.004

ing author. Tel.: +1405 271 7664;

7312.

ess: clarkem@omrf.ouhsc.edu (M. Clarke).

in the realms of music, art, and history. I count myself

fortunate to know him as colleague, mentor, and friend.

Introduction

Dictyostelium amoebae are professional phagocyteswhose primary food source in the wild is bacteria. Theamoebae are very efficient at capturing and digestingtheir prey. Mutants of Dictyostelium capable of rapiduptake of fluid by macropinocytosis also exist, havingbeen selected for growth on liquid nutrient medium inthe laboratory. Both types of endocytosis have been thesubject of many studies at the levels of biochemistryand/or microscopy (reviewed by Cardelli, 2001; Duhon

ARTICLE IN PRESSM. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–10101002

and Cardelli, 2002; Maniak, 2001, 2002). In general,studies using live-cell microscopy have employed fluo-rescent fluid-phase markers to visualize macropinocy-tosis, and large, easily tracked objects such as yeast cellsor latex beads to monitor phagocytosis. In the presentstudy, we have employed confocal microscopy ofindividual living cells to examine the uptake anddigestion of bacteria, the prey that Dictyostelium cellsnormally consume. Using fluorescent tags for proteinsthat function in phagocytosis, we have distinguished aseries of steps that characterize early endocytic transitfor phagosomes containing bacterial prey.

Materials and methods

Strains and culture conditions

Dictyostelium discoideum strain AX2 and its fluo-rescent derivatives were cultured at 22 1C in HL5(Clarke et al., 1980). As appropriate, 10 mg/ml G418and/or 10 mg/ml blasticidin were included for mainte-nance of plasmid(s) encoding fluorescent fusion pro-teins. AX2 cells expressing GFP-Arp3 (Insall et al.,2001), GFP-a-tubulin (Neujahr et al., 1998), or coronin-GFP (Maniak et al., 1995) were used. In some cases, asindicated, the cells were also expressing mRFP-LimE-delta-coil (Fischer et al., 2004), called LimED here.Escherichia coli B/r expressing the IPTG-induciblefluorescent markers DsRed-Express (Otto et al., 2004)or GFP (Hilbi et al., 2001) were cultured at 37 1C in LBcontaining chloramphenicol (0.5mg/ml) and IPTG(20–500 nM, depending on the desired expression levelof GFP or DsRed). Bacteria expressing these fluorescentproteins in their cytoplasm are referred to as E. coli-DsRed and E. coli-GFP in this report.

Phagocytosis and confocal microscopy conditions

Bacteria were washed twice in buffer containing10mM Na2HPO4, 10mM KH2PO4, 2mM MgCl2,0.2mM CaCl2 (pH 6.3) (PBMC), then diluted 50-foldin PBMC, and 0.5ml was plated in microscopechambers with 30-mm diameter circular cover slipbottoms (WillCo Wells, B.V., GWSt-5030). The bacteriawere allowed to settle at 37 1C for at least 10minutes, orup to 2 hours, before use. AX2 cells were harvested fromexponentially growing cultures and washed once withPBMC, after which they were incubated in lowfluorescence medium (LF, Liu et al., 2002) for up to4 h or in PBMC for 30–60minutes before use. For someexperiments, neutral red (25–125 mM) was present in theLF or PBMC. The cells (�2� 105 cells) were added to amicroscope chamber with previously plated E. coli,which had been shifted to the cold room a few minutes

earlier, and were allowed to settle in the cold for15–30minutes before being overlaid with a thin sheet of2% agarose and transferred to the microscope forviewing at room temperature. The cells were imagedwith a Zeiss LSM 510 confocal microscope using a Plan-Apochromat 63� 1.4 N.A. DIC objective. S65T-GFPwas excited with the 488-nm laser line of an argon laserwith a 505–530-nm filter for emission, and DsRed wasexcited with the 543-nm line of a HeNe laser, with a 560-nm long pass filter for emission. An HFT UV/488/543/633 beam splitter was used. The lasers were kept at verylow power to minimize photodamage to the cells.

Results

Visualization of actin dynamics during phagosome

formation and internalization

Using Dictyostelium amoebae expressing mRFP-LimED, a protein that brightly labels actin filaments(see Discussion), we visualized the phagocytosis of greenfluorescent bacteria. Figure 1 shows a cell that hadsettled on the cover slip near a clump of bacteria (E. coli-GFP). The cell proceeded to move into the clump,steadily ingesting the bacteria that were contacted by itsleading lamellipodium. The uptake of one bacterium (a)is shown in the top row of Figure 1. Here, the nascentphagosome was initially coated with actin filaments,labeled with mRFP-LimED, which disappeared after thephagosome sealed. About 15 seconds later, new mRFP-LimED appeared between the phagosome and theplasma membrane, and the phagosome moved awayfrom the site of uptake with a bright comet tail of actinfilaments. A succession of five such events is recorded inMovie 1. The duration of the actin-based movementranged from a few seconds to up to 30 seconds,whereupon the phagosome paused, and the comet tailof F-actin dissipated. The phagosome then remainedstationary for a period of time that ranged from secondsto minutes. The next movement of the phagosome was asudden displacement without any indication of actininvolvement. Two examples of this displacement areshown in the second row of Figure 1. Each set of threeimages shows first, the actin comet tail that accompa-nied the newly internalized phagosome, second, thephagosome at rest, and third (one frame later), thedisplaced phagosome. As we show later in this report,the second type of movement reflects the interaction ofthe phagosome with microtubules.

The dynamics of actin filament assembly duringphagocytic cup formation and phagosome internaliza-tion were visualized by pairing mRFP-LimED with asecond marker for actin filaments, coronin-GFP. Theinterplay of these two markers is illustrated in Figure

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Fig. 1. F-actin assembly during the formation and internalization of phagosomes containing bacteria. The images are frames from a

confocal time series (Movie 1) showing E. coli-GFP being phagocytosed by a Dictyostelium cell expressing mRFP-LimED. In all

figures, the numbers are seconds elapsed since the first frame. The top row shows F-actin (labeled with mRFP-LimED) surroundingthe phagocytic cup (a, 0 seconds) and just-completed phagosome (16 seconds); this circumferential labeling disappeared after the

phagosome sealed. A short while later, new F-actin appeared between the phagosome and the plasma membrane (43 seconds). This

F-actin formed a comet tail as the phagosome moved away from the site of entry (59 and 79 seconds), a process that lasted about

30 seconds. The bottom row shows three images each of two additional bacteria (b and c) that were phagocytosed by the same cell.

These phagosomes also displayed F-actin comet tails as they moved away from the site of entry (first image of each set of three). The

second image shows the stationary phagosome after the F-actin coat had disappeared. The third image, one frame later, shows

displacement of the phagosome without actin involvement. The marked phagosomes are the (a) first, (b) second, and (c) fifth

phagosomes internalized during Movie 1, which spans 6minutes. Bars, 5mm.

M. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–1010 1003

2A, which shows the spontaneous formation of atransient small bleb or protrusion of the plasmamembrane. Red mRFP-LimED marks the leading edgeof the membrane protrusion, but after one frame(�4 seconds later), there is overlapping binding bycoronin-GFP, which replaces mRFP-LimED completelywithin a few frames. Coronin-GFP binding disappearsas the actin filaments are disassembled and the protru-sion collapses. This event and several others like it areseen in Movie 2. The transition of a protrusion from redto green generally occurred within three frames(�12 seconds) and the green signal disappeared about1 frame later, suggesting a lifetime for the actin filamentsof about 16 seconds.

This same cell phagocytosed three bacteria during thecourse of the time series (Movie 2). The bacteria are onlyfaintly labeled; they had been induced to express a lowlevel of DsRed so as not to obscure the red signal frommRFP-LimED. At the top of the cell, a bacterium wasinitially the subject of a failed attempt at phagocytosis,but later it was successfully ingested. Two bacteria at thebottom of the cell were phagocytosed without hesita-tion. Figure 2B highlights several features of thesephagocytic events. The assembly of new actin, detectedwith mRFP-LimED, occurred at the leading edge of theextending phagocytic cup (arrows), with coronin-GFPbinding just behind. By the time the phagosome hadsealed, actin filaments had begun to disappear from the

side of the phagosome that was first internalized, andthis actin disassembly continued until the phagosomewas largely free of label. After a variable lag, newF-actin appeared between the phagosome and theplasma membrane, and the phagosome moved awayfrom the membrane with a comet tail of actin filaments.Often, and in two of the three phagocytic events shownin Movie 2, new actin assembly was initiated both at thesite of phagosome uptake, producing a small plasmamembrane protrusion, and also close to the phagosomeas it moved away. Similar mechanisms may account forboth types of movement (see Discussion).

An additional example of phagocytosis by a cellexpressing these two F-actin markers is shown in Figure2C. Like the cell in Figure 1, this cell had approached aclump of bacteria and begun ingesting bacteria one afteranother at its leading edge. The formation and inter-nalization of one phagosome is tracked in Figure 2C.The phagocytosed bacterium was only faintly red, so ithas been overlaid in each panel by an arrowheadshowing the position and direction of movement of thephagosome. The actin-based movement of this phago-some persisted through several frames, making it easy tosee that new F-actin was continually added just to therear of the moving phagosome. Additional examples areseen in Movie 3.

To test the possible involvement of the Arp2/3complex in the new actin assembly that accompanies

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Fig. 2. F-actin dynamics during membrane protrusion, phagocytic cup formation, and phagosome internalization. This figure shows

Dictyostelium cells expressing both coronin-GFP and mRFP-LimED. The behavior of these two markers with respect to actin

dynamics is illustrated in Figure 2A, which shows the appearance and disappearance of a small, transient plasma membrane

protrusion or bleb. mRFP-LimED labeled new F-actin at the leading edge of the growing protrusion (0 and 4 seconds). Beginning in

the second frame, coronin-GFP overlapped with and progressively replaced the mRFP-LimED, until only the green signal remained

(16 seconds). As the coronin-GFP signal disappeared (32 and 48 seconds), the protrusion gradually collapsed. The images are frames

from a confocal time series (Movie 2). Figure 2B shows actin dynamics during the phagocytosis of bacteria by the same cell. The

leading edge of a phagocytic cup displayed the red LimED signal, with coronin-GFP labeling the slightly older filaments just behind

(0 and 47 seconds). By the time the phagosome had sealed (95 seconds), neither actin marker was associated with the end of the

phagosome that had been first formed, and only coronin-GFP remained at the end that had just sealed. The coronin-GFP had

dwindled to a trace in the next image (122 seconds), and the phagosome membrane was devoid of label. At 130 seconds, there was

new actin assembly (detected with mRFP-LimED) at the plasma membrane (arrow) and a hint of red at the phagosome (second

arrow). At 138 seconds, new mRFP LimED could be clearly seen close to the phagosome (arrow) as it moved away from the site of

uptake; this signal persisted for only two frames (8 seconds). Panel B0 shows the same internalization step for the phagosome at the

top of the cell. Again, there were two sites of new actin assembly (marked by two arrows), one at the original site of entry and the

other next to the phagosome. The events shown in Figure 2A, B, and B0 are high-magnification views from Movie 2. Figure 2C

shows the uptake of a bacterium by a cell in the process of phagocytosing several bacteria from a clump contacted by its leading

lamellipodium. The dynamics of actin assembly were clearly displayed as each new phagosome moved away from the site of uptake

(Movie 3). Red mRFP-LimED always labeled the actin filaments closest to the moving phagosome, with coronin-GFP overlapping a

short distance behind (yellow) and becoming the sole label (green) further back in the comet tail. Bars, 2 mm (A, B, B0); 10mm (C).

M. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–10101004

phagosome internalization, we monitored phagocytosisof E. coli-DsRed by Dictyostelium cells expressing GFP-Arp3. As shown in Figure 3 and Movie 4, GFP-Arp3faintly labeled the phagocytic cup, but disappeared fromthe completed phagosome. Shortly thereafter, a brightcomet tail of GFP-Arp3 trailed the phagosome as itmoved away from the site of uptake. These resultssuggest that the Arp2/3 complex plays a role in the newactin assembly that powers phagosome internalization.Note that during the internalization step, phagosomemovement was often broadside to the direction of travel(also evident in Figure 1A). Also note that phagosome(a) in Figure 3A, after being internalized, moved fromthe cell center to the cell periphery and back to the cellcenter without any further GFP-Arp3 involvement. This

pattern of movement is consistent with microtubule-based transport.

Visualization of the digestion of bacteria

Information regarding early steps in phagosomematuration could be obtained by observing the fluo-rescent proteins expressed in the bacterial cytoplasm andthe contrasting backdrop of cytoplasmic fluorescence inthe Dictyostelium cell. For cells pressed rather flat by theagar overlay, it was possible to track individualphagosomes after their uptake and determine when thebacterial fluorescence was lost. Subsequent changes inphagosome morphology could also be observed in

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Fig. 3. Arp2/3 dynamics during phagosome internalization. This figure shows two Dictyostelium cells expressing GFP-Arp3 in the

process of phagocytosing E. coli-DsRed. The images in the top row (A) are frames from Movie 4. The cell in (A) ingested several

bacteria during the two and one-half minutes of observation. Each new phagosome moved away from the site of entry with a tail of

GFP-Arp3 (marked with parallel arrows). The cell shown in (B) expressed a higher level of GFP-Arp3, allowing better visualization

of the weaker GFP-Arp3 signal surrounding the phagocytic cup. Phagosomes (a) and (b), being formed in panel 0, were completed

by 20 seconds and moved inward with tails of GFP-Arp3 at 47 s; phagosomes (c) and (d) behaved similarly between 47 and

106 seconds. For all four phagosomes, there was brighter labeling of the comet tail than of the phagocytic cup. Bars, 10 mm.

M. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–1010 1005

negative contrast against the cytoplasm of the host cell.An example may be seen in Figure 4A, which shows acell expressing mRFP-LimED phagocytosing E. coli

expressing GFP. When we began our recording, theDictyostelium cell already contained several no-longer-fluorescent bacteria faintly visible in phagosomes withinthe cell. These phagosomes were at various stages ofmaturation, appearing as dark ovoid or round vacuolesin which a bacterium could occasionally be detected byDIC optics. The stages that could be observed were theinitial close apposition of the phagosome membrane to anewly ingested bacterium, the loss of bacterial fluores-cence, and the expansion of the phagosome, first to anovoid and then to a round morphology.

During our recording (Movie 5), the cell phagocy-tosed two new bacteria. The formation of the firstphagosome (arrowhead) is shown in panel 0. Thisphagosome was tracked until only a trace of bacterialfluorescence remained (153 seconds). The second phago-some (tailed arrow) was in the process of being formedat 153 seconds; it was also tracked until the fluorescenceof the bacterium had largely disappeared (384 seconds).Similar data were collected for several other phago-somes containing E. coli expressing GFP or DsRed. Ingeneral, bacterial fluorescence was lost within three tofour minutes after uptake of the phagosome, andexpansion of the phagosome from a snug fit to an ovoid

or irregular morphology occurred in a similar timeframe.

We used a second probe to examine the relationshipbetween loss of bacterial fluorescence and acidificationof the bacterial contents (Fig. 4B and Movie 6). Forthese experiments, we incubated AX2 cells with neutralred, a vital stain that fluoresces red in an acidicenvironment. The contents of endosomes in the acidicstage of endocytic transit are brightly labeled by thisstain. Figure 4B shows an AX2 cell that had beenincubated in LF medium containing neutral red (finalconcentration 125 mM) for about 30minutes beforebeing added to a glass cover slip coated with E. coli-GFP. At 0 seconds, the cell shown in Figure 4Bcontained a green bacterium that had recently beentransported to the center of the cell; that phagosome (a)was surrounded by acidic endosomes labeled withneutral red. A phagosome containing a second recentlyinternalized bacterium (b) had paused near the cellperiphery in the first panel, but was transported to thecell center about 40 seconds later (see Movie 6). At thecell center, each bacterium lost its green cytoplasmicfluorescence and presently became brightly stained withneutral red. These data and related experiments showedthat the bacterial contents became acidic and accessibleto neutral red within one to three minutes after thecytoplasmic bacterial fluorescence had disappeared.

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Fig. 4. Digestion of fluorescent bacteria and acidification of bacterial contents. (A) Dictyostelium cell expressing mRFP-LimEDphagocytosing E. coli-GFP. The images are frames fromMovie 5. The phagosome ingested at time 0 is marked by arrowheads, while

that ingested at 153 seconds is marked by arrows. Two to three minutes after a phagosome was ingested, the GFP fluorescence of the

bacterium abruptly began to decline, and thirty to forty seconds after that, the fluorescence was barely detectable. This point was

reached 169 seconds after uptake for the first phagosome and 216 seconds after uptake for the second. Note that an actin comet tail

was visible for each phagosome as it moved away from its entry point (51 seconds and 193 seconds, also Movie 5). (B) AX2 cell that

had been pre-incubated in medium containing neutral red phagocytosing E. coli-GFP. The images are frames from Movie 6. One

phagosome (a) has already been transported to the cell center at time 0 and is surrounded by acidic endosomes labeled with neutral

red. Another phagosome (b) is already internalized but still near the cell periphery at time 0. At 63 seconds, the bacterium in the first

phagosome has lost its fluorescence (a) and the second one (b) has moved to the cell center. At 220 seconds, the bacterium in the first

phagosome has become bright red, indicating that its contents have been acidified and become accessible to neutral red. At

307 seconds, the bacterium in the second phagosome has lost its color, and at 374 seconds, it too has become red. Bars, 10mm.

M. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–10101006

Visualization of microtubule-based phagosome

transport

As described in earlier sections, the initial movementof newly formed phagosomes away from the site ofuptake appeared to be powered by actin assembly. Thiswas followed, either immediately or after a pause ofseconds to minutes, by an abrupt movement, usuallytoward the cell center, that was not accompanied by anyactin signal. Examples can be seen in Figure 1/Movie 1and in Figure 3A/Movie 4. We show here that thissecond type of movement is due to the association of thephagosome with microtubules.

In order to visualize microtubules at the same time asE. coli-GFP and neutral red, we matched the intensity ofthe bacterial GFP fluorescence to that of microtubulesin cells expressing GFP-a-tubulin. The cells were pre-incubated with neutral red, chilled briefly, and added toE. coli-GFP already plated on a glass cover slip. Thecells were allowed to settle in the cold for 15minutes,then brought to the microscope, where they warmed toroom temperature. When we began recording a fewminutes later, the cell shown in Figure 5 and Movie 7had already phagocytosed several bacteria. In panel 0,the bacteria deep inside the cell were still green, althoughmany of the phagosomes containing these bacteria had

become associated with neutral-red-stained endosomes.Also in panel 0, there was a phagosome (a) movingalong a microtubule toward the centrosome, andanother phagosome (d) moving away from thecentrosome along a microtubule. Two other phago-somes, (b) and (c), had already been internalized, butwere paused at the cell periphery. In the second panel(17 s), both of those phagosomes had been picked up bymicrotubules and were being transported toward thecentrosome, where phagosome (a) had already arrived.(See Movie 7 for a more detailed view of these events.)Note that phagosomes (a) and (b) (and more faintly,(c)), recently internalized at the beginning of the timeseries, were coated with red endosomes at the end,152 seconds later. Also note that at the beginningof the time series, there were many phagosomesalready near the cell center containing green bacteriaassociated with red endosomes, and 152 secondslater, the majority of those phagosomes containedbright red (acidified) bacteria. One phagosome (e) thatcould be tracked during the green-to-red transition ismarked. Thus, microtubules appear to provide a meansof bringing together recently internalized phagosomeswith pre-existing acidic endosomes, leading to thepermeabilization and acidification of the phagosomalcontents.

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Fig. 5. Microtubule-based phagosome transport. Dictyostelium cells expressing GFP-a-tubulin were preincubated in LF medium

containing 125mM neutral red, then added to a cover slip coated with E. coli-GFP. Some bacteria that could be tracked from frame

to frame in the time series (Movie 7) are marked. Microtubule-based transport is evident for several of these, in particular those

labeled (a, b, c, and d). One can also observe the transition of the bacterium within the phagosome from purely green, to green

associated with red endosomes, to fully permeated with neutral red (e). Bar, 10mm.

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Discussion

Dictyostelium LimE specifically labels filamentousactin (Prassler et al., 1998; Bretschneider et al., 2004).A fusion of GFP to LimED (a truncated version ofLimE) is a sensitive probe for the rapid assembly of newactin filaments in the cortical actin network of Dictyo-

stelium cells (Diez et al., 2005). Images in (Diez et al.,2005) suggest that LimED labels newly polymerizedfilaments more strongly than older filaments, as do oursequential images of bleb formation. Diez et al. (2005)reported that in areas of active actin remodeling, thedisassembly of actin filaments occurred within 20 s afterthe filaments were formed. Similarly, our observationsof spontaneous blebs suggest a lifetime of about 16 s fordynamic actin filaments, based on images collected at4-second intervals.

In contrast to the very rapid association of LimED tonewly assembled actin filaments, Dictyostelium coronindid not bind to actin filaments until a few seconds afterthey had been formed. In confocal frames collected at4-second intervals, coronin-GFP binding appeared oneframe after that of LimED, in the same location whereLimED had bound in the previous frame. This timingmay reflect a role for coronin in the disassembly of actinfilaments mediated by its interaction with the Arp2/3complex. The Arp2/3 complex is a major regulator ofnew actin assembly; it binds to the sides of existing actinfilaments, creating new sites of barbed-end filamentgrowth. The resulting dendritic array of growingfilaments pushes the membrane forward at the leadingedge of the cell (Pollard and Borisy, 2003). Coronins inother cell types have been demonstrated to bind to theArp2/3 complex (Humphries et al., 2002; Cai et al.,2005) and alter the conformation of the complex so thatit can no longer nucleate actin assembly (Rodal et al.,2005). That property of coronin may allow it to promotethe disassembly of actin filaments, a possibility consis-tent with our images. Whether or not this is themechanism, the differential binding preferences of

LimED and coronin have provided a sensitive windowinto actin filament dynamics during phagocytic cupformation and phagosome internalization. A higher-resolution view of these two markers during thephagocytosis of yeast cells is in preparation (G. Gerischand J. Prassler, personal communication).

The pattern of actin assembly and disassemblydetected here during formation of the phagocytic cuphas been observed in earlier studies using single probes,coronin-GFP (Maniak et al., 1995), GFP-actin (Peraci-no et al., 1998; Konzok et al., 1999), and GFP-ABD(Rupper et al., 2001). Similarly, enrichment of GFP-Arp3 about the phagocytic cup was previously observedby Insall et al. (2001). However, these earlier studies didnot address the subsequent movements of the phago-some.

In the present study, the bright labeling of new actinfilaments by LimED has highlighted a second actin-mediated step in phagocytosis. After closure of thephagocytic cup and disappearance of all or most of theF-actin that had surrounded the nascent phagosome,new actin assembly was detected with LimED. Often, thenew actin appeared at two sites. One was at the plasmamembrane near the site where the phagosome had beenformed, leading to a small protrusion of the plasmamembrane there. The other was close to the phagosomeand correlated with movement of the phagosome. Thenew actin assembly was often very brief, lasting only afew seconds, as seen in Figure 2B. A brief increase incoronin-GFP fluorescence at the interface betweenyeast-containing phagosomes and the plasma membranewas previously noted by Dormann et al. (2004), usuallyaccompanied by an inward movement of the phago-some. In the present study, we found that in polarizedcells with an extended lamellipodium, such as the cellsshown in Figures 1, 2C, and 3, the trajectory of newphagosomes often remained close to the plasmamembrane for several seconds, displaying a longcomet-like tail of actin filaments. Use of the dual probesmRFP-LimED and coronin-GFP made it clear that the

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new actin assembly always occurred close to the movingphagosome.

Cells expressing GFP-Arp3 displayed a comet tailduring phagosome internalization similar to that ob-served with mRFP-LimED, strongly suggesting that thistype of propulsive actin assembly involves the Arp2/3complex. However, it should be noted that there areother regulators of actin assembly in Dictyostelium thathave not been tested here. These include formins, whichcan nucleate actin assembly to create filopodia in theabsence of Arp2/3 (Schirenbeck et al., 2005; Steffenet al., 2006) and capping protein, which binds to thebarbed end of actin filaments and limits polymerization(Hug et al., 1995).

We suspect that the brief burst of actin polymeriza-tion that moves new phagosomes away from the plasmamembrane is powered by addition of actin at the plasmamembrane, rather than by nucleation of actin assemblyat the phagosome membrane, as is true of pathogens(reviewed by Stevens et al., 2006) and has been proposedfor endosomes in other contexts (e.g., Merrifield et al.,1999; Taunton, 2001; Chang et al., 2005). We favor theformer model because of our observations concerningphagosome rocketing induced by mechanical pressure(Clarke, Muller-Taubenberger, Anderson, and Gerisch,under revision). We have discovered that when theplasma membrane of a Dictyostelium cell is pressedagainst large yeast-containing phagosomes, even thosein the acidic phase of endocytic transit, Arp2/3 isrecruited to the plasma membrane in the area of contact,and actin-mediated phagosome rocketing is initiated. Asin the present study, mRFP-LimED shows that newactin is added in the zone of contact between thephagosome and plasma membrane as the phagosomemoves along just under the plasma membrane. For largeyeast-containing phagosomes, one can distinguish thatactin assembly takes place only at or very close to theplasma membrane; actin is often not associated with themembrane of rocketing phagosomes viewed in deeperfocal planes. If the pressure is eased, allowing the largephagosome to move away from the plasma membrane,rocketing ceases, and the actin tail disappears. Forrocketing induced by mechanical pressure, it is evidentthat the Arp2/3 complex is more highly enriched at theplasma membrane than at the phagosome membrane,arguing that filament growth is occurring at the plasmamembrane.

Although bacteria-containing phagosomes are toosmall for pressure from an agar overlay to inducerocketing, newly sealed phagosomes, still in the midst ofthe cortical actin network that coats the plasmamembrane, are in a situation rather similar to that oflarge phagosomes mechanically pressed against theplasma membrane. This close proximity appears tocause a burst of actin polymerization at the interfacebetween the phagosome and the plasma membrane,

driving the phagosome away from the plasma mem-brane and deeper into the cell, thereby ending thereaction. We speculate that the addition of new actinmonomers occurs at the plasma membrane, as it doesduring leading edge extension (Pollard and Borisy,2003), during rocketing induced by mechanical pressure(discussed above), and during internalization of cla-thrin-coated vesicles in yeast (Kaksonen et al., 2005).However, at present our data cannot exclude thepossibility that the phagosome itself acquires themachinery for propulsive actin polymerization.

Following actin-powered internalization, a new pha-gosome usually pauses briefly, then begins to moveagain without any actin signal. This movement almostcertainly reflects contact of the phagosome with micro-tubules, which are continually sweeping through thecytoplasm. As we have shown elsewhere (Clarke et al.,2002b), endosomes, especially those in the early stagesof endocytic transit, undergo constant, highly dynamictransport along microtubules, and phagosomes too aretransported back-and-forth along microtubules (Lu andClarke, 2005). Microtubule-based transport appears topromote the merger of new phagosomes with acidicvesicles (labeled here with neutral red), leading topermeabilization and acidification of the bacteriumabout four to seven minutes after uptake.

We previously tracked the delivery to new phago-somes of the vacuolar H+-ATPase (V-ATPase), theenzyme responsible for acidifying endosomes andphagosomes (Clarke et al., 2002a). We found that pre-existing endosomes that contained the V-ATPase intheir membrane began clustering about a new phago-some �90 seconds after uptake. Within the next minuteor two, these endosomes fused with the phagosomemembrane, delivering their contents to the lumen andthe V-ATPase to the membrane of the phagosome. Incells depleted of acidic endosomes by the prior uptake ofmany yeast particles, delivery of the V-ATPase wasaccomplished more slowly by fusion of the newphagosome with other phagosomes already bearing theV-ATPase. The timing of the delivery of the V-ATPaseby endosome fusion corresponds well to the timing ofthe loss of bacterial fluorescence and acidification ofbacterial contents detected with neutral red in thepresent study. Furthermore, Souza et al. (1997) demon-strated that vesicles containing cysteine proteinases,enzymes that play a major role in the digestion ofbacteria, are also delivered to new bacteria-containingphagosomes about three minutes after uptake. Gerischand co-workers had demonstrated earlier that Dictyos-

telium amoebae contain hydrolases that efficientlydegrade the murein-lipoprotein complex of the bacterialcell wall (Braun et al., 1972).

The highly efficient initial processing of bacteriadescribed here may be compared with earlier studiesthat have analyzed the maturation of endosomes and

ARTICLE IN PRESSM. Clarke, L. Maddera / European Journal of Cell Biology 85 (2006) 1001–1010 1009

phagosomes at the population level. Aubrey et al. (1993)monitored the evolution of endosomal pH in aDictyostelium population that had taken up a pH-sensitive fluid-phase marker; they found that endosomalpH decreased over a 20-minute interval and thenincreased during the next 20–40minutes, after whichthe marker was exocytosed. Maselli et al. (2002) usedlive bacteria expressing DsRed to monitor the uptakeand degradation of bacteria by Dictytostelium cells.These workers observed extremely rapid uptake ofbacteria, but the DsRed signal from internalizedbacteria, measured using a plate fluorometer, decreasedslowly, with a half-life of 45minutes. In contrast to thepopulation data and consistent with our results, single-cell observations by Maniak using a pH-sensitive fluid-phase marker revealed acidification of endosomes within50–60 seconds after uptake, resulting from fusion withother vesicles (Maniak, 2001, 2003). We infer that themore gradual transitions found in population studiesreflect differences in the time of uptake of the endosomesas well as heterogeneity in the endocytic activity of cellsin the population. We are seeking to develop additionalmarkers that will allow us to track individual phago-somes through the later stages of the endocytic pathway,so that we can better distinguish the consistent andvariable aspects of endocytic transit.

Acknowledgements

We thank Annette Muller-Taubenberger and GuntherGerisch for plasmids or cells expressing mRFP-LimED,coronin-GFP, GFP-Arp3, and GFP-a-tubulin, and theOMRF Imaging Facility for access to microscopyequipment. This work was supported by grants fromthe National Science Foundation (MCB-0344541) andthe Oklahoma Center for the Advancement of Scienceand Technology (HR05-020) to M. Clarke, who alsoacknowledges support as the J.P. Hannigan Distin-guished Research Scientist.

Appendix A. Supplementary data

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.ejcb.2006.05.004.

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