A role for microtubule dynamics in phagosome movement

303Journal of Cell Science 111, 303-312 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS9679

A role for microtubule dynamics in phagosome movement

Ariel Blocker 1,*, Gareth Griffiths 1, Jean-Christophe Olivo 2, Anthony A. Hyman 1 and Fedor F. Severin 1

Cell Biology1 and Cell Biophysics2 Programmes, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,Germany*Author for correspondence at present address: Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex,France (e-mail: [email protected])

Accepted 21 November 1997: published on WWW 15 January 1998

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We have shown previously that intracellular phagosomemovement requires microtubules. Here we provideevidence that within cells phagosomes display two differentkinds of microtubule-based movements in approximatelyequal proportions. The first type occurs predominantly inthe cell periphery, often shortly after the phagosome isformed, and at speeds below 0.1 µm/second. The second isfaster (0.2-1.5 µm/second) and occurs mainly afterphagosomes have reached the cell interior. Treating cellswith nanomolar concentrations of taxol or nocodazolealters microtubule dynamics without affecting either totalpolymer mass or microtubule organisation. Suchtreatments slow the accumulation of phagosomes in theperinuclear region and reduce the number of slowmovements by up to 50% without affecting the frequencyof fast movements. This suggests that a proportion of slowmovements are mediated by microtubule dynamics while

fast movements are powered by microtubule motors. Inmacrophages, interphase microtubules radiate from themicrotubule organising centre with their plus-end towardsthe cell periphery. To understand the behaviour of ‘early’phagosomes at the cell periphery we investigated theirability to bind microtubule plus-ends in vitro. We show thatearly phagosomes have a strong preference for microtubuleplus-ends, whereas ‘late’ phagosomes do not, and that plusend affinity requires the presence of microtubule-associated proteins within cytosol. We suggest thatphagosomes can bind to the plus-ends of dynamicmicrotubules and move by following their shrinkage orgrowth.

Key words: Microtubule dynamics, Phagocytosis, Intracellularorganelle movement

SUMMARY

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INTRODUCTION

In vertebrates phagocytosis serves many functions, includclearance of invading parasites and dead cells followinfection or apoptosis. During phagocytosis a cell, oftenmacrophage, internalises particles too large to be endocytothereby forming a new membrane compartment, tphagosome (Silverstein et al., 1989). Once formed, phagosome is the site of a series of biochemical reactions usually lead to the killing of any live organism within it. Todegrade the internalised particle the phagosome acidifies acquires hydrolases by sequential fusions with endocycompartments (Muller et al., 1980; Pitt et al., 1992; Jahraual., 1994 and unpublished; Desjardins et al., 1994a).

Early evidence suggested that microtubules facilitate fusiobetween phagosomes and endocytic organelles (Goldsteial., 1973; Pesanti and Axiline, 1975; D’Arcy Hart et al., 1983We showed that the acquisition by phagosomes of membraprotein and fluid-phase markers of late endocytic organelleinhibited by microtubule depolymerisation (Desjardins et a1994a; Blocker et al., 1996). Phagosomes and a varietyendocytic compartments interact directly with microtubule(Gruenberg et al., 1989; Aniento et al., 1993; Hopkins et

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1990; Scheel and Kreis, 1991; Blocker et al., 1996). Recenwe showed that phagosomes move bidirectionally alomicrotubules in vivo and demonstrated in vitro that therequire the microtubule motors cytoplasmic dynein and kinesas well as motor accessory subunits and the transmembrprotein kinectin to do so (Blocker et al., 1997).

Yet, microtubules are not simply static structures alonwhich cytoplasmic cargoes dock and travel. Indeemicrotubules display a particular form of turnover in vitrotermed ‘dynamic instability’, in which filaments switchstochastically between growth and shrinkage (Mitchison aKirschner, 1984a,b). This switch is controlled by GThydrolysis within tubulin subunits. At the plus-end of growing microtubule, a cap of GTP-bound tubulin subunits thought to exist (reviewed in Carlier, 1989). The presencethe cap stabilises the microtubule and keeps it in the growphase.

Dynamic instability of microtubules also occurs in vivo(Sammak and Borisy, 1987; Sammak et al., 1987; Schulze Kirschner, 1986, 1987, 1988). A role for microtubule dynamicin organelle movement was discussed some time ago (Bikleal., 1966; Porter, 1973; Murphy and Tilney, 1974), yet sinthe discovery of cytoplasmic microtubule motors moving alon

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taxol-stabilised microtubules in vitro, the possibility of suchphenomenon has been somewhat forgotten. The measureof the parameters of microtubule dynamic instability in vivled to a paradox for those studying microtubule motomediated organelle transport. For instance, Rodionov et(1994) investigated microtubule dynamics in pigment granucontaining fish melanophores, the original model for studyiorganelle transport. In these cells, all the pigment granulesbe induced to aggregate at the microtubule organising ceor disperse to the cell periphery experimentally within minutRodionov and coworkers found that microtubule turnover wrapid in cells with both aggregated and dispersed granuMoreover, they found that stable, post-transcriptionamodified microtubules were absent in these cells. This led thto ask: ‘how can dynamic microtubules provide the tracks fpigment granule movement if the time of microtubule turnois comparable to the aggregation time and much less than of dispersion?’.

One clue may come from chromosomes within the mitospindle, which display two kinds of movements, one mediaby motors and the other dependent on microtubule dynamBoth kinds of movement appear to be mediated by the sastructure, the kinetochore (reviewed in Inoué and Salm1995). The motor-mediated movement of kinetochores occin prometaphase when the chromosomes need to posthemselves at the plus-end of spindle microtubules, and speed of transport varies from 20-50 µm/minute (cytoplasmicdynein) to 1.5-2 µm/minute (mitotic kinesins). Duringanaphase A, kinetochores lead sister chromatids towards respective spindle pole at speeds of 1.5-2 µm/minute by amechanism involving depolymerisation of kinetochomicrotubules from their plus-end. The movement chromosomes along depolymerising microtubule tips has breconstituted in vitro using microtubules grown off extracteTetrahymenapellicles (Coue et al., 1991; Lombillo et al.1995a,b). The question of whether membrane organelles also follow the growing or shrinking end of a microtubule currently under study.

The dynamics of the endoplasmic reticulum in vivo habeen correlated with those of the underlying microtubu(Terasaki et al., 1986; Waterman-Storer and Salmon, 1996vitro the tips of ‘tubules’ formed by crude membranes isolatfrom Xenopuseggs have been shown to follow the growth ashrinkage of microtubules in Xenopusextracts (Waterman-Storer et al., 1995). Moreover, electron microscopy usinegative staining of membrane tubules tethered to microtubhas revealed what may well be the membrane/microtubuleattachment complexes (TACs) as also being the site microtubule motor attachment (Allan and Vale, 1994). Furthdetails on the structure of TACs may come from the only otexample of membrane/microtubule tip attachment studiedany detail: the tip of the Chlamydomonasflagella with theplasma membrane. This structure (which is also found in otorganisms; reviewed in Dentler, 1990) morphologicalresembles a ‘kinetochore half’. One polypeptide of thcomplex shares antibody reactivity with some mammalikinetochores, suggesting that kinetochores and TACs somehow related structures.

Here we have used phagosomes to study the contributiomicrotubule dynamics to organelle movement. We routinemark phagosomes with 1 µm latex beads (Wetzel and Korn

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1969; Stossel et al., 1971; Muller et al., 1980). Our data shthat latex bead-containing phagosomes in fact display b‘fast’ and ‘slow’ movements in approximately equaproportions, depending on their position within cells. When waltered microtubule dynamics in vivo using low concentratioof taxol or nocodazole, the slow class of phagosommovements was selectively inhibited, suggesting thphagosomes might use microtubule dynamics to move in livicells. Since microtubule dynamics depend on a structudifference between the plus-ends of microtubules and the of the polymer, we also asked whether isolated phagosomshowed any preference for a particular part of the microtubwhen they bound in vitro. The low buoyant density of lateallows the purification of intact phagosomes, by flotation onsucrose step gradient, from J774 mouse macrophages that internalised beads (Desjardins et al., 1994a,b). We show purified phagosomes display a strong preference microtubule plus-ends, which is regulated during thematuration, and that microtubule-associated proteins (MAPare necessary but not sufficient cytosolic components support this binding.

MATERIALS AND METHODS

Cell cultureJ774 mouse macrophages and normal rat kidney (NRK) cells wmaintained as described in Blocker et al. (1996) and Jahraus e(1994), respectively. Phagosomes were prepared from J774 cellpreviously described in Blocker et al. (1996).

In vivo movement analysisFixed time point microscopyNRK cells were plated overnight on coverslips such that they woube semi-confluent 14 hours later. They were then pulsed with a 0.(w/v) solution of 1 µm latex beads (Seradyn, USA) for 30 minutes a37°C in culture medium (DMEM containing 5% fetal calf serumSigma) and chased in the same medium for up to 5 hours in presence or absence of either 100 nM nocodazole (Sigma) or 50taxol (Paclitacel, Sigma) or 50 nM cytochalasin D (Sigma).

Cells were then fixed in methanol at −20°C for 3 minutes.Microtubules were labelled using a mouse monoclonal anti-α tubulin(Amersham, USA) at 1:1000 (v:v) dilution and a rhodamineconjugated goat anti-mouse (Dianova, Germany) at 1:300 (vdilution. Coverslips were mounted in Mowiol and for each time poiapproximately 100 cells were scored for their phagosome distributby bright field microscopy using a 25× lens. Cells were considered todisplay a perinuclear phagosome distribution when over 50% of beads that they contained were found to be clustered aroundnucleus. The scores were averaged, and errors given are standeviations. This experiment was repeated a total of five times aalthough data from only 2 separate days was used to generate Fiall experiments demonstrated similar results. Cells were aphotographed on a Axiophot microscope (Zeiss) using 63× Plan-APOCHROMAT lens, a 1.25× magnifier by phase or using aRhodamine filter set (Zeiss) on 400 ASA Kodak black and white film

Video microscopyNRK cells were pulsed with a 0.01% (w/v) solution of 1 µm latexbeads for 15 minutes at 37°C in internalisation medium (IM: MEM10 mM Hepes, 5 mM D-glucose, pH 7.4) containing 10% newbocalf serum from (Gibco) and chased in the same medium withbeads for a further 15 minutes. They were then mounted inmicroscopy chambers (EMBL workshop, Heidelberg) containing t

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same medium in the presence of either 100 nM nocodazole or 50taxol. Cells were observed by differential interference contrmicroscopy at 37°C (using stage, condenser and lens heating devEMBL workshop, Heidelberg) with a 100× Plan-APOCHROMATlens and matching Wollaston on a Zeiss Axiovert 10 using a 50mercury lamp and Hamamatsu CCD camera model C5405(Hamamatsu Photonics, Japan). The video signal was fed throuHamamatsu camera control, into an image processor (Argus Hamamatsu) and saved using a Sony OMDR and large optical dThe image processor and the OMDR were controlled by a compprogram (PC) available from Dr E. Stelzer (EMBL, Heidelberg). 3different cells were observed per coverslip, each for 10 minutes, for each field 300 frames were captured at 2 second intervals. field size was calibrated by measuring the number of pixels in aµm pitch on a 1 mm graticule. Movements were analysed eitherhand using the NIH Image programme on a MacIntosh Quadramore efficiently and precisely using our previously describcomputer programme (Blocker et al., 1997; Nguyen Ngoc et 1997). t-tests were performed to assess the significance of a differebetween two frequencies.

End-binding assayPreparation of microtubulesPolarity-marked guanylyl-(α,β)-methylene diphosphonate(GMP-CPP) microtubules were generated as described in Severin e(1997) 1 day before performing an end-binding assay and with following modifications: 1 µM Oregon green tubulin was added to 0.mM GMP-CPP in BRB80 (80 mM K-Pipes, 1 mM MgCl2, 1 mMEGTA, pH 6.8) and incubated at 37°C for 3-4 hours. 2 µl of theseOregon green-labelled microtubule seeds were mixed with 10 µl of0.3 µM cycled unlabelled tubulin, 0.2 µM rhodamine tubulin and 1mM GMP-CPP in BRB80. The mixture was incubated at 37°C fototal of 6-8 hours and replenished four times with 2 µl of 0.3 µMcycled unlabelled tubulin and 0.2 µM rhodamine tubulin in BRB80 atregular intervals during this period. The length of the microtubuwas checked periodically and the reaction was stopped by placemat room temperature when the microtubules were about 10-20 µmlong. They were stored overnight in the dark at room temperaturealways pipetted with cut micropipette tips to reduce breakage.

Assay protocol and data acquisitionThe principle of this assay is to mix phagosomes with microtubuin very dilute form so that when the reaction is pipetted onto a sand flattened by a coverslip, one can count each phagosomicrotubule binding event on the coverslip and assume it represa biological interaction rather than a simple physical juxtapositiThus, each assay reaction contained: 0.25 µl of purified phagosomescontaining 1 µm carboxylated blue latex beads (Seradyn) coupledfish skin gelatine (FSG) (Sigma) as described in Blocker et al. (190.5 µl of macrophage cytosol at approximately 30 mg/ml preparedin Blocker et al. (1996; the preparation of MAP and mock deplecytosol as well as MAPs are also described in this publication; MAwere eluted from microtubules using 150 mM NaCl in one tenththe initial volume of macrophage cytosol); 1 µl of polarity-markedGMP-CPP microtubules previously diluted 1:20 (v:v) in BRB80; 1 µlof 8× antifade prepared as in Blocker et al. (1997) but omitting taxol; 7.25 µl of 2 mg/ml casein (Sigma) in PMEE (35 mM K-Pipes5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, pH 7.4). Reactions wermixed by finger flicking, immediately pipetted into a glass slide (KT360, Propper Ltd., UK) and covered with a glass coverslip (22×22mm; No. 1 Gold Seal, Clay Adams, USA). Slides were observeda Zeiss Axioscope microscope fitted with a 100 W mercury bulb aa 63× PlanApo 1.6 lens. Slides were systematically scanned and ephagosome found bound to a microtubule of clear polarity wphotographed. Images were captured via a Colour Cool View cocamera (Photonic Science, East Sussex, UK) controlled by the IPLprogramme for the MacIntosh.

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RESULTS

Phagosomes display both fast and slow intracellularmovements, depending on their position within cellsOur goal was to investigate phagosome movement in vivomore detail than in our previous work. For this we observecells that had internalised latex beads by computer-enhandifferential interference contrast microscopy (DIC). Since thcells we used to generate phagosomes, J774 momacrophages, are poorly adherent and not suited for dirobservation, we used NRK cells, which we have also usedprevious studies for such purposes (Jahraus et al., 19Desjardins et al., 1994a; Blocker et al., 1997). SpecificalNRK cells were grown on coverslips and then pulsed withµm carboxylated latex beads for 15 minutes and chased forminutes at 37°C. The cells were then observed by DIC (in tplane of the coverslip to ensure that all the beads that wobserved inside cells had been internalised and were not simbound to the outer cell membrane) at 37°C for 10 minutes ea

As shown in Fig. 1 and in Table 1, during the time intervaof observation about 20% of phagosomes displayed linemovements of several µm at speeds varying betweenapproximately 1.5 and 0.05 µm/second. We had earlier notedspeeds of 1.5-0.2 µm/second for phagosomes moving in vivoand in vitro (Blocker et al., 1997), but were suprised to find a more detailed analysis that approximately 50% phagosomes moving in vivo displayed no instantaneous speabove 0.1 µm/second along the entire length of theidisplacement. On a few occasions we saw phagosominitiating their displacement at slow speed right behind the zoof plasma membrane ruffling where internalisation took placIn these cases, after a slow movement of several µm, oftenfollowed by a pause of many seconds or a change in directiwe saw a number of phagosomes then move much faster aover several µm. When we investigated the distribution ophagosome speeds within cells, we found that approximat90% of movements with speeds of below 0.1 µm/second startedwhen phagosomes were within 3 µm of the cell periphery, while90% of movements occurring within the cell interior displayespeeds above 0.1 µm/second (Table 1). These data suggestedus that phagosomes were displaying two different kinds movements within cells: a ‘slow’ kind (at or below 0.1µm/second), occurring mostly right after their formation in thcell periphery, and a ‘fast’ kind (above 0.1 µm/second) morefrequently seen when these organelles reached the cell inte

Altering microtubule (but not actin) turnover inhibitsslow phagosome movements onlyWe have already documented that most phagosom

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Fig. 1.Fast and slow phagosomemovements occur in NRK cells. (Aand B) The first and last images,respectively, of a 10-minute videosequence of an NRK cell havingbeen pulsed with latex beads andchased as described in Materialsand methods for 30 minutes andobserved by computer enhanceddifferential interference microscopyat 37°C. The sequence has beenanalysed by a dedicated computerprogramme which recognises andmarks the position of eachphagosome at each frame (taken at2 second intervals) to generate theblack tracks seen in B. Threephagosomes which movedsignificantly have their startingpositions marked in A and in B bywhite arrowheads. The density ofthe points marking the tracks is a reflection of how fast the phagosomes moved. The leftmost phagosome displayed brownian movementfollowed by a rapid (approximately 1 µm/second) movement of a few µm and more brownian oscillations. The rightmost phagosome displayeda long slow movement (average of 0.05 µm/second) directed towards the cell centre immediately after having become internalised. Thephagosome in the middle displayed brownian movement initially and then reached the nuclear periphery by a movement of several µm at anaverage speed of approximately 0.2 µm/second (still classified as ‘fast’). Bar, 3 µm.

movements are abolished by treatment of cells with 10 µMnocodazole (Blocker et al., 1997). Under in vitro conditiowe have also reconstituted the fast type of in vivo movemwhere phagosomes move bidirectionally along microtubuusing the microtubule motors kinesin and cytoplasmic dyn(Blocker et al., 1997). We were therefore curious about nature of the second type of movement that we observed hIts average speed of below 0.1 µm/second made it unlikely tobe mediated by microtubule motors. Such ranges of spehave been reported, however, for some myosin-type mo(Wolenski et al., 1993), for endosomes or membrane vesic(Heuser and Morisaki, 1992; Marchand et al., 1995) aintracellular pathogens (Theriot et al., 1992; Cudmore et 1995) moving via dynamic ‘actin comets’, and also fochromosomes moving by microtubule turnover within thmitotic spindle (reviewed by Inoué and Salmon, 1995). Wtherefore investigated whether actin or microtubule dynammight be involved in propelling the slow phagosommovements. For this we treated NRK cells with nanomo

Table 1. Parameters of p% of internalised %

Condition beads moving n m

Control 17.5 154100 nM nocodazole 13.3 19550 nM taxol 11.7 206

For each condition approximately 25 cells were observed at 37°C by DIhave moved if they showed a directed displacement greater than one beavery broad distribution of speeds (including pauses of many seconds) typmeasurements (NIH Image) on time intervals long enough (pauses excludmeasurement errors. For statistical analysis movements were classified atheir maximal velocity was above 0.1 µm/second (usually between to 0.2-0.5µmwere separated by a pause of more than 30 seconds and/or demonstrateoccurred in the cell periphery if they were initiated less than 3 µm from the cell

n, total number of latex bead-containing phagosomes observed.

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concentrations of drugs known to affect actin or microtubulecytochalasin D, nocodazole and taxol. At concentratiobelow 200 nM in vivo cytochalasin D selectively binds to actfilament barbed ends (Cooper, 1987). Since 200 ncytochalasin D rapidly inhibited NRK cell membrane rufflingas observed by DIC microscopy, we titrated the drug dowuntil its effects on the cell cortex only became visible aftmore than 30 minutes of incubation with the drug and fouthis concentration to be 50 nM (not shown). The effects of nconcentrations of the microtubule stabilising drug taxol anthe destabilising drug nocodazole on microtubule dynamhave been well studied (Wilson and Jordan, 1994; Vasqueal., 1997). In NRK cells in particular, such concentrations these drugs do not qualitatively or quantitatively alter thmicrotubule network over a period of 2 hours (Liao et a1995). Instead they appear to increase the percentage of microtubules are ‘paused’ (from approximately 30% to 75%and decrease their growth rate and ‘catastrophe’ freque(with little effect on the shrinkage rate and none on th

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C microscopy for 10 minutes. Latex bead-containing phagosomes were considered tod diameter (approximately 1 µm) during this time. Individual movements displayed theical for saltatory motion. Particle speeds were measured using computerised distanceed) to lead to displacements greater than 0.2 mm on the screen so as to minimises ‘slow’ if they showed speeds below 0.1 µm/second along their entire course or ‘fast’ if /second). Movements of a given phagosome were considered independent if theyd a change in direction of an angle above 90°. Movements were considered to haveborder.


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‘rescue’ frequency; G. Gundersen, personal communicatioWe therefore pulsed NRK cells with latex beads and chathem in the presence or absence of 50 nM cytochalasinnocodazole or 100 nM taxol, while monitoring thaccumulation of phagosomes in the perinuclear region of cas a measure of the ability of these organelles to move. F2 and 3 show that nanomolar concentrations of taxol nocodazole slowed the perinuclear accumulation phagosomes approximately twofold (nocodazole beisomewhat less effective) over control cells after 2 hourschase with little effect on microtubule organisation while 5nM cytochalasin had no effect. We conclude that alterimicrotubule but not actin dynamics affects phagosommotility within cells.

We next wanted to investigate in more detail which aspeof phagosome motility these low concentrations microtubule-active drugs were affecting. For this we studithe motility of individual phagosomes in NRK cells treate

Fig. 2. Low levels of nocodazole ortaxol alter phagosome distributionwithout significantly perturbing themicrotubule network. NRK cells werepulsed with latex beads for 30minutes at 37°C, then fixed andstained for microtubules using ananti-α tubulin monoclonal antibodyfollowed by an anti-mousemonoclonal antibody coupled torhodamine. The same cells werephotographed using phase contrast(A) and fluorescence (B) microscopy,to show the positions of latex bead-containing phagosomes, the cellmembrane and nucleus or theorganisation of the microtubules,respectively. (C) NRK cells werepulsed with latex beads and chasedfor 2 hours. At this time all the beadsare observed to be in the perinuclearregion of the cells. (D) The same cellsas in C photographed by fluorescencemicroscopy to show their microtubulenetwork. (E) NRK cells were pulsedwith latex beads and chased for 2hours in the presence of 100 nMnocodazole. Many cells showphagosomes only partially clusteredat the nucleus or even with a totallyrandom distribution. (F) The samecells as in E photographed byfluorescence microscopy. Themicrotubules in these cells most oftenappear normal, occasionally they areslightly sparser. (G) NRK cells werepulsed with latex beads and chasedfor 2 hours in the presence of 50 nMtaxol. Most cells have theirphagosomes randomly distributed. (F)The same cells as in G photographedby fluorescence microscopy. Themicrotubules in these cells most oftenappear normal, if perhaps slightlydenser. Bar, 15 µm.

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with 100 nM nocodazole or 50 nM taxol. Table 1 shows thaat such concentrations these drugs reduce the numberphagosomes moving by 25% and 45%, respectively (significaat P=0.2 and 0.05, respectively, by t-test; 50 nM cytochalasinD had no effect, not shown). In particular, Table 1 shows ththe total ‘slow’ phagosome movements are reduced by 20%the presence of 100 nM nocodazole and by 40% with 50 ntaxol. In fact it is essentially the slow movements occurrinwithin the cell periphery that are reduced (by 20% and 70%respectively). In agreement with the results shown in Fig. nocodazole was less effective than taxol at reducing slomovements. The sum of these data indicate that the slowdoin the perinuclear accumulation of phagosomes upon treatmof cells with nanomolar concentration of taxol and nocodazois mainly due to a decrease in the number of slow movemewithin the cell periphery. We thus conclude that a proportioof the ‘slow’ phagosome movements we observe are somehpowered by microtubule dynamics.

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Fig. 3. Low levels of taxol or nocodazole, but not cytochalasin D,significantly delay the accumulation of phagosomes in theperinuclear region. NRK cells were pulsed with latex beads for 30minutes and chased in the presence or absence (control) of 100 nnocodazole (nocodazole), 50 nM taxol (taxol) or cytochalasin D(cytochalasin D) for up to 5 hours. In each sample approximately cells were scored for their phagosome distribution. Cells were scoas having a perinuclear distribution if over 50% of their phagosomwere located near the nucleus. This experiment was repeatedindependently a total of five times (always with similar results). Togenerate this graph, the scores from at least two independentexperiments were averaged, errors are sample standard deviationAll points have error bars, those where they are invisible were simtoo small to be represented clearly.

Phagosomes preferentially bind to microtubule plus-ends in vitroWe next performed in vitro experiments aimed understandhow phagosomes could move using microtubule dynamicsvivo. In the case of chromosomes it has been shown that attach via their kinetochores to the microtubule wall prometaphase and subsequently move to the microtubule where they remain throughout mitosis (Rieder and Alexand1990; Skibbens et al., 1993). It has thus been postulated kinetochores are able to follow the growing and shrinking eof a microtubule and that this is one way in which they mowithin the microtubule spindle. We have recently demonstrathe preferential binding of kinetochores to microtubule pluends in vitro (Severin et al., 1997) and we thus wished to whether phagosomes were also able to discriminate betwmicrotubule ends. For this we generated stable, polarmarked microtubules made entirely of tubulin in its GTconformation. We polymerised short green microtubule seby incubating Oregon green-labelled tubulin with the slowhydrolysable GTP analogue GMP-CPP. We then proceedeelongate these microtubules by repeated addition of a concentration of rhodamine-labelled tubulin in the presenceadditional GMP-CPP. As the rate of microtubule growth approximately threefold faster at the plus- than at the minend, this generates stable red microtubules with a short gsegment located near their minus end (Fig. 4A; the polaritythe microtubules was checked as in Severin et al., 1997).next mixed these microtubules with phagosomes containbeads which fluoresce dimly within the rhodamine range (Materials and methods) and macrophage cytosol, and sc

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the position of each phagosome found to be bound to microtubule of clear polarity by fluorescence microscopy. Fig4C shows that phagosomes purified after 1 hour of pulse a1 hour of chase within cells bind microtubules with strongpreference for their plus-end (the probability of getting thiresult randomly is less than 0.0005; t-test). These data directlyshow that phagosomes, in the presence of cytosol, are abledistinguish between the wall of the microtubule and its plusend.

The ability of phagosomes to bind microtubule plus-ends is regulated during phagosome maturation andrequires a MAP-like factor(s)As the ability of phagosomes to display ‘slow’ movements incells appeared to correspond to an early stage in thematuration, we wished to test whether the affinity ophagosomes for the microtubule plus-end was also regulatin vivo by testing phagosomes of different ‘ages’ in our in vitrobinding assay. For this we isolated phagosomes after a 2minute pulse or a 1-hour pulse followed by a 24-hour chasand tested their ability to bind microtubule plus-ends. Fig4B,D shows that 20-minute pulse phagosomes also displaypreference for microtubule plus-ends while 1-hour pulse/24hour chase phagosomes bind randomly along the microtubuwall. This suggests that the ability of phagosomes to interawith microtubule-plus ends is regulated during phagosommaturation and is an early requirement in the life of thiorganelle.

We have previously characterised not only the bidirectionamotility of phagosomes along microtubules (the frequency owhich increases with phagosome maturation; Blocker et a1997), but also the static interaction of phagosomes wimicrotubules. We showed that the latter interaction wamediated by a single cytosolic factor, sized at approximate200 kDa by gel filtration and displaying properties of amicrotubule-associated protein (MAP; Blocker et al., 1996)The static interaction of phagosomes with microtubules via thMAP linker appeared strongest for ‘early’ phagosomes anweakest for ‘late’ phagosomes. Thus, we asked whether tMAP linker might be responsible for the preference of earlphagosomes for microtubule plus-ends. For this, we measurthe ability of 1-hour phagosomes to bind to microtubule plusends in the presence of cytosol which had been depletedMAPs. Fig. 5B shows that MAP-depleted cytosol no longesupported phagosome binding to microtubule plus-ends, whmock-depleted cytosol was unaffected (Fig. 5A). Fig. 5Cshows that re-addition of MAPs to MAP-depleted cytosorestored the ability to phagosomes to recognise microtubuplus-ends. However, by itself the MAP fraction was not ablto support microtubule plus-end binding (Fig. 5D). Takentogether, these data indicate that MAPs are necessary, but sufficient, for early phagosomes to bind preferentially tomicrotubule plus-ends.

DISCUSSION

In this study, we provide evidence that a cytoplasmimembrane organelle other than the endoplasmic reticulum cuse microtubule dynamics to move around in cells. Our dafurther suggest that this ability can be explained, at least in pa

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Observing phagosomes in vivo we found that they dispmovements of very different speeds. We have defined as ‘sl

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movements those that show no speeds above 0.1 µm/second(often 0.05-0.02 µm/second) and as ‘fast’ those with any speeabove 0.1 µm/second (generally between 0.2 and 1µm/second). This is a somewhat arbitrary boundary since movements are saltatory and often display some speeds inrange of slow movements. Thus, when the speeds are plothe classes of movements are not distinctly separatHowever, we believe that this classification reflects a trbiological difference since altering microtubule dynamicreduces the occurrence of slow movements without affectthe speed or frequency of fast ones.

It is unlikely that actin dynamics play an important role islow phagosome movements as 50 nM cytochalasin D, whstops most membrane ruffling (not shown), had no effect slow movements. However, the speeds reported for somyosin-mediated movements are well within the range of thoof the slow movements we observe (Wolenski et al., 1993).addition, nanomolar amounts of either taxol or nocodazole opartially inhibit slow movements. The latter result might havtwo explanations: nanomolar concentrations of taxol anocodazole only incompletely dampen microtubule dynamand/or the organelles have an additional means of getting abwhich is not inhibitable in this manner. While there is direexperimental support of the first possiblity (G. Gundersepersonal communication), the latter explanation is also velikely. Indeed, we have results showing that in the in vivo assdescribed in Fig. 2, 10 mM butanedione monoxime, (the onavailable relatively specific inhibitor of myosin ATPasesCramer and Mitchison, 1995) inhibits phagosome accumulatat the perinuclear region at least as effectively as 50 nM taxIn addition, Kuznetsov and co-workers have shown that perinuclear accumulation of phagosomes is strongly inhibitby µM amounts of cytochalasin D (M. Shonn et alunpublished). Thus we believe that phagosomes have a lthree modes of locomotion within cells: (1) microtubule-basmotors, (2) actin-based motors and (3) microtubule dynamFurther work should reveal why this is so and how the differemodes of transport relate to the life cycle of this organelle.

Fig. 4. Phagosomes display a regulated preference for bindingmicrotubules at their plus-end. (A) Polarity-marked GMP-CPPmicrotubules were generated as described in Materials and methodsand mixed with phagosomes (purified after 1 hour of pulse with fishskin gelatine (FSG)-coupled blue latex beads into J774 mousemacrophages and 1 hour of chase, Blocker et al., 1996, 1997) andmacrophage cytosol at 2 mg/ml. A typical phagosome-microtubuleend-binding event is shown here, where the microtubule minus-end ismarked by an Oregon green-labelled segment approximately onefourth into its length (the rest of the microtubule being labelled withrhodamine). The latex bead containing-phagosome appears as a redspherical structure obscuring the plus-end of the microtubule becausethe blue latex bead fluoresces dimly in the rhodamine range. Thescale of this micrograph is given by the latex bead diameter, which isapproximately 1 µm. Polarity-marked microtubules were mixed inthe presence of macrophage cytosol with phagosomes purified after a20-minute latex bead pulse (B), or a 1-hour bead pulse followed by a1- (C) or 24- (D) hour bead chase. The graphs are histograms of thepercentage of phagosomes found bound within each tenth percentileof microtubule length. For each graph a total of 50-100 bindingevents were scored in at least three independent experiments. Thedifference observed in plus-end binding between 20-minute or 1hour/1 hour- and 1 hour/24-hour phagosomes is significant atP=0.0005 (t-test).

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We have no data to indicate how phagosomes move usmicrotubule dynamics. One possibility, however, is that thare able to latch on to the end of a microtubule and follow growth and shrinkage. We have preliminary evidence thphagosomes can move by following the depolymerising eof a microtubule in a modified version of the in vitroTetrahymenapellicle assay of McIntosh and co-workers (ABlocker, F. Severin, G. Griffiths and A. Hyman, unpublishedata), and this is supported by the speed of phagosome smovements. 6 µm/minute or less is considerably slower thathe rate of growth or shrinkage of Golgi or ER tubules in vitor in vivo (20-70 µm/minute; Waterman-Storer et al., 1995Lee and Chen, 1988; Cooper et al., 1990). Yet, the rate of sphagosome movements is in the same range as thakinetochore poleward movements during anaphase in vivoµm/minute), which have been directly shown to be powerby microtubule dynamics (Mitchison et al., 1986)Interestingly, the rate of microtubule shrinkage in vivo at least an order of magnitude faster, suggesting tphagosomes, like kinetochores, can alter parameters microtubule dynamics.

To be able to follow the end of a dynamic microtubulphagosomes must somehow recognise and stay at the plustip of a dynamic microtubule. We therefore designed an asto measure such an interaction. This system is based onprevious work showing that phagosomes interact in a stamanner with microtubules in vitro (Blocker et al., 1996) anon work showing that kinetochores can recognise microtubplus ends (Severin et al., 1997). By using far more dilumicrotubules and phagosomes than in our earlier studiescould examine more precisely the position within thmicrotubule where the phagosomes bind. Reassuringly, new system showed similar requirements (cytosolic MAPbest for ‘young’ phagosomes) for phagosome-microtubuinteractions. The phagosome-microtubule end-binding asalso provided new information. Indeed it allowed us demonstrate that in the presence, but not in absence shown) of cytosol, phagosomes preferentially recognise plus end of microtubules polymerised entirely from tubulibound to the slowly hydrolysable GTP analogue GMP-CPThis indicates that phagosomes and kinetochores (Severial., 1997) can differentiate between the plus- and minus-eof microtubules even when both are in the same nucleotstate. This, in turn, suggests that what the organelles recognising is not the GTP structure of the tubulin that is liketo be at the plus-end of growing microtubules in cells (Carlie1989; Dreschel and Kirschner, 1994; Mitchison, 1993), brather the structure of the plus-end per se. Unfortunately,

Fig. 5.MAPs are necessary but not sufficient for phagosomes torecognise microtubule plus ends. Blue latex beads coupled to fishskin gelatine were pulsed and chased for 1 hour into J774 mousemacrophages; phagosomes were purified and mixed with polarity-marked GMP-CPP microtubules in the presence of (A) mock-depleted macrophage cytosol at 2 mg/ml, (B) MAP-depleted cytosolat 2 mg/ml, (C) MAP-depleted cytosol plus MAPs at 300 µg/ml and(D) MAPs alone at 300 µg/ml. The graphs are histograms of thepercentage of phagosomes found bound within each tenth percentileof microtubule length. For each graph a total of 50-100 bindingevents were scored in at least three independent experiments. Thedifference observed in plus-end binding between mock-depletedcytosol and MAP-depleted cytosol is significant (P=0.0005; t-test).


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precise structure of GMP-CPP microtubules as well as thamicrotubule plus-ends in cells or even in cytoplasmic extraremains unknown.

Our data show that the presence of MAPs within cytosoessential for early phagosomes to recognise microtubule pends. We were unable to establish, but cannot exclude, thastill unidentified 200 kDa binding factor we previouslcharacterised is part of the cytosolic activity which mediatthe preference of phagosomes for microtubule plus-enIndeed, when we tried to complement MAP-depleted cytowith the 200 kDa fraction from macrophage cytosol thcontained the phagosome-microtubule binding activity widentified previously (Blocker et al., 1996), we had no succedespite the fact that this fraction was able to complement MAdepleted cytosol in our previous assay (not shown). Thusseems that macrophage MAPs alone are not able to medthe recognition of microtubule plus-ends by early phagosomalthough we previously reported that MAPs alone supporstatic phagosome-microtubule binding (Blocker et al., 199We conclude that ‘binding’ factor(s) are necessary but nsufficient for microtubule end-recognition.

The sum of these data provides the first evidence preferential microtubule plus-end binding of a membraorganelle. We have as yet no data to support the notion microtubule plus-end binding is required for following dynamic microtubule. However, our work does providinteresting correlative evidence this may be the case. Indewe showed that 90% of the phagosomes that move sloinitiate their movement within 3 µm of the cell membrane.Phagosomes become internalised at the cell periphery aonce within the cell, they often move faster. That the slomovements represent early phagosomes following shrinking ends of dynamic microtubules is supported by ourvitro finding that 20-minute and 1-hour pulsed/1-hour chasphagosomes but not 1-hour pulsed/24-hours chaphagosomes recognise microtubules plus-ends. This switcend affinity, which we measure in vitro between early and laphagosomes, may represent a means for these organelldeal with the in vivo behaviour of microtubules known a‘tempered dynamic instability’. Indeed, most microtubules cells have a highly dynamic plus-end extremity on a mostable minus-end (possibly post-transcriptionally modified microtubule-associated protein-coated; reviewed in Wordemand Mitchison, 1994). Thus phagosomes would use motormove along the relatively stable microtubules in the centre pof the cell and follow their dynamics where they rapidly tuover, that is in the cell periphery.

Although we have shown that MAPs are required fphagosomes to prefer microtubule plus-ends, their precise in the motility of organelles following dynamic microtubuleis still an open question. McIntosh and co-workers have shothat kinesin and kinesin-like proteins are involved in this kinof movement in vitro (Lombillo et al., 1995a,b). Future woris required to determine the respective role of motors aMAPs in microtubule motor- and dynamics-mediated organemovement.

This work is dedicated to the memory of Mary S. Tilney.

We thank Gregg Gundersen for communication of results priorpublication, Philippe Sansonetti for understanding patience a

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Rebecca Heald, Sigrid Reinsch, Trina Schroer, Scott Schuyler aMike Sheetz for stimulating discussions. A.B. is particularly indebteto Irmgard Sinning for helping her obtain, from the EMBLAdministration, the maternity leave that she was entitled to and whallowed her to return to EMBL for the few months necessary to finithe experiments presented in this article. This work was also suppoby grants from the Human Frontiers Science Programme to GarGriffiths and Tony Hyman.

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A role for microtubule dynamics in phagosome movement