IntroductionMelanosomes are organelles unique to melanocytes thatfunction in the synthesis of melanin, a complex pigmentinvolved in photoprotection of the skin through its ability toabsorb and scatter light and reduce reactive oxygen species(Marks and Seabra, 2001). In order to accomplish this,melanosomes must be transferred to epidermal keratinocytes,where they are found in autophagic vacuoles in a perinuclearor cap-like distribution in vitro and in vivo (Corcuff et al.,2001). Melanosomes are elliptical or spheroidal organelles thatcontain melanogenic enzymes and co-factors, including thetyrosinase gene family of proteins and have been categorizedas stage I-IV on the basis of their electron microscopicappearance and degree of melanization (Kushimoto et al.,2001). Recent studies suggest that premelanosomes (stage Iand II) and late stage melanosomes (stage III and IV) representa distinct lineage of organelles that are separable fromconventional endosomes and lysosomes within pigmented cells(Raposo et al., 2001).

It is now known that melanosome trafficking is mediatedin part by microtubular motor myosin Va, the product of thedilute locus, which traps melanosomes at the actin-richperiphery of the dendrite, and rab27a, the product of theashenlocus (Mercer et al., 1991; Provance et al., 1996; Wuet al., 1997; Wu et al., 1998; Wei et al., 1997; Wilson et al.,2000; Bahadoran et al., 2001; Wu et al., 2001). Rab27a isinvolved in the transport of melanosomes through its abilityto recruit myosin Va to the tip of the melanocyte dendrite(Hume et al., 2001). These important and relatively recentinsights into melanosome trafficking were made possiblethrough the use of mutant mouse strains and time-lapse video

microscopy of cultured cells, which allowed directvisualization of melanosome movement and modifiers ofactin, microtubules and their motor proteins. In contrast withmelanosome trafficking, much less is known aboutmelanosome transfer. A major hurdle that has severelylimited progress in understanding the molecular basis ofmelanosome transfer has been the lack of a model system.The majority of studies of melanosome transfer tokeratinocytes have been based on co-cultures of non-humancells observed by electron microscopy. Studies performedutilizing time-lapse video microscopy have been limited bythe relatively poor resolution achieved (Mottaz andZelickson, 1967; Cohen and Szabo, 1968; Wolff, 1973).Other more recent studies have utilized gold particle uptakeby keratinocytes, melanin uptake or transfer of cytoplasmicdyes from melanocytes to keratinocytes to measure transfer(Seiberg et al., 2000a; Seiberg et al., 2000b; Sharlow et al.,2000; Minwalla et al., 2001). In toto, these prior studies ledto important observations that suggested phagocytosis ofmelanocyte dendrites by keratinocytes as the major mode ofmelanosome transfer, although exocytosis of melanosomesinto the extracellular space with uptake by keratinocytes andinsertion of melanocyte dendrites and transfer ofmelanosomes to keratinocytes have also been proposed(Yamamoto and Bhawan, 1994). Although the more recentstudies using particle uptake provide insight into the role ofthe keratinocyte in granule uptake, the use of a model systemin which melanosome transfer is being studied directlyprovides an opportunity to examine the potential role of themelanocyte in melanosome transfer.

It is well established that Cdc42, a member of the Rho

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Melanosomes are specialized melanin-synthesizingorganelles critical for photoprotection in the skin.Melanosome transfer to keratinocytes, which involveswhole organelle donation to another cell, is a uniquebiological process and is poorly understood. Time-lapsedigital movies and electron microscopy show that filopodiafrom melanocyte dendrites serve as conduits formelanosome transfer to keratinocytes. Cdc42, a smallGTP-binding protein, is known to mediate filopodiaformation. Melanosome-enriched fractions isolated fromhuman melanocytes expressed the Cdc42 effector proteinsPAK1 and N-WASP by western blotting. Expression ofconstitutively active Cdc42 (Cdc42V12) in melanocytes co-

cultured with keratinocytes induced a highly dendriticphenotype with extensive contacts between melanocytesand keratinocytes through filopodia, many of whichcontained melanosomes. These results suggest a unique rolefor filopodia in organelle transport and, in combinationwith our previous work showing the presence of SNAREproteins and rab3a on melanosomes, suggest a novel modelsystem for melanosome transfer to keratinocytes.

Movies available on-line

Key words: Melanosome, Melanocyte, Cdc42, Filopodia,Keratinocyte

Summary

Filopodia are conduits for melanosome transfer tokeratinocytesGlynis Scott, Sonya Leopardi, Stacey Printup and Brian C. MaddenDepartment of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USAAuthor for correspondence (e-mail: Glynis_Scott@urmc.rochester.edu)

Accepted 4 January 2002Journal of Cell Science 115, 1441-1451 (2002) © The Company of Biologists Ltd

Research Article

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family of GTP-binding proteins, is involved in filopodia andmicrospike formation in many cell types. Filopodia are actin-based structures that arise from neuronal growth cones andfunction in neuronal pathfinding (Davenport et al., 1993;Rosentreter et al., 1998). The recent demonstration that Cdc42is associated with coatamer proteins in the Golgi apparatus,that it regulates exit of apical and basolateral proteins from theGolgi network and is involved in exocytosis of secretorygranules in mast cells is indicative of the diverse roles thatCdc42 plays in cells (Brown et al., 1998; Hong-Geller andCerione, 2000; Wu et al., 2000; Müsch et al., 2001). Thedownstream effectors of Cdc42 fall into six families most ofwhich contain a CRIB-binding domain and include Cdc42-binding kinase, myotonic dystrophy kinase-related Cdc42-binding kinase, mixed lineage kinase, p21-activated kinase(PAK), WASP (Wiscot-Aldrich Syndrome Protein), IQGAPand MSE55/BORG/CEP (Burbelo et al., 1995). At least fourclosely related isoforms of PAK (PAK1, PAK2, PAK3 andPAK4) exist in mammalian cells (Manser et al., 1994; Manseret al., 1995; Martin et al., 1995; Dan et al., 2001). PAK-familykinases are activated by GTP-Cdc42 or GTP-Rac1 as well asG-protein-coupled receptors and cytokines and phosphotidyl-inositol 3-kinase (PI3-kinase); this leads to a change inconformation of the kinase inducing autophosphorylation onmultiple serine and threonine residues and activation (Knauset al., 1995; Manser et al., 1997; Wang et al., 1999; Chung etal., 2001). Activation of PAK results in effects that mimicRac1 and Cdc42 and include lamellipodia and filopodiaformation, activation of the c-Jun N-terminal kinase MAPkinase cascade and NKκB, alteration in cell motility andinhibition of apoptosis and stimulation of macropinocytosis(Sells 1997; Sells et al., 1999; Frost et al., 1998; Frost et al.,2000; Dharmawardhane, 2000). Non-kinases that interact withCdc42 include the WASP family, which consist of WASp, N-WASP and related Scar proteins isolated in Dictyostelium.WASP, in concert with WIP (WASP-interacting protein)participates with the Arp2/3 complex to induce actinnucleation and filopodia formation (Symons et al., 1996; Mikiet al., 1996; Miki et al., 1998; Rohatgi et al., 1999; Banzai etal., 2000; Martinez-Quiles et al., 2001). WASP is onlyexpressed in hematopoietic cells and is mutated in patientswith Wiscot-Aldrich syndrome, whereas N-WASP isubiquitously expressed but is enriched in the brain (Fukuokaet al., 1997). In a cell-free system, addition of active Cdc42significantly stimulates neuronal-WASP (N-WASP) byexposure of N-WASPs’ actin depolymerizing region, creatingfree barbed ends from which actin polymerization can takeplace (Suzuki et al., 1998).

In this report we used have high resolution movies madefrom digital images to directly observe melanosome transfer tokeratinocytes in human cells. These movies, along withelectron microscopy of cells in vitro and skin in vivo provideevidence that suggests that melanosome delivery tokeratinocytes occurs along filopodia. We show that expressionof activated Cdc42 in human melanocytes accentuatesfilopodia formation and melanosome transport and thatmelanosomes are enriched in PAK1 and N-WASP, Cdc42-effector proteins. In combination with previous data showingSNARE and rab proteins on melanosomes (Scott and Zhao,2001), these observations suggest a novel model formelanosome transfer to keratinocytes.

Materials and MethodsAntibodies and reagentsPolyclonal antibodies to PAK1 were purchased from ZymedLaboratories (San Francisco, CA); polyclonal antibodies to Cdc42were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA); polyclonal antibodies to N-WASP were a generous gift of DrRohatgi (Harvard Medical School, Boston, MA) and have beendescribed previously (Rohatgi et al., 1999); polyclonal antibodies tochick brain myosin 5a tail domain (clone 32a) were a generous giftof Richard Cheney (Chapel Hill, NC) and have been describedpreviously (Espreafico et al., 1992); monoclonal antibodies to TRP-1(mel-5) were purchased from Signet Laboratories (Dedham, MA);monoclonal antibodies to transferrin receptor were purchased fromZymed Laboratories (San Francisco, CA); polyclonal antibodies totubulin were purchased from Sigma Co (St. Louis, MO); fluoresceinisothiocyanate and Texas Red goat anti-rabbit and anti-mouseantibodies were purchased from Molecular Probes (Eugene, OR);horseradish-peroxidase-conjugated goat anti-rabbit and anti-mouseantibodies and normal rabbit serum were purchased from Sigma Co;vitrogen was purchased from Cohesion (Palo Alto, CA). Membranedyes DiI and DiO and Alexa Fluor 594 phalloidin were purchasedfrom Molecular Probes. Nocodazole and cytochalasin D werepurchased from Sigma Co.

Cell cultureNeonatal foreskins were obtained according to the University ofRochester’s Research Subject Review Board. Co-cultures of humanmelanocytes and keratinocytes were initiated from human foreskinsas previously described (Scott and Haake, 1991) and maintained inKeratinocyte Growth Media (KGM, Gibco BRL, Gaithersburg, PA).In primary skin cultures this media sustains melanocyte growththrough the production of melanocyte growth factors by proliferatingkeratinocytes (Halaban et al., 1988). For growth of melanocytes, cellswere cultured in Melanocyte Growth Media (MGM, Gibco-BRL).

Time-lapse digital microscopy and image processingCo-cultures of melanocytes and keratinocytes (approximately 105

cells total) were subcultured on vitrogen-coated 25 mm glasscoverslips for 1-2 days and placed in a closed heated chamber (WarnerInstruments, New Haven, CT) maintained at 37°C. The cells wereviewed on a Nikon Eclipse Microscope 800 under differentialinterference contrast (DIC) optics with a 100× objective. The chamberwas perfused with KGM maintained at a constant temperature of 37°Cby an in-line heater (Warner Instruments) using gravity flow. The rateof flow was approximately 166 µl/min and imaging lasted 45 minutes.Cell viability was checked following experiments with trypan blueand no cytotoxicity was observed.

Sequential images were obtained at 8 second intervals using thegreen filter of a Spot digital camera (Diagnostic Instruments, SterlingHeights, MI). The resulting 8 bit/pixel megapixel (1315x1033) imagesyielded a resolution of 10 pixels/micron when combined with the100× microscope objective. A series of operations to reduce noise andartefacts were performed on the images using the Matlab GUI facility(Mathworks, Natick, MA). To further reduce size, the movies werecreated using QuickTime Pro (Apple Computer Inc, Cupertino, CA).

Labeling of melanocyte and keratinocyte membranes andevaluation of membrane fusionMelanocytes grown in MGM and keratinocytes grown in KGM werelabeled with DiI (0.6 µM) and DiO (0.2 µM) respectively for 10minutes at 37°C followed by extensive washing. 24 hours later,melanocytes were trypsinized from the dish and added tokeratinocytes on vitrogen-coated 100 mm glass coverslips at an

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1443Role of Cdc42 in melanosome transfer

approximate ratio of 1:1 in KGM. To stimulate melanosome transfer,co-cultures were irradiated with a single dose of ultraviolet (UV)irradiation using a solar simulator at a dose of 4 J/cm2 as previouslydescribed (Scott and Zhao, 2001). 24 hours after irradiation, live cellswere viewed on a Nikon Eclipse Microscope 800 and images werecaptured with a Spot digital camera. To arrive at an approximatepercentage of cells with membrane fusion, the number ofkeratinocytes with yellow fluorescence viewed under a filter to detectboth DiI and DiO was counted in 10 random fields (100× objective).Experiments were repeated three times. Digital images were post-processed using Adobe PhotoShop 5.0.

Melanosome isolation and western blottingMelanosomes were isolated from human melanocytes essentially asdescribed for isolation of melanosomes from cultured B16F1 cells,with a few modifications (Scott and Zhao, 2001). The postnuclearsupernatant was centrifuged for 10 minutes at 10,000 g at 4°C toobtain a large granule and a small granule fraction. The large granulefraction, which is enriched in melanosomes, was then layered onto asucrose gradient and centrifuged at 85,000 g at 4°C for 1 hour. Themelanosome-rich fraction was collected from the 2M layer of thegradient and lysed in buffer (150 mM NaCl, 10 mM Tris-HCL, pH7.8, 1% Triton-X) plus protease inhibitors (Complete TM Mini,Boehringer Mannheim, GmbH, Germany). Protein samples werequantified using the Bio-Rad Dc protein assay kit (Bio-RadLaboratories, Hercules, CA) with bovine serum albumin as standard.Equal amounts of protein were electrophoresed on 10% or 15%precast SDS gels (Jule Inc, New Haven, CT) and blotted tonitrocellulose membranes (Bio-Rad Laboratories) using standardprocedures. Full range rainbow molecular weight markers werepurchased from Amersham Life Sciences (Arlington Heights, Ill).Visualization of the immunoreactive proteins was accomplished usingan enhanced chemiluminescence reaction (Amersham Life Sciences).Positive controls for Cdc42 and N-WASP consisted of mouse brainextracts (StresGen Biotechnologies, Victoria, Canada); positivecontrols for PAK1 consisted of Jurkat cell lysates (UpstateBiotechnology, Lake Placid, NY).

Immunofluorescence stainingMelanocytes grown in MGM were subcultured onto vitrogen-coated2-well glass chamber slides (Nalge Nunc International Corp.,Naperville, IL). Cell monolayers were fixed in cold methanol/acetone(1/1) followed by permeabilization in 0.5% triton-X-100 instabilization buffer (PBS, 100 mM MgCl2, 1 mM CaCl2) for 15minutes, and non-specific binding of antibody was blocked byincubation of the slides in 10% normal goat serum. Primary antibodieswere applied overnight at 4°C followed by incubation withappropriate Texas-Red- or fluorescein-conjugated secondaryantibodies for one hour at room temperature. For double labelingexperiments, the second primary antibody was applied for one hourat room temperature followed by the appropriate secondary antibody.DAPI (Vector Laboratories, Burlingame, CA) was used to stain nuclei.To stain actin, cells were fixed in formalin, permeabilized as describedabove and incubated with Alexa Fluor 595 for 2 hours at roomtemperature. Images were captured with a Spot digital camera andpost-processed using Adobe PhotoShop 5.0.

Electron microscopyMelanocyte-keratinocyte co-cultures were grown on vitrogen-coatedglass chamber slides, as described above, in KGM. Cells were fixedfor 30 minutes in 2.5% glutaraldehyde in Sorensen’s phosphate bufferpH 7.4 and were post-fixed in 1.0% osmium tetroxide in Sorensen’sphosphate buffer. Cell membranes were enhanced by incubation ofcells for 45 minutes in 0.5% uranyl acetate diluted in 25% ethanol.

After dehydrating in a graded series of ethanol, the cells wereinfiltrated for 30 minutes with a 1:1 solution of 100% ethanol and100% Spurr epoxy resin and were then infiltrated overnight in 100%Spurr epoxy resin. The next day the slides were inverted onto Spurrepoxy resin filled BEEM capsules and allowed to polymerize.Capsules were trimmed and thin-sectioned with 2.0% uranyl acetateand Reynolds lead citrate and viewed with a Hitachi 7100 electronmicroscope. To assess purity of melanosome fractions, melanosomeswere fixed in glutaraldehyde and post-fixed in osmium tetroxide asdescribed above. Melanosomes were captured in 4% warm agaroseand were embedded in Spurr epoxy resin and thin sectioned asdescribed above except that thin sections were not stained with uranylacetate or lead citrate. Electron microscopy of human skin wasaccomplished by fixation of a 2 mm punch biopsy of skin from a malesubject in glutaraldehyde in Sorensen’s buffer overnight. The tissuewas processed identically to the cells in culture except that cellmembranes were not enhanced by uranyl acetate.

Fig. 1.Melanocytes extend long filopodia from dendrite tips thattransport melanosomes to keratinocytes. (a) Two melanocytedendrites with prominent filopodia (fp; arrowhead) are shown inimages taken at 8 seconds. The arrowheads point to thin structuresconsistent with filopodia. (b) A melanosome (circle) is present in afilopodia (fp) and moves towards the keratinocyte membrane (KM;outlined in hatched line). Over the course of 16 seconds themelanosome has moved along a filopodia towards the keratinocytemembrane. (c) The tip of a melanocyte dendrite is shown withmultiple connections with a keratinocyte membrane (KM; outlined inhatched line). A melanosome (circle) moves towards the KM overthe course of 40 seconds. (d) Two sequential images captured 5minutes after treatment of cells with nocodazole are shown.Melanosomes have redistributed towards the melanocyte cell body,leaving a dendrite that appears empty. Filopodia (arrowhead) werenot affected by nocodazole treatment.

Infection of cells with adenovirusRecombinant adenovirus capable of expressing constitutively activeCdc42 (Cdc42V12) and green fluorescence protein (GFP) in the AdEasyvector (Quantum Biotechnologies, Montreal, Canada), and emptyvector expressing GFP alone, were a kind gift of Dr Bambera(Colorado State University, CO) and have been described previously(Brown et al., 2000). Infection efficiency was monitored by viewingthe cells in an inverted phase microscope (Nikon Diaphot) equippedwith a filter to detect GFP. To assess the effect of Cdc42V12 onmelanosome transfer to keratinocytes, pure populations of melanocytes(105 cells) grown in MGM were infected with either Adeasy Cdc42V12

or empty vector with a multiplicity of infection (MOI) of 30. 18 hourslater keratinocytes were added (105) and the co-culture was allowed togrow in KGM for at least 5 days prior to imaging.

Cdc42 GTPase activity assayCell lysates were incubated with GST-PAK-PBD fusion proteinaccording to the manufacturer’s instructions (Cytoskeleton Inc.,Boulder, CO), and GTP-bound Cdc42 was captured by incubation ofthe lysate with glutathione beads (BD pharMingen, San Diego, CA).Positive controls consisted of lysates pre-loaded with GTPγS (200µM). The beads and proteins bound to the fusion protein were washedin lysis buffer, eluted in Laemmli sample buffer, resolved on 15% gelsand blotted with antibodies against Cdc42.

ResultsMelanosome filopodia are utilized to attach tokeratinocyte membranes To better define the mechanism of melanosome transfer, we

utilized DIC optics and time-lapse digital microscopy todirectly visualize melanosome movement in humanmelanocyte-keratinocyte co-cultures and to better define themechanism of melanosome transfer. The most striking featureobserved from time-lapse digital microscopy was the presenceof long (up to 16 microns) dynamically active filopodia arisingfrom melanocyte dendrite tips and the melanocyte cell body,many of which contained melanosomes that were easilyvisualized under DIC optics. Shown in Fig. 1a are images oftwo melanocyte dendrite tips in which numerous filopodiawere observed. Movies of these co-cultures viewed at highmagnification allowed one to see the rapid motion of thefilopodia. When melanocytes were cultured in the absence ofkeratinocytes, filopodia moved in a random fashion and madelittle contact with neighboring cells, although some contactwith other melanocytes was seen. Filopodia attached tokeratinocyte membranes in many instances and remainedattached for the duration of image acquisition (approximately45 minutes; Fig. 1b,c). Melanosomes were observed to movetowards the keratinocyte membrane along filopodia, althoughin the majority of cases melanosomes remained in filopodiaand were not transferred to keratinocytes. Movement ofmelanosomes along filopodia from one melanocyte to anothermelanocyte was also observed (not shown). Real timeobservations confirmed that the structures were filopodia andnot retraction fibers, although some retraction fibers werealso present. Melanosomes in retraction fibers movedbidirectionally, which is consistent with the presence ofmicrotubules in these structures. To further verify that the

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Fig. 2.Melanosomes are transported to the keratinocytealong filopodia. (a) A scanning view of a melanocytedendrite (MD) contacting a keratinocyte (KC) is shown.The boxed area is shown in detail in sequential imagestaken every 8 seconds from movies made from this area(jcs.biologists.org/supplemental orwww.urmc.rochester.edu/derm/scottmovies.html). A filopodia arising from the lateral aspect of the dendriteis either attached or inserted into the keratinocytemembrane. A string of melanosomes (approximately sixof them) moves in single file toward the keratinocyte (2-5). The arrowhead indicates the leading melanosome.The last image (6) shows the same melanocyte in whichthe melanocyte dendrite (arrowhead) is markedlyattenuated. A long filopodia (arrow) is shown in whichthree melanosomes are present. (b) A scanning view of amelanocyte dendrite (MD) adjacent to a keratinocyte(KC) is shown. The hatched line delineates the KCmembrane in the upper right hand corner. The boxed areais shown in detail in images that span 152 seconds. Astring of melanosomes (approximately four of them;arrowhead) is present within a projection arising from theside of the body of the melanocyte dendrite. These

projections were frequently observed in melanocytes and were shorter and thickerthan filopodia. Other similar projections are present (asterisks). (c) Melanocytesand keratinocytes separately labeled with DiI (red fluorescence) and DiO (greenfluorescence), respectively, were co-cultured and irradiated. Representative imagestaken with filters to detect both green and red fluorescence in the range of the twodyes are shown. Sham-irradiated cells showed approximately 1% of keratinocyteswith yellow fluorescence (arrows; 1). 24 hours after irradiation approximately10% of keratinocytes show yellow fluorescence (arrow) when viewed with filtersto detect DiI and DiO, indicating membrane fusion (2). Bar, 50 µm.

1445Role of Cdc42 in melanosome transfer

structures observed were filopodia, co-cultures were imaged inthe presence of nocodazole (10 µg/ml), which would not beexpected to alter filopodia movement. Preliminary experimentsof nocodazole-treated melanocytes stained with anti-tubulinantibodies established that this dose results in total dissolutionof microtubules within 5 minutes; re-establishment ofmicrotubules after washout took up to 30 minutes (data notshown). Fig. 1d shows images of a melanocyte dendrite froma co-culture after 5 minutes of treatment with nocodazole.There was rapid re-distribution of melanosomes to the cellbody, with retention of some melanosomes in a cap-likedistribution at the actin-rich dendrite tip. These observationsare similar to those reported by Wu et al. (Wu et al., 1998) inmurine melanocytes treated with nocodazole and are consistentwith the role of microtubules in melanosome trafficking to thedendrite tip. The rapid movement of the filopodia was notaffected by nocodazole treatment. We attempted to assess theeffect of cytochalasin D on filopodia; however theseexperiments were uninformative because even very low dosesof cytochalasin D resulted in rapid collapse of the melanocyteactin network, with retraction of dendrites, as determined bothmorphologically and by staining of the cells for actin (data notshown).

Melanosome transfer to keratinocytes in culture is anuncommon event; however Fig. 2a shows a sequence of imagesin which melanosome transfer to keratinocytes has occurred.A melanocyte and keratinocyte viewed at 100× magnificationis shown; next to it are enlarged views of sequential images ofan area of melanocyte-keratinocyte contact (images 2-5). Afilopodia arising from the lateral aspect of the tip of thedendrite overlays or is attached to the keratinocyte membrane.Sequential images demonstrate melanosomes moving upwardtowards the keratinocyte in single file over the course of 80seconds. Melanosomes were also frequently observed infilopodia that arose from the body of the dendrite, even in theabsence of contact with a keratinocyte (Fig. 2a; image 6). Inaddition to filopodia, which were easily recognizable owing totheir dynamic motion and thin diameter, we also observedshorter thicker projections arising predominantly from thesides of melanocyte dendrites, which contacted keratinocytes(Fig. 2b). Melanosomes were transported along thesestructures singly or in pairs towards the keratinocytemembrane.

Because the optical properties of the filopodia and thekeratinocyte membrane are similar, we were unable todefinitively determine whether membrane fusion occurredusing this technique. In an initial attempt to address thisquestion we utilized two lipophilic fluorescent membrane dyesto separately label melanocyte and keratinocytes, followed byco-culture of the cells after a single dose of irradiation tostimulate melanosome transfer. Lipophilic dyes have beencommonly used to assess cell fusion in other cell types(Sowers, 1985; Spotl et al., 1995) and show little leakage ofone dye to another. DiI absorbs maximally at 546 nm and hasa maximum emission at 563 nm. DiO, a closely relatedcompound, absorbs maximally at 489 nm and its peak emissionis at 499 nm (Sims et al., 1974; Montecucco et al., 1979; Honigand Hune, 1986). Our preliminary experiments showed thatthese lipophilic dyes are rapidly incorporated into melanocyteand keratinocyte cell membranes where they persist for weeksin culture and show little if any cytotoxicity. 24 hours after

Fig. 3.Electron micrographs of melanocyte-keratinocyte co-culturesand human skin in vivo demonstrate melanosomes within filopodia.Electron micrographs of co-cultures of melanocyte and keratinocytesrevealed numerous long thin projections arising from melanocyte(MC) dendrites (a,b), most of which were cut in cross section(arrowhead; c,d). A longitudinal section of a filopodia is shown in(b). Occasionally we detected osmophilic structures consistent withmelanosomes within cross sections of filopodia (c,d). Melanosomeswere aligned along the base of filopodia (e) and were present alongthe length of areas of contact between melanocytes and keratinocyte(KC; f). In human skin in vivo thin structures arising frommelanocyte dendrites (MD), consistent with filopodia (fp), wereeasily identified (g,h) (arrows), many of which containedmelanosomes (*). Figures (i,j) show enlarged images from the boxedarea of parts (g,h), respectively. The arrows show the presence ofmelanosomes within filopodia. Magnification ×5000 (a); ×40,000 (b-d); ×17,000 (e); ×15,000 (f), ×4000 (g,h).

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irradiation of co-cultures that had been separately labeled withDiI and DiO, approximately 10% of keratinocytes exhibitedyellow fluorescence when viewed with a filter to detect bothdyes (Fig. 2c). Yellow fluorescence was observed in anintracellular vesicular pattern, which resembled endosomes, aswell as in larger deposits in the Golgi area. The presence ofyellow fluorescence within endosome-like structures isconsistent with membrane fusion between melanocytes andkeratinocytes, with subsequent recycling of the fusedmembranes into recycling endosomes and transport to theGolgi apparatus. We cannot exclude, however, the possibilitythat keratinocyte-phagocytosis of melanocyte dendritesresulted in the presence of yellow fluorescence inkeratinocytes. In sham-irradiated cells evidence of membranefusion was observed in approximately 1% of keratinocytes,indicating membrane fusion even in unstimulated cells.

Electron microscopy performed on human melanocyte-keratinocyte co-cultures demonstrated thin projectionsconsistent with filopodia arising from the tips and sides ofmelanocytes (Fig. 3a). In many cases filopodia were cut incross section; a longitudinal section of a filopodia is shown inFig. 3b. Melanosomes aligned themselves near the base of thefilopodia and osmophilic membrane-bound bodies consistentwith melanosomes were observed within the filopodia (Fig.

3c,d). In some cases we observed direct connection betweenmelanocytes and keratinocytes in the form of thin projectionsthat spanned a small space between the two cells (Fig. 3e,f).Melanosomes appeared to be passing between the melanocyteand the keratinocyte along these projections (Fig. 3f). In humanskin in vivo we detected structures consistent with filopodiaarising from the sides and tips of dendritis which containedmelanosomes within their lumina (Fig. 3g-j).

Expression of a Cdc42V12 in human melanocytes resultsin melanocyte dendricity and filopodia formation Because of the well known role of Cdc42 in mediatingfilopodia formation, we next examined the effect of expressionof constitutively active Cdc42 on melanocyte morphology,filopodia formation and melanosome transfer to keratinocytes.To investigate whether the virus-expressed Cdc42V12 wouldexhibit the expected properties of a constitutively active mutantconstruct in melanocytes, in vitro binding assays of lysates ofcells infected with Adeasy virus expressing Cdc42V12 andempty vector-infected cells with the PBD were performed (Fig.4a). The PBD is conserved in several effector proteins ofCdc42 and Rac1 and mediates their interactions in a GTP-dependent manner (Sander et al., 1998). Five days after

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Fig. 4. Expression of Cdc42V12 by human melanocytesinduces dendricity and multiple melanosome-containingfilopodia. (a) Cdc42V12 expressed using adenovirus isfunctionally active. Melanocytes (106 cells) were infectedwith adenovirus vector expressing Cdc42V12 (lane 3) orvirus alone (lane 2) for 5 days at 37°C. Infection efficiencywas approximately 50%. Whole cell lysates were incubatedwith GST-PBD, and bound proteins were analyzed bywestern blotting with anti-Cdc42 antibodies. Positivecontrols consisted of cell lysates pre-loaded with GTPγS(lane 1). Cdc42V12-expressing cells show increased levelsof PBD-bound Cdc42. The band migrating at ~35 kDarepresents the GST-fusion protein. Total cell lysates blottedfor Cdc42 show increase Cdc42 in Cdc42V12-expressingcells. (b) Cells expressing Cdc42V12 (detected by greenfluorescence) show a highly dendritic morphology reminiscent of neural cells (1-3). Cells infected with empty vector show a bipolarmorphology typical of melanocytes grown in keratinocyte growth medium, which lack phorbol esters (4). Bar, 20 µm (1-3); bar, 5 µm (4). (c)An Cdc42V12-infected cell is identified by expression of GFP tag (1). Examination of the same cell stained with antibodies to TRP-1 (2) showsnumerous melanosomes within the filopodia (arrowheads) compared with cells expressing empty vector (3,4). Bar, 20µm. (d) The imagesshown were captured under DIC optics and show tips of melanocyte dendrites with numerous filopodia (1,2). Co-culture of Cdc42V12-expressing melanocytes and non-infected keratinocytes (3) shows multiple filopodia (arrowheads) arising from a Cdc42V12-expressingmelanocyte in contact with a keratinocyte (KC). Images (4,5) show empty-vector-expressing melanocyte dendrite tips with a normalcomplement of filopodia (arrowhead).

1447Role of Cdc42 in melanosome transfer

infection of melanocytes (1×106 cells) with 30 MOI ofCdc42V12AdEasy vector, levels of PBD-bound Cdc42 wereincreased in Cdc42V12 expressing cells compared with emptyvector-infected cells. These results indicate that Cdc42V12

expressed from adenovirus is functionally active inmelanocytes. The amount of Cdc42 activation is likely to be

underestimated in this experiment because infection efficiencywas only 60% and therefore Cdc42 from non-infected cellsdiluted the amount of activated Cdc42. Because the GFP andCdc42V12 cDNAs are driven from separate CMV promoters,we confirmed that GFP-expressing cells also overexpressedCdc42 by staining infected cells with antibodies to Cdc42.Virtually all cells that expressed GFP also overexpressedCdc42 (not shown).

Melanocytes were infected with 30 MOI ofCdc42V12Adeasy virus or 30 MOI of Adeasy virus (emptyvector), and 1 day later keratinocytes were added at a ratio of1:1. The morphological features of melanocytes 5 days afterinfection with Cdc42V12Adeasy vector, viewed underimmunofluorescence microscopy, are shown in Fig. 4b (images1-3). Melanocytes exhibited multiple arborizing dendrites andsome cells displayed a growth-cone like morphology. Cellsinfected with empty vector maintained a bipolar morphologytypical of melanocytes grown in the absence of phorbol esters

Fig. 5.PAK1, Cdc42 and myosin Va localization in humanmelanocytes. (a) Immunofluorescence microscopy for Cdc42 (1) andPAK1 (2) was performed on cultured human melanocytes. Cdc42displayed a vesicular pattern with localization along the length of themelanocyte dendrite as well as in the Golgi area. PAK1, in contrast,was distributed diffusely in the cytosol, in the Golgi area as well as inthe dendrites. Bar, 30 µm. (b) The results of double labeling ofmelanocytes with antibodies to TRP-1 (mel-5) and PAK1 are shown.Images (1,4) show mel-5 localization; images (2,5) show PAK1localization, images (3,6) are cells viewed with filters to detect bothfluorescein-isothiocyanate and Texas Red. PAK1 and TRP-1colocalize predominantly in the peri-nuclear area (3); howevercolocalization is also observed along the melanocyte dendrite (6;arrowhead). Bar, 30 µm (1-3); bar, 10 µm (4-6). (c) Melanocytesstained with antibodies to myosin Va show the expected localizationof myosin Va at the tips of melanocyte dendrites, consistent with thepresence of melanosomes at this site. Myosin Va staining was alsodetected along the length and at the tips of melanocyte filopodia. Bar,2 µm.

Fig. 6.PAK1 and N-WASP are present on enriched melanosomefractions. (a,b) Electron microscopy on human melanosomes (HMS)showed that enriched melanosome factions contained primarily stageIII (arrows) and stage IV (arrowhead) melanosomes (a;Magnification, 20,000). Western blotting on HMS for transferrinreceptor (b) was performed to assess purity of the preparation. HMSextracts (20 µg) run on a 10% gel for transferrin receptor arenegative, suggesting that few contaminating endosomes are presentin the melanosome preparation. Positive controls consisted ofmelanocyte whole cell lysate. (c) Western blots of lysates run on15% gels (20 µg/lane; PAK1, 70 µg/lane; N-WASP) of humanmelanosome fraction (HMS) and whole cell lysates of humanmelanocytes (HMC) for PAK1 and N-WASP are shown. Positivecontrols consisted of Jurkat cell lysates (PAK1) and rat brain lysates(N-WASP). A strong immunoreactive band for PAK1 and N-WASPat the expected molecular weights are present.

1448

(image 4). To determine whether expression of Cdc42V12

resulted in increased numbers of melanosome-containingfilopodia, infected cells were stained with antibodies againstTRP-1 (Fig. 4c; images 1 and 2). Filopodia were clearly visiblein infected cells owing to the presence of numerousmelanosomes within them. Vector-expressing cells stained forTRP-1 showed some melanosomes in filopodia but they wereless numerous than in Cdc42V12-expressing cells. (Fig. 4c;images 3 and 4). Time-lapse digital microscopy of thesecultures viewed under DIC optics confirmed that Cdc42V12-infected cells exhibited multiple long filopodia arising fromdendrite tips (Fig. 4d, images 1-3) and in many cases filopodiacontained melanosomes. Cdc42V12 expressing melanocytesshowed more extensive contacts with keratinocytes throughfilopodia. As expected, empty vector expressing melanocytesviewed under DIC optics showed filopodia arising from the tipsof dendrites, but the number of filopodia was dramaticallyfewer than in Cdc42V12-expressing cells (images 4 and 5).

Melanosomes express the Cdc42 effector proteins PAK1and N-WASPWe next examined the expression and localization of Cdc42and PAK1 in human melanocytes by immunofluorescencemicroscopy (Fig. 5). We were unable to performimmunofluorescence staining with the antibodies to N-WASPavailable to us. Cdc42 was present in a vesicular pattern withprominent localization to the melanocyte cell membrane, aswell as in the peri-nuclear area (Fig. 5a; image 1). Thelocalization of Cdc42 to the peri-nuclear area (presumed to bethe Golgi apparatus) is consistent with previous reportsshowing that Cdc42 localizes to the Golgi (Erickson et al.,1996). PAK1 was heavily concentrated in the peri-nuclear areawith some vesicular staining in the dendrites (Fig. 5a; image2). Staining of cells with normal rabbit serum instead of aprimary antibody failed to show any labeling (not shown). Todetermine if Cdc42 or PAK1 colocalized with melanosomes,double labeling with antibodies to the melanosome-specificprotein mel-5 (TRP-1) and either Cdc42 or PAK1 wasperformed (Fig. 5b). PAK1 and TRP-1 colocalized in the peri-nuclear region as well as focally in the melanocyte dendrites(images 1-3). Because melanosomes are heavily concentratedin the peri-nuclear area, it is difficult to determine whether thisstaining pattern represents true colocalization or an artefact ofoverlay of melanosomes and other PAK1-expressing structuresin this area. Higher power images of melanocyte dendritesfrom double-labeled cells (lower panel, Fig. 5b) shows clearcolocalization of PAK1 and TRP-1 within the dendrites;however colocalization was not 100%. Cdc42 did notcolocalize with melanosomes but did colocalize with thetransferrin receptor, indicating a component of Cdc42 inrecycling endosomes (not shown). Myosin Va in melanocytescolocalizes with melanosomes, the endoplasmic reticulum,Golgi apparatus and mitochondria (Nascimento et al., 1997;Tabb et al., 1998), and myosin Va has been shown to play animportant role in filopodia extension in neuronal cells, and inmurine melanocytes myosin Va has been identified at the tipsof filopodia (Wang et al., 1996; Tsakraklides et al., 1999).Myosin Va was also present in a punctate or dot-like pattern atthe tips of filopodia and along the length of the filopodia (Fig.5c; image 1 and 2).

Western blots were performed for analysis of expression ofCdc42, PAK1 and N-WASP in melanosome fractions (Fig. 6).The purity of the melanosome isolate was assessed by electronmicroscopy, which demonstrated a relatively homogeneouspopulation of stage III and stage IV melanosomes with few ifany contaminating elements such as mitochondria (Fig. 6a).Transferrin receptor expression, used as a marker for recyclingendosomes, was not detected in melanosome-enrichedfractions, indicating low amounts of contaminating recyclingendosomes in the preparation (Fig. 6b). PAK1 was heavilyenriched in melanosome extracts; a single strongimmunoreactive band was detected (Fig. 6c; 20 µg/lane).Western blotting for N-WASP showed an immunoreactive bandat the expected molecular weight for N-WASP in melanosomeextracts (Fig. 6c; 70 µg/lane). Cdc42 was not detected inmelanosome-enriched fractions even when large amounts ofprotein (up to 80 µg) were loaded onto the gel (not shown).

DiscussionThrough the use of time-lapse digital imaging of melanocyte-keratinocyte co-cultures, we identified filopodia as a conduitfor melanosome transfer to keratinocytes. Our ability to creategreatly enlarged movies of areas of interest from digital imagesacquired with a 100× objective allowed us to directly visualizemelanosomes, which have an approximate diameter of 0.5-1.0µm, in real time. These movies, in combination with DICoptics, also allowed us to detect filopodia through their rapidmovement and enhanced appearance under phase contrast.Even though time-lapse video microscopy has been used toexamine melanosome transfer in the past, we believe that therelatively low resolution of these movies, compared with thehigh resolution of digital images, prevented detection offilopodia and the role they play in melanosome transfer.Electron microscopy demonstrated melanosomes withinstructures consistent with filopodia, confirming observationsmade from time lapse imaging. In human skin in vivo wedetected structures with morphologic features consistent withfilopodia, many of which contained melanosomes within them.Expression of constitutively active Cdc42 protein inmelanocytes resulted in a marked increase in melanosome-containing filopodia and in filopodia attachment tokeratinocytes. We also demonstrate the presence of N-WASPand PAK1, Cdc42 effector proteins, on enriched melanosomefractions. Myosin Va, which is an actin-based motor protein formelanosome transport and for filopodia extension, wasdetected at the tips of melanocyte filopodia.

The well known role of Cdc42 in filopodia formation, aswell as data showing increased filopodia formation inmelanocytes expressing a constiutively active Cdc42 mutant,are consistent with a role for Cdc42 in mediating filopodiaformation in human melanocytes. Although at this point we canonly speculate on the motors involved in moving melanosomesalong the length of the filopodia, the presence of myosin Va atthe tips of filopodia and the fact that filopodia contain actin butnot microtubules makes myosin Va a strong candidate. Therapid movement and attachment of melanocyte filopodia tokeratinocyte membranes is highly analogous to growth conefilopodia, which contact nearby axons with subsequent synapseformation and synaptic vesicle transmission. In neuronal cellsthe primary role of growth cone filopodia is to sample the

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1449Role of Cdc42 in melanosome transfer

immediate environment and translate environmental cues to thegrowth cone, which in turn affects growth cone behavior(Rosentreter et al., 1998; Harris, 1999). Recent reports showthat neuronal filopodia respond to exogenous growth factorssuch as fibroblast growth factor by doubling their length(Szebenyi et al., 2001). In a similar manner, melanocytesrespond to keratinocyte-derived growth factors throughdendrite extension and possibly melanosome transfer(Imokawa et al., 1995; Hara et al., 1995). It is likely thatmelanocyte filopodia respond to a gradient of keratinocyte-derived growth factors that direct filopodia growth andattachment and subsequent transfer of melanosomes, althoughfurther experiments are needed to establish this. Sabry et al.(Sabry et al., 1991) showed that microtubules invade neuronalfilopodia during growth cone steering events subsequent tofilopodia attachment to guidepost cells. Therefore, anotherpotential function of melanocyte filopodia may be to serve asa vanguard for dendrite extension. We also frequently observedblunt, short projections arising from the shafts of melanocytedendrites, which also functioned as conduits for melanosometransfer to keratinocytes. These projections may be analogousto dendritic spines, which, in neuronal cells, are actin-containing, short bulbous projections that arise from the sidesof neuronal dendrites (Harris, 1999; Kaech et al., 2001).

Activated Cdc42 induced a markedly dendritic morphologyin human melanocytes. Because activation of Rac1 results indendrite extension in melanocytes (Scott and Cassidy, 1998)and because others have shown that inhibition of RhoA resultsin dendrite extension in melanocytes (Busca et al., 1998),signaling for melanocyte dendrite extension may be analogousto N1E-115 cells in which activated Rac1 inhibits RhoA withsubsequent dendrite extension (Altun-Gultekin and Wagner,1996; Kozma et al., 1997). A hierarchical, unidirectionalcascade of activation of Cdc42, Rac and Rho has beendescribed in a variety of cell types (for a review, see Kjollerand Hall, 1999). In most cell types, Cdc42 activates Rac1,which leads to inhibition of Rho activity (Sander et al., 1999;Reid et al., 1999). These studies suggest that in melanocytesCdc42 may be upstream of Rac1 in dendrite formation throughactivation of Rac1, which in turn inhibits Rho A. Cdc42 isactivated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1 (Wojciak-Stothard et al., 1998; Puls et al.,1999), both of which are released by keratinocytes followingUV irradiation, a potent stimulus for melanosome transfer(Pathak et al., 1978; Sturm, 1998; Kondo, 1999). Therefore thewell known effect of UV irradiation on melanocyte dendriteformation and melanosome transfer may be mediated in partby interleukin-1- and tumor-necrosis-α-induced activation ofCdc42.

The role of PAK1 and N-WASP, which were enriched inmelanosome fractions, in melanosome movement is unclearand must await further experiments analyzing the effect ofmutants of these proteins on melanosome transport andtransfer. A strong association between PAK1 activation andlamellipodia formation, loss of stress fibers, disassembly offocal adhesions and increased cell motility has beendemonstrated (Frost et al., 1998; Sells et al., 1999). Daniels etal. (Daniels et al., 1998) have shown that nerve-growth-factor-induced neurite outgrowth in PC10 cells is mediated by PAK1.We have shown previously that Rac1 mediates melanocytedendrite formation in response to growth factors and to UV

irradiation (Scott and Cassidy, 1998). It is possible that inmelanocytes, which, similar to PC10 cells, are neuronallyderived cells, PAK1 is a downstream effector for Rac1-mediated dendrite extension. Although PAK1 is heavilyenriched on melanosomes, activated PAK1, as identified byantibodies to phosphorylated PAK1 (a generous gift of DrChernoff, Fox Chase Cancer Center, Philadelphia, PA) was notassociated with melanosomes but with the small granulefraction of melanocytes as demonstrated by western blotting(G.S., unpublished). Therefore activation of PAK1 is unlikelyto occur on the melanosome membrane.

It is likely that melanosome transfer is accomplished throughmultiple mechanisms, including phagocytosis of dendrite tipsand possibly exocytosis of the melanosome into theextracellular space with uptake by keratinocytes (Yamamotoand Bhawan, 1994). Although we did not directly observephagocytosis of melanocyte dendrites in time-lapse movies,this may have been due to the relative infrequency ofmelanosome transfer in culture, lack of appropriate stimulus orboth. Although the digital movies presented (jcs.biologists.org/supplemental or www.urmc.rochester.edu/derm/scottmovies.html) provide intriguing evidence of a role for filopodia inmelanosome transport, we are unable to definitively concludethat melanosome transfer occurred because of the opticalproperties of the melanocyte and keratinocyte membranes. Ourinitial attempt to circumvent this problem using membranedyes is suggestive of keratinocyte-melanocyte membranefusion but is not conclusive. We believe that definitiveevaluation of melanosome transfer will require in vivo labelingof melanosomes with a marker that would allow one to observemovement of melanosomes from the melanocyte to thekeratinocyte, in combination with high resolution digitalmovies. This would allow one to assess transferredmelanosomes within keratinocytes both through directvisualization, and through biochemical means, in response toexpression of mutants of a variety of candidate proteins,including PAK1, N-WASP and Cdc42. At the present time weare evaluating the ability of a GFP-Pmel17 fusion protein tolabel human melanocyte melanosomes.

This work was supported by 1R01AR45427(GS). We thank KarenJensen for her assistance with electron microscopy.

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