J Cell Sci-1993-Perou-99-107 (1)

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

1/9

INTRODUCTION

Chediak-Higashi Syndrome (CHS) is an autosomal recessivedisorder of humans, mice, cows and mink. Homozygotes forthe defective gene suffer from a variety of symptoms includ-ing partial albinism, neurological abnormalities, and recur-rent bacterial infections (for review see Barak and Nir, 1987).A characteristic feature of this disorder is the presence ofgiant granules or vesicles within most cells of the body.This phenotype is most pronounced within granule-contain-ing cells, such as melanocytes and phagocytes. Although thebasic defect responsible for CHS has remained elusive,studies have demonstrated a variety of cellular abnormali-ties. Alterations have been reported in membrane fluidity(Haak et al., 1979), cyclic nucleotide levels (Oliver et al.,1975), and impaired microtubule function (Oliver et al.,1976; Boxer et al., 1979). Oliver et al. (1975) demonstratedthat in polymorphonuclear leukocytes (PMNs) from the beigemouse (an animal model of CHS), addition of concanavalinA results in the formation of a concanavalin A cap.Whereas, in normal murine PMNs, capping required bothconcanavalin A and colchicine. While this study implicatedthe involvement of microtubules in this disorder, further workdemonstrated that CHS cells possess normal numbers,lengths and distributions of microtubules (Frankel et al.,1978; White and Clawson, 1979; Pryzwansky et al., 1985).These findings suggest that the molecular defect responsiblefor CHS is not a defect in tubulin or microtubules.

Various studies have shown that lysosomal morphologyis dependent upon microtubules and microtubule motors(Matteoni and Kreis, 1987; Swanson et al., 1987; Hollen-beck and Swanson, 1990; Swanson et al., 1992). Hollen-beck and Swanson (1990) demonstrated that anti-kinesinantibodies scrape-loaded into macrophages caused theirlysosomes to collapse around the nucleus. This altered dis-tribution of lysosomes mimics the natural distribution oflysosomes within a CHS cell. In many, but not all cell types,intact microtubules appear to be required for the deliveryof endocytosed ligands to lysosomes in vivo (Oka andWeigel, 1983; Gruenberg et al., 1989). These results, whichimplicate the microtubule-based motor system in the for-mation of lysosomes, suggest that the abnormal size anddistribution of lysosomes in CHS cells may be due to analtered lysosome-microtubule motor interaction. Morespecifically, CHS may be caused by a defect in a micro-tubule-associated motor protein, resulting in a net inwardshift in the lysosome equilibrium. This would cause anaccumulation of lysosomes within the perinuclear region.

It is possible to alter the lysosomal distribution inmacrophages by treating cells with phorbol myristateacetate (Phaire-Washington et al., 1980) or by acidifyingthe cytoplasm (Heuser, 1989). In treated macrophages, thedistribution of lysosomes changes from a perinuclear tubu-lar array to a peripheral distribution of many small vesi-cles, which are often located close to the plasma membrane.When the acidification medium is removed, lysosomes

99Journal of Cell Science 106, 99-107 (1993)Printed in Great Britain The Company of Biologists Limited 1993

Chediak-Higashi Syndrome is an autosomal recessivedisorder that affects intracellular vesicle formation. The

diagnostic feature of Chediak-Higashi Syndrome is thepresence of giant lysosomes clustered near the nucleus.Lysosome morphology in macrophages is maintained bymicrotubules and microtubule-based motors, such askinesin. Dramatic changes in lysosome morphology canbe induced by lowering cytoplasmic pH or by addingphorbol esters. When macrophages from beige mice (amurine homolog of Chediak-Higashi Syndrome) weresubjected to these protocols they were able to alter their

lysosomal distribution and morphology to the samedegree as macrophages from control mice. These results

indicate that lysosomes in Chediak cells are capable ofinteracting with the microtubule-based motor system,suggesting that the defective gene product is not analtered microtubular element involved in lysosomalmovement.

Key words: lysosomes, microtubules, Chediak-Higashi Syndrome,macrophages

SUMMARY

Chediak-Higashi Syndrome is not due to a defect in microtubule-based

lysosomal mobility

Charles M. Perou and Jerry Kaplan*

Division of Cell Biology and Immunology, Department of Pathology, University of Utah College of Medicine, Salt Lake City,Utah, USA

*Author for correspondence

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

2/9

100 C. M. Perou and J. Kaplan

Fig. 1. Lysosome distribution and morphology in treated and untreated macrophages. Control macrophages (A,C,E) and beige mousemacrophages (B,D F) were incubated in complete medium plus 1 mg/ml LY for 1 hour, and then chased in normal medium for anadditional hour. Cells were then examined either directly (A,B), or after incubation in either Acetate Ringers medium for 20 minutes(C,D) or in complete medium plus 1 g/ml PMA for 20 minutes (E,F). The arrows denote large lysosomes, which appeared immobile(C,D,F). Bar, 10 m.

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

3/9

101Chediak-Higashi Syndrome

move back towards the nucleus and regain their normal con-figuration. The movement of lysosomes is thought to resultfrom a motor protein-microtubule interaction. We have uti-lized these protocols to test the hypothesis that the abnor-mal morphology and distribution of CHS lysosomes resultsfrom an altered lysosome microtubule-motor interaction.We observed that lysosomes from control and beige mice

behaved similarly when exposed to acidification medium orphorbol myristate acetate (PMA). We also observed that innormal cells, large perinuclear lysosomes are sometimesunable to move in response to acidification or PMA, whilethe rest of the cells lysosomes respond. Lack of movementof large lysosomes occurs in both normal and CHS cells,but is more prevalent in CHS cells because they have ahigher proportion of large lysosomes. These results indicatethat the molecular defect responsible for CHS is not a defectin a microtubule motor.

MATERIALS AND METHODS

CellsBone marrow-derived macrophages were obtained by the methodof Swanson (1989) from C57BL/6 mice and from C57BL/6 bg/bg(beige mice) (obtained from Jackson Lab., Bar Harbor, ME).Macrophages were maintained in complete bonemarrow/macrophage medium (Dulbeccos Modified EaglesMedium plus 30% L-cell conditioned medium plus 20% FCS).Typically, cells were subcultured by using ice-cold divalentcation-free phosphate-buffered saline (PD), with 7105 cellsplated onto 18 mm glass coverslips, which were contained withina 60 mm plate.

Experimental treatments

Macrophages were labeled with Lucifer Yellow (LY) as described

by Swanson et al. (1987), with the following modifications. Cellson glass coverslips were incubated in complete bone marrowmacrophage medium plus 1 mg/ml Lucifer Yellow CH (AldrichChemical Co., Milwaukee, WI) or 2 mg/ml Dextran-Texas Red(Molecular Probes, Eugene, OR) for 1-2 hours at 37C. The cellswere washed three times with PBS, and then incubated in com-plete medium for at least 30 minutes to chase the fluorescentmarker out of the early endocytic pathway. Cells were eitherdirectly examined, or exposed to further treatments. Acid-treatedcells were incubated in Acetate Ringers solution (80 mM NaCl,70 mM sodium acetate, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,2 mM NaH2PO4, 10 mM Hepes, 10 mM glucose, and 0.5 mg/mlBSA, pH 6.9; Heuser, 1989) for 20-30 minutes at 37C. Phorbolmyristate acetate (PMA)-treated cells were incubated in completemedium plus 1 g/ml PMA (Sigma Chemical Co., St. Louis, MO)

for 20-30 minutes at 37C. Nocodazole-treated cells were incu-bated in complete medium plus 5 M nocodazole (Sigma Chem-

ical Co., St. Louis, MO) for the indicated times at 37C. Cellswere incubated in complete medium plus FITC-coupled 0.15,0.46, or 1.0 m latex beads (Polysciences, Inc., Warrington, PA)for 2 hours.

Microscopy

After the stated treatments, macrophages on coverslips wereinverted onto a glass microscope slide into the same medium asused for their last incubation, sealed with nail polish, andobserved. Macrophages used for indirect immunoflourescencewere prepared as described by Karsenti et al. (1984) using a mousemonoclonal antibody directed against -tubulin (ICN, Costa Mesa,

CA). The cells were examined for fluorescence using a Zeiss pho-tomicroscope with a 100 Zeiss Plan-Neofluar oil immersionobjective, and photographed using Kodak EktachromeP800/P1600 Professional color reversal film.

RESULTS

Beige mouse macrophage lysosomes are able toredistribute to the same magnitude as normalmacrophages lysosomes

Cultured macrophages were incubated with LY in order tovisualize their lysosomes (see Materials and Methods), andthen incubated in either complete medium plus 1 g/mlPMA (Phaire-Washington et al., 1980) or Acetate Ringer(Heuser, 1989) for 20-30 minutes. In untreated control cells,there is an array of tubular lysosomes emanating from a

Fig. 2. Effects of removal of Acetate Ringer on the distribution oflysosomes in control and beige macrophages. Control and beigemacrophages were incubated with LY as described for Fig. 1.After labeling with LY, the cells were incubated in AcetateRinger for 20 minutes. The cells were then washed three times inculture medium and incubated in culture medium. (A) Controland (B) beige mouse macrophages after a 1 hour recovery inculture media.

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

4/9

102

perinuclear location, presumably the microtubule organiz-ing center (Fig. 1A). Vesicular lysosomes can be found inthe perinuclear region and also scattered throughout the cell.There are, however, very few lysosomes near the plasmamembrane. These observations agree with previously pub-lished data (Swanson et al., 1987; Hollenbeck and Swan-son, 1990). A similar distribution of lysosomes is seen inthe beige mouse-derived macrophages, except for anincrease in the number of giant lysosomes clustered nearthe nucleus (Fig. 1B). These enlarged lysosomes are char-acteristic of CHS.

When either control or beige mouse macrophages weretreated with Acetate Ringer for 20 minutes, there was a dis-

appearance of tubular lysosomes and a large increase in thenumber of smaller vesicles (Fig. 1C,D). The small lyso-somes were scattered throughout the cell, but were pre-dominantly located at the cell periphery, and were oftenclustered in lamellipodia (Fig. 1C). Similar changes in lyso-some morphology were seen when cells were incubated inmedium that contained 1 g/ml PMA for 20 minutes (Fig.1E,F). Macrophages incubated in lower concentrations ofPMA also responded by redistributing their lysosomes. AtPMA concentrations of 0.01 g/ml to 0.1 g/ml the fre-quency of responding cells was lower, and it took a longertime for these cells to respond (data not shown). The major-ity of lysosomes, in both control and beige macrophages,

C. M. Perou and J. Kaplan

Fig. 3. Effects of nocodazole on the acid-induced redistribution of lysosomes in beige mouse macrophages. Lysosomes in beigemacrophages were labeled with LY as described in Materials and Methods. One set of macrophages was incubated with 5 M nocodazolefor 40 minutes only. Other sets of macrophages were treated with nocodazole before, during or after incubation in Acetate Ringer.(A,B) Macrophage treated with Acetate Ringer. (C,D) Nocodazole-treated macrophage. (E,F) Macrophage incubated in Acetate Ringerplus 5 M nocodazole. (G,H) Macrophage incubated with nocodazole during the lysosome recovery period following Acetate Ringertreatment. Bar, 10 m.

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

5/9


responded in a similar manner. Occasionally, large perinu-clear lysosomes did not move after either treatment (Fig.1C,D,F). These large immobile lysosomes occurred in con-trol macrophages, as well as in beige mouse macrophages,

but were far more numerous in the beige mouse-derivedcells.Upon removal of the Acetate Ringer, the normal distri-

bution of tubular lysosomes was recovered after 1-2 hoursfor both control (Fig. 2A) and beige mouse macrophages(Fig. 2B); changes induced by PMA were not reversible.While both cell types showed tubular lysosomes, qualita-tively CHS cells appeared to have more tubular lysosomes.At this point, the beige mouse macrophage appeared phe-notypically normal, except for the increased number ofimmobile lysosomes. The giant lysosomes characteristicof CHS, however, required an additional 4-8 hours to re-form fully.

Movement of lysosomes is dependent upon intactmicrotubules

We employed nocodazole to verify that microtubules wereinvolved in the maintenance of lysosomal distributions.

Beige mouse macrophages were loaded with LY and thentreated with nocodazole before, during or after incubationin Acetate Ringer. Macrophages incubated with nocodazolecontained no intact microtubules as determined by indirectimmunoflourescence of an anti--tubulin monoclonal anti-body (data not shown). Beige cells incubated in AcetateRinger responded by redistributing their lysosomes to thecell periphery (Fig. 3A). For this and the following exper-imental conditions the corresponding phase-contrast pic-tures are included to show the position of lysosomes rela-tive to the cell periphery (Fig. 3B,D,F,H). Macrophagesincubated with 5 M nocodazole for 40 minutes did notexhibit the normal tubular array of lysosomes. Rather,

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

6/9

104

vesicular lysosomes were clustered near and around thenucleus (Fig. 3C). This is in accord with the observationsof Hollenbeck and Swanson (1990) in mouse bone marrow-derived macrophages. Cells that received nocodazole plus

Acetate Ringer appeared similar to cells that had receivednocodazole alone (Fig. 3E). These cells did not contain anyperipheral lysosomes but, instead, contained only aggre-gates of perinuclear vesicles.


Fig. 4. Effects of cytoplasmic acidification on the movement of phagolysosomes containing beads of different diameters. Lysosomes incontrol and beige macrophages were labeled with Dextran-Texas Red as described in Materials and Methods. The cells were thenincubated with 1.0 m or 0.46 m FITC-coupled latex beads for 2 hours, washed several times with culture medium, and then incubatedin culture medium for an additional hour. The cells were then further incubated in Acetate Ringer for 20 minutes. The cells werephotographed using two-color optics, which allowed for the simultaneous visualization of the FITC and Texas Red. (A) Control and (B)beige macrophage loaded with 1.0 m beads after Acetate Ringer treatment. (C) Control and (D) beige macrophage loaded with 0.46 mbeads after Acetate Ringer treatment.

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

7/9


To determine if the microtubule-based motor system isrequired for the movement of lysosomes back to the centerof a cell, beige macrophages were labeled with LY and thenincubated in Acetate Ringer for 20 minutes. At the end ofthat incubation, 5 M nocodazole was added for ten moreminutes. The cells were then incubated in normal medium

plus 5 M nocodazole for 40 minutes before being exam-ined. In about 70% of the cells, aggregates of vesicular lyso-somes were observed throughout the cell. These cellsresponded to nocodazole by rounding up. This alterationin cell shape made it difficult to define lysosomal distrib-ution. In about 30% of the population the cells remained

Fig. 5. Effects of cytoplasmic acidification on the mobility of phagolysosomes containing 0.15 m beads. Control and beige macrophageswere treated as described for Fig. 4, except that they were loaded with 0.15 m FITC-latex beads. In contrast to the larger beads, thesmaller beads could not be visualized using two-color optics. Two separate photographs of each cell are provided to visualize the FITC-latex beads (A,C) and the lysosomes labeled with Dextran-Texas Red (B,D). (A,B) Control macrophage and (C,D) beige macrophageafter Acetate Ringer treatment.

8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

8/9

106

spread out in nocodazole. In these spread cells, there wasno apparent recovery from the Acetate Ringer-induced dis-tribution of lysosomes (Fig. 3G). This observation suggeststhat if microtubules are disrupted while the lysosomes areat the periphery, the lysosomes are unable to return to theiroriginal position. Similar results were obtained using con-trol macrophages. These results demonstrate that intact

microtubules are required for the movement of lysosomesto the periphery, and from the periphery back to the perin-uclear region.

Large perinuclear lysosomes in macrophagessometimes remain immobile

Large lysosomes, the main distinguishing characteristic ofCHS cells, are also found within normal cells, although ata much lower frequency. Both control and beige mousebone marrow-derived macrophages possess large perinu-clear lysosomes, which sometimes failed to move inresponse to either Acetate Ringer or PMA. We reasonedthat large lysosomes do not redistribute because they aretoo large to move through the crowded cytoplasm using

molecular motors. To determine whether it is simply thesize of a vesicle that influences its ability to move we exam-ined the movement of vesicles containing latex beads ofvarious sizes.

Control and beige macrophages were incubated inmedium containing 2 mg/ml Dextran-Texas Red for 2hours, followed by a 1 hour chase. This allows for visual-ization of the cells lysosomes. The cells were then incu-bated in medium that contained 1.0 m FITC-coupled latexbeads for 2 hours, followed by a 1 hour chase in the absenceof beads. The beads were initially internalized into phago-somes, which, with time, fused with the Dextran-TexasRed-containing lysosomes, resulting in phagolysosomescontaining both markers (data not shown). When thesemacrophages were exposed to Acetate Ringer, the lyso-somes containing 1.0 m latex beads failed to move whilemost of the other, smaller lysosomes within the same cellresponded (Fig. 4A,B).

In contrast to cells containing 1.0 m beads, cells withvesicles containing 0.46 m beads showed a limited degreeof movement. Most of the beads showed a perinuclear dis-tribution, but between 20 and 30% of the beads were ableto show some movement. The beads that moved assumeda position intermediate between the nucleus and the cellperiphery; only rarely was a bead found at the cell periph-ery (Fig. 4C,D). Vesicles containing the 0.15 m beadsbehaved identically to vesicles containing Dextran-TexasRed. These beads responded to acidification and weremoved to the cell periphery (Fig. 5A-D). In all of the beadexperiments, control and beige mouse macrophagesresponded in a similar manner; no difference in bead mobil-ity was seen when these two macrophage populations werecompared. These experiments demonstrate that size may bea major factor influencing lysosome mobility.

DISCUSSION

Studies on the surface mobility of membrane proteins ledto the view that an alteration in microtubules was respon-

sible for the defect in granule maturation seen in CHS(Oliver et al., 1975). Since that initial observation, studieshave looked for a defect in microtubules without success.The discovery that vesicle movement is mediated not onlyby microtubules, but also by a set, or sets, of proteins thatfunction as motors (Hollenbeck and Swanson, 1990),inspired us to re-examine the role of microtubule involve-

ment. We proposed that CHS may result from an alterationin either a microtubule motor or the coupling between vesi-cle and motor protein. To examine this hypothesis, we uti-lized protocols that result in a microtubule and microtubulemotor-dependent alteration in lysosomal vesicle movement(Phaire-Washington et al., 1980; Heuser, 1989). We demon-strated that beige mouse macrophages, in response to theaddition of PMA or cytosolic acidification, were able toredistribute their lysosomes within the same time span andto the same degree as control macrophages.

Upon exposure to Acetate Ringer or PMA, beige mousemacrophages alter the distribution of their lysosomes froma perinuclear tubular array to being located in small periph-eral vesicles. The original distribution of tubular lysosomes

was recovered when Acetate Ringers medium wasremoved, but changes induced by PMA were irreversible.Large, perinuclear lysosomes sometimes remained immo-bile, while other lysosomes within the same cell changedlocation and morphology. Although this phenomenonoccurred in control cells (i.e. C57BL/6), it was far morefrequent in beige mouse-derived macrophages, due to theincreased number of large lysosomes. On the basis of thisobservation, we reasoned that one factor that may influencethe mobility of lysosomes may be the size of the vesicle.

To test this hypothesis, we generated vesicles of differ-ent sizes using fluorescent latex beads. As demonstrated inFig. 4A and B, phagolysosomes containing 1.0 m beadsdid not move when the cells were exposed to AcetateRinger. This observation extends the results obtained byHeuser (1989), who demonstrated that phagosomes formedby a 5 minute exposure of macrophages to latex beads wereunable to move in cells incubated with Acetate Ringer.Experiments were also performed using beads of smallerdiameter. Vesicles containing 0.46 m beads showed lim-ited movement. While most of these vesicles did not move,a small proportion showed some movement; 0.46 m beadswere rarely found at the cell periphery and were generallyobserved at positions intermediate between the peripheryand the nucleus. The possibility exists that the beads thatdid move were of a smaller size than the ones that did not,since the diameter of the beads reflects the population aver-age. The ability of smaller beads (0.15 m) to move wasidentical to that of the Dextran-Texas Red-containing lyso-somes. These studies indicate that size may play a role inregulating lysosome mobility; large lysosomes, which canbe 2-4 m in diameter, may simply be too large to movethrough the network of filaments present within a cell.

CHS neutrophils are typified by delayed phagosome-lysosome fusion (Root et al., 1972; White and Clawson,1980) and impaired degranulation (Stossel et al., 1972). Theinability to move giant vesicles could be responsible forthese defects. The large lysosomes may be excluded fromthe crowded filamentous cytosol. This exclusion maydelay or inhibit their fusion with newly formed phago-


8/11/2019 J Cell Sci-1993-Perou-99-107 (1)

9/9


somes; the phagosomes must now move to the large lyso-somes, rather than the phagosome and lysosomes movingtowards one another. Support for this hypothesis comes

from the observation that the occasional small lysosomespresent in CHS PMNs fuse normally with newly formedphagosomes, whereas fusion with the large lysosomes isdelayed (White and Clawson, 1980).

These results suggest that the defect responsible for

Chediak-Higashi Syndrome is not a mutation within a generequired for microtubule-based lysosomal movements.Lysosomes in beige mouse-derived macrophages were ableto undergo microtubule-based alterations in lysosome mor-phology. The observation that the giant lysosomes charac-teristic of beige mouse macrophages required more time to

re-form than the tubular portion of the lysosomal array aftercytoplasmic acidification suggests that factors other thanmicrotubules are involved in defining lysosome morphol-ogy and number.

It has been suggested that the giant lysosomes form as

a result of lysosome-lysosome fusion (White and Clawson,

1980; Spicer et al., 1981). Large lysosomes, present inmature monocytes, neutrophils and eosinophils in the beigemouse, appear to arise from inappropriate lysosomal fusionsthat occur during cellular maturation (Oliver and Essner,

1975). It has recently been demonstrated that GTP-bindingproteins are involved in the regulation of vesicular traffic(Bacon et al., 1989; Walworth et al., 1989; Zahraoui et al.,1989; Chavrier et al., 1990). Gorvel et al. (1991) directlydemonstrated that alterations in rab5 expression can result

in enlarged vesicles. As yet, no GTP-binding protein hasbeen identified that is specifically associated with lyso-somes. One attractive hypothesis is that an uncharacterizedGTP-binding protein may be involved in regulating lyso-somal fusion. A defective GTP-binding protein could

account for the CHS phenotype by altering the regulation

of lysosomal fusions due to a mutation that changes itsnormal GTP hydrolytic cycle.

This work was supported by a grant from the NIH (H.L. 26922).C. M. Perou is the recipient of a NIH training grant(2T32GM07464). The authors thank Don Morse for his help in

preparing this manuscript and Dr M. Rechsteiner for reviewingthis manuscript.

REFERENCES

Bacon, R. A., Salminen, A., Ruohola, H., Novick, P. and Ferro-Novick,

S. (1989). The GTP-binding protein Ypt1 is required for transport in vitro:The golgi apparatus is defective in ypt1 mutants.J. Cell Biol. 109, 1015-

1022.

Barak, Y. and Nir, E. (1987). Chediak-Higashi Syndrome.Amer. J. Pediat.

Hematol./Oncol.9, 42-55.

Boxer, L. A., Albertini, D. E., Baehner, R. L. and Oliver, J. M. (1979).

Impaired microtubule assembly and polymorphonuclear leukocyte

function in the Chediak-Higashi Syndrome correctable by ascorbic acid.

Br. J. Haematol.43, 207-213.

Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K. and Zerial, M.(1990). Localization of low molecular weight GTP binding proteins to

exocytic and endocytic compartments. Cell 62, 317-329.

Frankel, F. R., Tucker, R. W., Bruce, J. and Stenberg, R. (1978).

Fibroblasts and macrophages of mice with the Chediak-Higashi

Syndrome have microtubules and actin cables.J. Cell Biol.79, 401-408.

Gorvel, J.-P., Chavrier, P., Zerial, M. and Gruenberg, J. (1991). rab5controls early endosome fusion in vitro. Cell 64, 915-925.

Gruenberg, J., Griffiths, G. and Howell, K. E. (1989). Characterization ofthe early endosome and putative endocytic carrier vesicles in vivo andwith an assay of vesicle fusion in vitro.J. Cell Biol.108, 1301-1316.

Haak, R. A., Ingraham, L. A., Baehner, R. L. and Boxer, L. A. (1979).

Membrane fluidity in humans and mouse Chediak-Higashi leukocytes.J.Clin. Invest. 64, 138-141.

Heuser, J. (1989). Changes in lysosome shape and distribution correlatedwith changes in cytoplasmic pH.J. Cell Biol.108, 855-864.

Hollenbeck, P. J. and Swanson, J. A. (1990). Radial extension ofmacrophage tubular lysosomes supported by kinesin. Nature 346, 864-

866.Karsenti, E., Kobayashi, S., Mitchison, T. and Kirshner, M. (1984). Role

of the centrosome in organizing the interphase microtubule array:properties of cytoplasts containing or lacking centrosomes.J. Cell Biol.

98, 1763-1776.Matteoni, R. and Kreis, T. E. (1987). Translocation and clustering of

endosomes and lysosomes depends on microtubules. J. Cell Biol. 105,

1253-1265.Oka, J. A. and Weigel, P. H. (1983). Microtubule-depolymerizing agents

inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis

or degradation in isolated rat hepatocytes. Biochim. Biophys. Acta 763,368-376.

Oliver, C. and Essner, E. (1975). Formation of anomalous lysosomes in

monocytes, neutrophils, and eosinophils from bone marrow of mice withChediak-Higashi Syndrome.Lab. Invest. 32, 17-27.

Oliver, J. M., Krawiec, J. A. and Berlin, R. D. (1976). Carbamylcholineprevents giant granule formation in cultured fibroblasts from beige

(Chediak-Higashi) mice.J. Cell Biol.69, 205-210.Oliver, J. M., Zurier, R. B. and Berlin, R. D. (1975). Concanavalin A cap

formation on polymorphonuclear leukocytes of normal and beige

(Chediak-Higashi) mice.Nature 253, 471-473.Phaire-Washington, L., Silverstein, S. C. and Wang, E. (1980). Phorbol

myristate acetate stimulates microtubule and 10-nm filament extensionand lysosome redistribution in mouse macrophages.J. Cell Biol.86, 641-

655.Pryzwansky, K. B., Schliwa, M. and Boxer, L. A. (1985). Microtubule

organization of unstimulated and stimulated adherent human neutrophils

in Chediak-Higashi Syndrome.Blood66, 1398-1403.Root, R. K., Rosenthal, A. S. and Balestra, D. J. (1972). Abnormal

bactericidal, metabolic and lysosomal functions of Chediak-Higashi

Syndrome leukocytes.J. Clin. Invest.51, 649-665.Spicer, S. S., Sato, A., Vincent, R., Eguchi, M. and Poon, K. C. (1981).

Lysosome enlargement in the Chediak-Higashi Syndrome. Fed. Proc.Fed. Amer. Socs Exp. Biol. 40, 1451-1455.

Stossel, T. P., Root, R. K. and Vaughan, M. (1972). Phagocytosis inchronic granulomatous disease and the Chediak-Higashi Syndrome.New

Engl. J. Med286, 120-123.

Swanson, J. A. (1989). Phorbol esters stimulate macropinocytosis andsolute flow through macrophages.J. Cell Sci.94, 135-142.

Swanson, J. A., Bushnell, A. and Silverstein, S. C. (1987). Tubularlysosome morphology and distribution within macrophages depend on

the integrity of cytoplasmic microtubules. Proc. Nat. Acad. Sci. USA 84,1921-1925.

Swanson, J. A., Locke, A., Ansel, P. and Hollenbeck, P. J. (1992). Radial

movement of lysosomes along microtubules in permeabilizedmacrophages.J. Cell Sci.103, 201-209.

Walworth, N. C., Gould, B., Kabcenell, A. K. and Novick, P. J. (1989).

Mutational analysis of Sec4 suggests a cyclical mechanism for theregulation of vesicular traffic.EMBO J.8, 1685-1693.

White, J. G. and Clawson, C. C. (1979). The Chediak-Higashi Syndrome:Microtubules in monocytes and lymphocytes.Amer. J. Hematol. 7, 349-

356.White, J. G. and Clawson, C. C. (1980). The Chediak-Higashi Syndrome:

The nature of the giant neutrophil granules and their interactions with

cytoplasm and foreign particulates.Amer. J. Pathol.98, 151-194.Zahraoui, A., Touchot, N., Chardin, P. and Tavitian, A. (1989). The

human Rab genes encode a family of GTP-binding proteins related toyeast YPT1 and SEC4 products involved in secretion.J. Biol. Chem. 264,

12394-12401.

(Received 7 December 1992 - Accepted, in revised form,27 May 1993)

Documents

J Cell Sci-1993-Perou-99-107 (1)