Research Article 1441 are conduits for melanosome transfer to

Glynis Scott, Sonya Leopardi, Stacey Printup and Brian C. Madden Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Author for correspondence (e-mail: [email protected])

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

Summary Melanosomes are specialized -synthesizing cultured with keratinocytes induced a highly dendritic critical for photoprotection in the skin. phenotype with extensive contacts between Melanosome transfer to keratinocytes, which involves and keratinocytes through filopodia, many of which whole donation to another cell, is a unique contained melanosomes. These results suggest a unique role biological process and is poorly understood. Time-lapse for filopodia in organelle transport and, in combination digital movies and electron microscopy show that filopodia with our previous work showing the presence of SNARE from dendrites serve as conduits for proteins and rab3a on melanosomes, suggest a novel model melanosome transfer to keratinocytes. Cdc42, a small system for melanosome transfer to keratinocytes. GTP-binding protein, is known to mediate filopodia formation. Melanosome-enriched fractions isolated from Movies available on-line human melanocytes expressed the Cdc42 effector proteins PAK1 and N-WASP by western blotting. Expression of Key words: Melanosome, Melanocyte, Cdc42, Filopodia, constitutively active Cdc42 (Cdc42V12) in melanocytes co-

Introduction microscopy of cultured cells, which allowed direct Melanosomes are organelles unique to melanocytes that visualization of melanosome movement and modifiers of function in the synthesis of melanin, a complex pigment actin, and their motor proteins. In contrast with involved in photoprotection of the skin through its ability to melanosome trafficking, much less is known about absorb and scatter light and reduce reactive oxygen species melanosome transfer. A major hurdle that has severely (Marks and Seabra, 2001). In order to accomplish this, limited progress in understanding the molecular basis of melanosomes must be transferred to epidermal keratinocytes, melanosome transfer has been the lack of a model system. where they are found in autophagic in a perinuclear The majority of studies of melanosome transfer to or cap-like distribution in vitro and in vivo (Corcuff et al., keratinocytes have been based on co-cultures of non-human 2001). Melanosomes are elliptical or spheroidal organelles that cells observed by electron microscopy. Studies performed contain melanogenic enzymes and co-factors, including the utilizing time-lapse video microscopy have been limited by tyrosinase gene family of proteins and have been categorized the relatively poor resolution achieved (Mottaz and as stage I-IV on the basis of their electron microscopic Zelickson, 1967; Cohen and Szabo, 1968; Wolff, 1973). appearance and degree of melanization (Kushimoto et al., Other more recent studies have utilized gold particle uptake 2001). Recent studies suggest that premelanosomes (stage I by keratinocytes, melanin uptake or transfer of cytoplasmic and II) and late stage melanosomes (stage III and IV) represent dyes from melanocytes to keratinocytes to measure transfer a distinct lineage of organelles that are separable from (Seiberg et al., 2000a; Seiberg et al., 2000b; Sharlow et al., conventional and within pigmented cells 2000; Minwalla et al., 2001). In toto, these prior studies led (Raposo et al., 2001). to important observations that suggested phagocytosis of It is now known that melanosome trafficking is mediated melanocyte dendrites by keratinocytes as the major mode of in part by microtubular motor myosin Va, the product of the melanosome transfer, although exocytosis of melanosomes dilute locus, which traps melanosomes at the actin-rich into the with uptake by keratinocytes and periphery of the dendrite, and rab27a, the product of the insertion of melanocyte dendrites and transfer of ashen locus (Mercer et al., 1991; Provance et al., 1996; Wu melanosomes to keratinocytes have also been proposed et al., 1997; Wu et al., 1998; Wei et al., 1997; Wilson et al., (Yamamoto and Bhawan, 1994). Although the more recent 2000; Bahadoran et al., 2001; Wu et al., 2001). Rab27a is studies using particle uptake provide insight into the role of involved in the transport of melanosomes through its ability the keratinocyte in uptake, the use of a model system to recruit myosin Va to the tip of the melanocyte dendrite in which melanosome transfer is being studied directly (Hume et al., 2001). These important and relatively recent provides an opportunity to examine the potential role of the insights into melanosome trafficking were made possible melanocyte in melanosome transfer. through the use of mutant mouse strains and time-lapse video It is well established that Cdc42, a member of the Rho 1442 Journal of Cell Science 115 (7) family of GTP-binding proteins, is involved in filopodia and Materials and Methods microspike formation in many cell types. Filopodia are actin- Antibodies and reagents based structures that arise from neuronal growth cones and Polyclonal antibodies to PAK1 were purchased from Zymed function in neuronal pathfinding (Davenport et al., 1993; Laboratories (San Francisco, CA); polyclonal antibodies to Cdc42 Rosentreter et al., 1998). The recent demonstration that Cdc42 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, is associated with coatamer proteins in the , CA); polyclonal antibodies to N-WASP were a generous gift of Dr that it regulates exit of apical and basolateral proteins from the Rohatgi (Harvard Medical School, Boston, MA) and have been Golgi network and is involved in exocytosis of secretory described previously (Rohatgi et al., 1999); polyclonal antibodies to granules in mast cells is indicative of the diverse roles that chick brain myosin 5a tail domain (clone 32a) were a generous gift Cdc42 plays in cells (Brown et al., 1998; Hong-Geller and of Richard Cheney (Chapel Hill, NC) and have been described previously (Espreafico et al., 1992); monoclonal antibodies to TRP-1 Cerione, 2000; Wu et al., 2000; Müsch et al., 2001). The (mel-5) were purchased from Signet Laboratories (Dedham, MA); downstream effectors of Cdc42 fall into six families most of monoclonal antibodies to transferrin receptor were purchased from which contain a CRIB-binding domain and include Cdc42- Zymed Laboratories (San Francisco, CA); polyclonal antibodies to binding kinase, myotonic dystrophy kinase-related Cdc42- tubulin were purchased from Sigma Co (St. Louis, MO); fluorescein binding kinase, mixed lineage kinase, p21-activated kinase isothiocyanate and Texas Red goat anti-rabbit and anti-mouse (PAK), WASP (Wiscot-Aldrich Syndrome Protein), IQGAP antibodies were purchased from Molecular Probes (Eugene, OR); and MSE55/BORG/CEP (Burbelo et al., 1995). At least four horseradish-peroxidase-conjugated goat anti-rabbit and anti-mouse closely related isoforms of PAK (PAK1, PAK2, PAK3 and antibodies and normal rabbit serum were purchased from Sigma Co; PAK4) exist in mammalian cells (Manser et al., 1994; Manser vitrogen was purchased from Cohesion (Palo Alto, CA). Membrane et al., 1995; Martin et al., 1995; Dan et al., 2001). PAK-family dyes DiI and DiO and Alexa Fluor 594 phalloidin were purchased from Molecular Probes. Nocodazole and cytochalasin D were kinases are activated by GTP-Cdc42 or GTP-Rac1 as well as purchased from Sigma Co. G-protein-coupled receptors and cytokines and phosphotidyl- inositol 3-kinase (PI3-kinase); this leads to a change in conformation of the kinase inducing autophosphorylation on Cell culture multiple serine and threonine residues and activation (Knaus Neonatal foreskins were obtained according to the University of et al., 1995; Manser et al., 1997; Wang et al., 1999; Chung et Rochester’s Research Subject Review Board. Co-cultures of human al., 2001). Activation of PAK results in effects that mimic melanocytes and keratinocytes were initiated from human foreskins Rac1 and Cdc42 and include lamellipodia and filopodia as previously described (Scott and Haake, 1991) and maintained in formation, activation of the c-Jun N-terminal kinase MAP Keratinocyte Growth Media (KGM, Gibco BRL, Gaithersburg, PA). kinase cascade and NKκB, alteration in cell motility and In primary skin cultures this media sustains melanocyte growth inhibition of apoptosis and stimulation of macropinocytosis through the production of melanocyte growth factors by proliferating (Sells 1997; Sells et al., 1999; Frost et al., 1998; Frost et al., keratinocytes (Halaban et al., 1988). For growth of melanocytes, cells were cultured in Melanocyte Growth Media (MGM, Gibco-BRL). 2000; Dharmawardhane, 2000). Non-kinases that interact with Cdc42 include the WASP family, which consist of WASp, N- WASP and related Scar proteins isolated in Dictyostelium. Time-lapse digital microscopy and image processing WASP, in concert with WIP (WASP-interacting protein) Co-cultures of melanocytes and keratinocytes (approximately 105 participates with the Arp2/3 complex to induce actin cells total) were subcultured on vitrogen-coated 25 mm glass nucleation and filopodia formation (Symons et al., 1996; Miki coverslips for 1-2 days and placed in a closed heated chamber (Warner et al., 1996; Miki et al., 1998; Rohatgi et al., 1999; Banzai et Instruments, New Haven, CT) maintained at 37°C. The cells were al., 2000; Martinez-Quiles et al., 2001). WASP is only viewed on a Nikon Eclipse Microscope 800 under differential expressed in hematopoietic cells and is mutated in patients interference contrast (DIC) optics with a 100× objective. The chamber with Wiscot-Aldrich syndrome, whereas N-WASP is was perfused with KGM maintained at a constant temperature of 37°C by an in-line heater (Warner Instruments) using gravity flow. The rate ubiquitously expressed but is enriched in the brain (Fukuoka µ et al., 1997). In a cell-free system, addition of active Cdc42 of flow was approximately 166 l/min and imaging lasted 45 minutes. Cell viability was checked following experiments with trypan blue significantly stimulates neuronal-WASP (N-WASP) by and no cytotoxicity was observed. exposure of N-WASPs’ actin depolymerizing region, creating Sequential images were obtained at 8 second intervals using the free barbed ends from which actin polymerization can take green filter of a Spot digital camera (Diagnostic Instruments, Sterling place (Suzuki et al., 1998). Heights, MI). The resulting 8 bit/pixel megapixel (1315x1033) images In this report we used have high resolution movies made yielded a resolution of 10 pixels/micron when combined with the from digital images to directly observe melanosome transfer to 100× microscope objective. A series of operations to reduce noise and keratinocytes in human cells. These movies, along with artefacts were performed on the images using the Matlab GUI facility electron microscopy of cells in vitro and skin in vivo provide (Mathworks, Natick, MA). To further reduce size, the movies were evidence that suggests that melanosome delivery to created using QuickTime Pro (Apple Computer Inc, Cupertino, CA). keratinocytes occurs along filopodia. We show that expression of activated Cdc42 in human melanocytes accentuates Labeling of melanocyte and keratinocyte membranes and filopodia formation and melanosome transport and that evaluation of membrane fusion melanosomes are enriched in PAK1 and N-WASP, Cdc42- Melanocytes grown in MGM and keratinocytes grown in KGM were effector proteins. In combination with previous data showing labeled with DiI (0.6 µM) and DiO (0.2 µM) respectively for 10 SNARE and rab proteins on melanosomes (Scott and Zhao, minutes at 37°C followed by extensive washing. 24 hours later, 2001), these observations suggest a novel model for melanocytes were trypsinized from the dish and added to melanosome transfer to keratinocytes. keratinocytes on vitrogen-coated 100 mm glass coverslips at an Role of Cdc42 in melanosome transfer 1443 approximate ratio of 1:1 in KGM. To stimulate melanosome transfer, co-cultures were irradiated with a single dose of (UV) irradiation using a solar simulator at a dose of 4 J/cm2 as previously described (Scott and Zhao, 2001). 24 hours after irradiation, live cells were viewed on a Nikon Eclipse Microscope 800 and images were captured with a Spot digital camera. To arrive at an approximate percentage of cells with membrane fusion, the number of keratinocytes with yellow fluorescence viewed under a filter to detect both 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 blotting Melanosomes were isolated from human melanocytes essentially as described for isolation of melanosomes from cultured B16F1 cells, with a few modifications (Scott and Zhao, 2001). The postnuclear supernatant was centrifuged for 10 minutes at 10,000 g at 4°C to obtain a large granule and a small granule fraction. The large granule fraction, which is enriched in melanosomes, was then layered onto a sucrose gradient and centrifuged at 85,000 g at 4°C for 1 hour. The melanosome-rich fraction was collected from the 2M layer of the gradient and lysed in buffer (150 mM NaCl, 10 mM Tris-HCL, pH 7.8, 1% Triton-X) plus protease inhibitors (Complete TM Mini, Boehringer Mannheim, GmbH, Germany). Protein samples were quantified using the Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, 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 to nitrocellulose membranes (Bio-Rad Laboratories) using standard procedures. Full range rainbow molecular weight markers were Fig. 1. Melanocytes extend long filopodia from dendrite tips that purchased from Amersham Life Sciences (Arlington Heights, Ill). transport melanosomes to keratinocytes. (a) Two melanocyte Visualization of the immunoreactive proteins was accomplished using dendrites with prominent filopodia (fp; arrowhead) are shown in an enhanced chemiluminescence reaction (Amersham Life Sciences). images taken at 8 seconds. The arrowheads point to thin structures Positive controls for Cdc42 and N-WASP consisted of mouse brain consistent with filopodia. (b) A melanosome (circle) is present in a extracts (StresGen Biotechnologies, Victoria, Canada); positive filopodia (fp) and moves towards the keratinocyte membrane (KM; controls for PAK1 consisted of Jurkat cell lysates (Upstate outlined in hatched line). Over the course of 16 seconds the Biotechnology, Lake Placid, NY). melanosome has moved along a filopodia towards the keratinocyte membrane. (c) The tip of a melanocyte dendrite is shown with Immunofluorescence staining multiple connections with a keratinocyte membrane (KM; outlined in hatched line). A melanosome (circle) moves towards the KM over Melanocytes grown in MGM were subcultured onto vitrogen-coated the course of 40 seconds. (d) Two sequential images captured 5 2-well glass chamber slides (Nalge Nunc International Corp., minutes after treatment of cells with nocodazole are shown. Naperville, IL). Cell monolayers were fixed in cold methanol/acetone Melanosomes have redistributed towards the melanocyte cell body, (1/1) followed by permeabilization in 0.5% triton-X-100 in leaving a dendrite that appears empty. Filopodia (arrowhead) were stabilization buffer (PBS, 100 mM MgCl2, 1 mM CaCl2) for 15 not affected by nocodazole treatment. minutes, and non-specific binding of antibody was blocked by incubation of the slides in 10% normal goat serum. Primary antibodies were applied overnight at 4°C followed by incubation with After dehydrating in a graded series of ethanol, the cells were appropriate Texas-Red- or fluorescein-conjugated secondary antibodies for one hour at room temperature. For double labeling infiltrated for 30 minutes with a 1:1 solution of 100% ethanol and experiments, the second primary antibody was applied for one hour 100% Spurr epoxy resin and were then infiltrated overnight in 100% at room temperature followed by the appropriate secondary antibody. Spurr epoxy resin. The next day the slides were inverted onto Spurr DAPI (Vector Laboratories, Burlingame, CA) was used to stain nuclei. epoxy resin filled BEEM capsules and allowed to polymerize. To stain actin, cells were fixed in formalin, permeabilized as described Capsules were trimmed and thin-sectioned with 2.0% uranyl acetate above and incubated with Alexa Fluor 595 for 2 hours at room and Reynolds lead citrate and viewed with a Hitachi 7100 electron temperature. Images were captured with a Spot digital camera and microscope. To assess purity of melanosome fractions, melanosomes post-processed using Adobe PhotoShop 5.0. were fixed in glutaraldehyde and post-fixed in osmium tetroxide as described above. Melanosomes were captured in 4% warm agarose Electron microscopy and were embedded in Spurr epoxy resin and thin sectioned as described above except that thin sections were not stained with uranyl Melanocyte-keratinocyte co-cultures were grown on vitrogen-coated glass chamber slides, as described above, in KGM. Cells were fixed acetate or lead citrate. Electron microscopy of human skin was for 30 minutes in 2.5% glutaraldehyde in Sorensen’s phosphate buffer accomplished by fixation of a 2 mm punch biopsy of skin from a male pH 7.4 and were post-fixed in 1.0% osmium tetroxide in Sorensen’s subject in glutaraldehyde in Sorensen’s buffer overnight. The tissue phosphate buffer. Cell membranes were enhanced by incubation of was processed identically to the cells in culture except that cell cells for 45 minutes in 0.5% uranyl acetate diluted in 25% ethanol. membranes were not enhanced by uranyl acetate. 1444 Journal of Cell Science 115 (7)

Fig. 2. Melanosomes are transported to the keratinocyte along filopodia. (a) A scanning view of a melanocyte dendrite (MD) contacting a keratinocyte (KC) is shown. The boxed area is shown in detail in sequential images taken every 8 seconds from movies made from this area (jcs.biologists.org/supplemental or www.urmc.rochester.edu/derm/scottmovies.html). A filopodia arising from the lateral aspect of the dendrite is either attached or inserted into the keratinocyte membrane. A string of melanosomes (approximately six of 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 which the melanocyte dendrite (arrowhead) is markedly attenuated. A long filopodia (arrow) is shown in which three melanosomes are present. (b) A scanning view of a melanocyte dendrite (MD) adjacent to a keratinocyte (KC) is shown. The hatched line delineates the KC membrane in the upper right hand corner. The boxed area is shown in detail in images that span 152 seconds. A string of melanosomes (approximately four of them; arrowhead) is present within a projection arising from the side of the body of the melanocyte dendrite. These projections were frequently observed in melanocytes and were shorter and thicker than filopodia. Other similar projections are present (asterisks). (c) Melanocytes and keratinocytes separately labeled with DiI (red fluorescence) and DiO (green fluorescence), respectively, were co-cultured and irradiated. Representative images taken with filters to detect both green and red fluorescence in the range of the two dyes are shown. Sham-irradiated cells showed approximately 1% of keratinocytes with yellow fluorescence (arrows; 1). 24 hours after irradiation approximately 10% of keratinocytes show yellow fluorescence (arrow) when viewed with filters to detect DiI and DiO, indicating membrane fusion (2). Bar, 50 µm.

Infection of cells with adenovirus utilized DIC optics and time-lapse digital microscopy to Recombinant adenovirus capable of expressing constitutively active directly visualize melanosome movement in human Cdc42 (Cdc42V12) and green fluorescence protein (GFP) in the AdEasy melanocyte-keratinocyte co-cultures and to better define the vector (Quantum Biotechnologies, Montreal, Canada), and empty mechanism of melanosome transfer. The most striking feature vector expressing GFP alone, were a kind gift of Dr Bambera observed from time-lapse digital microscopy was the presence (Colorado State University, CO) and have been described previously of long (up to 16 microns) dynamically active filopodia arising (Brown et al., 2000). Infection efficiency was monitored by viewing from melanocyte dendrite tips and the melanocyte cell body, the cells in an inverted phase microscope (Nikon Diaphot) equipped with a filter to detect GFP. To assess the effect of Cdc42V12 on many of which contained melanosomes that were easily melanosome transfer to keratinocytes, pure populations of melanocytes visualized under DIC optics. Shown in Fig. 1a are images of (105 cells) grown in MGM were infected with either Adeasy Cdc42V12 two melanocyte dendrite tips in which numerous filopodia or empty vector with a multiplicity of infection (MOI) of 30. 18 hours were observed. Movies of these co-cultures viewed at high later keratinocytes were added (105) and the co-culture was allowed to magnification allowed one to see the rapid motion of the grow in KGM for at least 5 days prior to imaging. filopodia. When melanocytes were cultured in the absence of keratinocytes, filopodia moved in a random fashion and made little contact with neighboring cells, although some contact Cdc42 GTPase activity assay with other melanocytes was seen. Filopodia attached to Cell lysates were incubated with GST-PAK-PBD fusion protein keratinocyte membranes in many instances and remained according to the manufacturer’s instructions ( Inc., Boulder, CO), and GTP-bound Cdc42 was captured by incubation of attached for the duration of image acquisition (approximately the lysate with glutathione beads (BD pharMingen, San Diego, CA). 45 minutes; Fig. 1b,c). Melanosomes were observed to move Positive controls consisted of lysates pre-loaded with GTPγS (200 towards the keratinocyte membrane along filopodia, although µM). The beads and proteins bound to the fusion protein were washed in the majority of cases melanosomes remained in filopodia in lysis buffer, eluted in Laemmli sample buffer, resolved on 15% gels and were not transferred to keratinocytes. Movement of and blotted with antibodies against Cdc42. melanosomes along filopodia from one melanocyte to another melanocyte was also observed (not shown). Real time observations confirmed that the structures were filopodia and Results not retraction fibers, although some retraction fibers were Melanosome filopodia are utilized to attach to also present. Melanosomes in retraction fibers moved keratinocyte membranes bidirectionally, which is consistent with the presence of To better define the mechanism of melanosome transfer, we microtubules in these structures. To further verify that the Role of Cdc42 in melanosome transfer 1445 structures observed were filopodia, co-cultures were imaged in the presence of nocodazole (10 µg/ml), which would not be expected to alter filopodia movement. Preliminary experiments of nocodazole-treated melanocytes stained with anti-tubulin antibodies established that this dose results in total dissolution of microtubules within 5 minutes; re-establishment of microtubules after washout took up to 30 minutes (data not shown). Fig. 1d shows images of a melanocyte dendrite from a co-culture after 5 minutes of treatment with nocodazole. There was rapid re-distribution of melanosomes to the cell body, with retention of some melanosomes in a cap-like distribution at the actin-rich dendrite tip. These observations are similar to those reported by Wu et al. (Wu et al., 1998) in murine melanocytes treated with nocodazole and are consistent with the role of microtubules in melanosome trafficking to the dendrite tip. The rapid movement of the filopodia was not affected by nocodazole treatment. We attempted to assess the effect of cytochalasin D on filopodia; however these experiments were uninformative because even very low doses of cytochalasin D resulted in rapid collapse of the melanocyte actin network, with retraction of dendrites, as determined both morphologically and by staining of the cells for actin (data not shown). Melanosome transfer to keratinocytes in culture is an uncommon event; however Fig. 2a shows a sequence of images in which melanosome transfer to keratinocytes has occurred. A melanocyte and keratinocyte viewed at 100× magnification is shown; next to it are enlarged views of sequential images of an area of melanocyte-keratinocyte contact (images 2-5). A filopodia arising from the lateral aspect of the tip of the dendrite overlays or is attached to the keratinocyte membrane. Sequential images demonstrate melanosomes moving upward towards the keratinocyte in single file over the course of 80 seconds. Melanosomes were also frequently observed in filopodia that arose from the body of the dendrite, even in the absence of contact with a keratinocyte (Fig. 2a; image 6). In addition to filopodia, which were easily recognizable owing to their dynamic motion and thin diameter, we also observed shorter thicker projections arising predominantly from the sides of melanocyte dendrites, which contacted keratinocytes (Fig. 2b). Melanosomes were transported along these structures singly or in pairs towards the keratinocyte membrane. Because the optical properties of the filopodia and the keratinocyte membrane are similar, we were unable to definitively determine whether membrane fusion occurred Fig. 3. Electron micrographs of melanocyte-keratinocyte co-cultures using this technique. In an initial attempt to address this and human skin in vivo demonstrate melanosomes within filopodia. Electron micrographs of co-cultures of melanocyte and keratinocytes question we utilized two lipophilic fluorescent membrane dyes revealed numerous long thin projections arising from melanocyte to separately label melanocyte and keratinocytes, followed by (MC) dendrites (a,b), most of which were cut in cross section co-culture of the cells after a single dose of irradiation to (arrowhead; c,d). A longitudinal section of a filopodia is shown in stimulate melanosome transfer. Lipophilic dyes have been (b). Occasionally we detected osmophilic structures consistent with commonly used to assess cell fusion in other cell types melanosomes within cross sections of filopodia (c,d). Melanosomes (Sowers, 1985; Spotl et al., 1995) and show little leakage of were aligned along the base of filopodia (e) and were present along one dye to another. DiI absorbs maximally at 546 nm and has the length of areas of contact between melanocytes and keratinocyte a maximum emission at 563 nm. DiO, a closely related (KC; f). In human skin in vivo thin structures arising from compound, absorbs maximally at 489 nm and its peak emission melanocyte dendrites (MD), consistent with filopodia (fp), were easily identified (g,h) (arrows), many of which contained is at 499 nm (Sims et al., 1974; Montecucco et al., 1979; Honig melanosomes (*). Figures (i,j) show enlarged images from the boxed and Hune, 1986). Our preliminary experiments showed that area of parts (g,h), respectively. The arrows show the presence of these lipophilic dyes are rapidly incorporated into melanocyte melanosomes within filopodia. Magnification ×5000 (a); ×40,000 (b- and keratinocyte cell membranes where they persist for weeks d); ×17,000 (e); ×15,000 (f), ×4000 (g,h). in culture and show little if any cytotoxicity. 24 hours after 1446 Journal of Cell Science 115 (7)

Fig. 4. Expression of Cdc42V12 by human melanocytes induces dendricity and multiple melanosome-containing filopodia. (a) Cdc42V12 expressed using adenovirus is functionally active. Melanocytes (106 cells) were infected with adenovirus vector expressing Cdc42V12 (lane 3) or virus alone (lane 2) for 5 days at 37°C. Infection efficiency was approximately 50%. Whole cell lysates were incubated with GST-PBD, and bound proteins were analyzed by western blotting with anti-Cdc42 antibodies. Positive controls consisted of cell lysates pre-loaded with GTPγS (lane 1). Cdc42V12-expressing cells show increased levels of PBD-bound Cdc42. The band migrating at ~35 kDa represents the GST-fusion protein. Total cell lysates blotted for Cdc42 show increase Cdc42 in Cdc42V12-expressing cells. (b) Cells expressing Cdc42V12 (detected by green fluorescence) show a highly dendritic morphology reminiscent of neural cells (1-3). Cells infected with empty vector show a bipolar morphology 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) shows numerous melanosomes within the filopodia (arrowheads) compared with cells expressing empty vector (3,4). Bar, 20 µm. (d) The images shown 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-expressing melanocyte in contact with a keratinocyte (KC). Images (4,5) show empty-vector-expressing melanocyte dendrite tips with a normal complement of filopodia (arrowhead). irradiation of co-cultures that had been separately labeled with 3c,d). In some cases we observed direct connection between DiI and DiO, approximately 10% of keratinocytes exhibited melanocytes and keratinocytes in the form of thin projections yellow fluorescence when viewed with a filter to detect both that spanned a small space between the two cells (Fig. 3e,f). dyes (Fig. 2c). Yellow fluorescence was observed in an Melanosomes appeared to be passing between the melanocyte intracellular vesicular pattern, which resembled endosomes, as and the keratinocyte along these projections (Fig. 3f). In human well as in larger deposits in the Golgi area. The presence of skin in vivo we detected structures consistent with filopodia yellow fluorescence within -like structures is arising from the sides and tips of dendritis which contained consistent with membrane fusion between melanocytes and melanosomes within their lumina (Fig. 3g-j). keratinocytes, with subsequent recycling of the fused membranes into recycling endosomes and transport to the Golgi apparatus. We cannot exclude, however, the possibility Expression of a Cdc42V12 in human melanocytes results that keratinocyte-phagocytosis of melanocyte dendrites in melanocyte dendricity and filopodia formation resulted in the presence of yellow fluorescence in Because of the well known role of Cdc42 in mediating keratinocytes. In sham-irradiated cells evidence of membrane filopodia formation, we next examined the effect of expression fusion was observed in approximately 1% of keratinocytes, of constitutively active Cdc42 on melanocyte morphology, indicating membrane fusion even in unstimulated cells. filopodia formation and melanosome transfer to keratinocytes. Electron microscopy performed on human melanocyte- To investigate whether the virus-expressed Cdc42V12 would keratinocyte co-cultures demonstrated thin projections exhibit the expected properties of a constitutively active mutant consistent with filopodia arising from the tips and sides of construct in melanocytes, in vitro binding assays of lysates of melanocytes (Fig. 3a). In many cases filopodia were cut in cells infected with Adeasy virus expressing Cdc42V12 and cross section; a longitudinal section of a filopodia is shown in empty vector-infected cells with the PBD were performed (Fig. Fig. 3b. Melanosomes aligned themselves near the base of the 4a). The PBD is conserved in several effector proteins of filopodia and osmophilic membrane-bound bodies consistent Cdc42 and Rac1 and mediates their interactions in a GTP- with melanosomes were observed within the filopodia (Fig. dependent manner (Sander et al., 1998). Five days after Role of Cdc42 in melanosome transfer 1447

Fig. 6. PAK1 and N-WASP are present on enriched melanosome fractions. (a,b) Electron microscopy on human melanosomes (HMS) showed that enriched melanosome factions contained primarily stage III (arrows) and stage IV (arrowhead) melanosomes (a; Magnification, 20,000). Western blotting on HMS for transferrin receptor (b) was performed to assess purity of the preparation. HMS extracts (20 µg) run on a 10% gel for transferrin receptor are negative, suggesting that few contaminating endosomes are present in the melanosome preparation. Positive controls consisted of Fig. 5. PAK1, Cdc42 and myosin Va localization in human melanocyte whole cell lysate. (c) Western blots of lysates run on melanocytes. (a) Immunofluorescence microscopy for Cdc42 (1) and 15% gels (20 µg/lane; PAK1, 70 µg/lane; N-WASP) of human PAK1 (2) was performed on cultured human melanocytes. Cdc42 melanosome fraction (HMS) and whole cell lysates of human displayed a vesicular pattern with localization along the length of the melanocytes (HMC) for PAK1 and N-WASP are shown. Positive melanocyte dendrite as well as in the Golgi area. PAK1, in contrast, controls consisted of Jurkat cell lysates (PAK1) and rat brain lysates was distributed diffusely in the , in the Golgi area as well as in (N-WASP). A strong immunoreactive band for PAK1 and N-WASP the dendrites. Bar, 30 µm. (b) The results of double labeling of at the expected molecular weights are present. melanocytes with antibodies to TRP-1 (mel-5) and PAK1 are shown. Images (1,4) show mel-5 localization; images (2,5) show PAK1 localization, images (3,6) are cells viewed with filters to detect both underestimated in this experiment because infection efficiency fluorescein-isothiocyanate and Texas Red. PAK1 and TRP-1 was only 60% and therefore Cdc42 from non-infected cells colocalize predominantly in the peri-nuclear area (3); however diluted the amount of activated Cdc42. Because the GFP and colocalization is also observed along the melanocyte dendrite (6; Cdc42V12 cDNAs are driven from separate CMV promoters, arrowhead). Bar, 30 µm (1-3); bar, 10 µm (4-6). (c) Melanocytes we confirmed that GFP-expressing cells also overexpressed stained with antibodies to myosin Va show the expected localization Cdc42 by staining infected cells with antibodies to Cdc42. of myosin Va at the tips of melanocyte dendrites, consistent with the Virtually all cells that expressed GFP also overexpressed presence of melanosomes at this site. Myosin Va staining was also Cdc42 (not shown). detected along the length and at the tips of melanocyte filopodia. Bar, µ Melanocytes were infected with 30 MOI of 2 m. Cdc42V12Adeasy virus or 30 MOI of Adeasy virus (empty vector), and 1 day later keratinocytes were added at a ratio of 1:1. The morphological features of melanocytes 5 days after infection of melanocytes (1×106 cells) with 30 MOI of infection with Cdc42V12Adeasy vector, viewed under Cdc42V12AdEasy vector, levels of PBD-bound Cdc42 were immunofluorescence microscopy, are shown in Fig. 4b (images increased in Cdc42V12 expressing cells compared with empty 1-3). Melanocytes exhibited multiple arborizing dendrites and vector-infected cells. These results indicate that Cdc42V12 some cells displayed a growth-cone like morphology. Cells expressed from adenovirus is functionally active in infected with empty vector maintained a bipolar morphology melanocytes. The amount of Cdc42 activation is likely to be typical of melanocytes grown in the absence of phorbol esters 1448 Journal of Cell Science 115 (7) (image 4). To determine whether expression of Cdc42V12 Western blots were performed for analysis of expression of resulted in increased numbers of melanosome-containing Cdc42, PAK1 and N-WASP in melanosome fractions (Fig. 6). filopodia, infected cells were stained with antibodies against The purity of the melanosome isolate was assessed by electron TRP-1 (Fig. 4c; images 1 and 2). Filopodia were clearly visible microscopy, which demonstrated a relatively homogeneous in infected cells owing to the presence of numerous population of stage III and stage IV melanosomes with few if melanosomes within them. Vector-expressing cells stained for any contaminating elements such as mitochondria (Fig. 6a). TRP-1 showed some melanosomes in filopodia but they were Transferrin receptor expression, used as a marker for recycling less numerous than in Cdc42V12-expressing cells. (Fig. 4c; endosomes, was not detected in melanosome-enriched images 3 and 4). Time-lapse digital microscopy of these fractions, indicating low amounts of contaminating recycling cultures viewed under DIC optics confirmed that Cdc42V12- endosomes in the preparation (Fig. 6b). PAK1 was heavily infected cells exhibited multiple long filopodia arising from enriched in melanosome extracts; a single strong dendrite tips (Fig. 4d, images 1-3) and in many cases filopodia immunoreactive band was detected (Fig. 6c; 20 µg/lane). contained melanosomes. Cdc42V12 expressing melanocytes Western blotting for N-WASP showed an immunoreactive band showed more extensive contacts with keratinocytes through at the expected molecular weight for N-WASP in melanosome filopodia. As expected, empty vector expressing melanocytes extracts (Fig. 6c; 70 µg/lane). Cdc42 was not detected in viewed under DIC optics showed filopodia arising from the tips melanosome-enriched fractions even when large amounts of of dendrites, but the number of filopodia was dramatically protein (up to 80 µg) were loaded onto the gel (not shown). fewer than in Cdc42V12-expressing cells (images 4 and 5). Discussion Melanosomes express the Cdc42 effector proteins PAK1 Through the use of time-lapse digital imaging of melanocyte- and N-WASP keratinocyte co-cultures, we identified filopodia as a conduit We next examined the expression and localization of Cdc42 for melanosome transfer to keratinocytes. Our ability to create and PAK1 in human melanocytes by immunofluorescence greatly enlarged movies of areas of interest from digital images microscopy (Fig. 5). We were unable to perform acquired with a 100× objective allowed us to directly visualize immunofluorescence staining with the antibodies to N-WASP melanosomes, which have an approximate diameter of 0.5-1.0 available to us. Cdc42 was present in a vesicular pattern with µm, in real time. These movies, in combination with DIC prominent localization to the melanocyte , as optics, also allowed us to detect filopodia through their rapid well as in the peri-nuclear area (Fig. 5a; image 1). The movement and enhanced appearance under phase contrast. localization of Cdc42 to the peri-nuclear area (presumed to be Even though time-lapse video microscopy has been used to the Golgi apparatus) is consistent with previous reports examine melanosome transfer in the past, we believe that the showing that Cdc42 localizes to the Golgi (Erickson et al., relatively low resolution of these movies, compared with the 1996). PAK1 was heavily concentrated in the peri-nuclear area high resolution of digital images, prevented detection of with some vesicular staining in the dendrites (Fig. 5a; image filopodia and the role they play in melanosome transfer. 2). Staining of cells with normal rabbit serum instead of a Electron microscopy demonstrated melanosomes within primary antibody failed to show any labeling (not shown). To structures consistent with filopodia, confirming observations determine if Cdc42 or PAK1 colocalized with melanosomes, made from time lapse imaging. In human skin in vivo we double labeling with antibodies to the melanosome-specific detected structures with morphologic features consistent with protein mel-5 (TRP-1) and either Cdc42 or PAK1 was filopodia, many of which contained melanosomes within them. performed (Fig. 5b). PAK1 and TRP-1 colocalized in the peri- Expression of constitutively active Cdc42 protein in nuclear region as well as focally in the melanocyte dendrites melanocytes resulted in a marked increase in melanosome- (images 1-3). Because melanosomes are heavily concentrated containing filopodia and in filopodia attachment to in the peri-nuclear area, it is difficult to determine whether this keratinocytes. We also demonstrate the presence of N-WASP staining pattern represents true colocalization or an artefact of and PAK1, Cdc42 effector proteins, on enriched melanosome overlay of melanosomes and other PAK1-expressing structures fractions. Myosin Va, which is an actin-based motor protein for in this area. Higher power images of melanocyte dendrites melanosome transport and for filopodia extension, was from double-labeled cells (lower panel, Fig. 5b) shows clear detected at the tips of melanocyte filopodia. colocalization of PAK1 and TRP-1 within the dendrites; The well known role of Cdc42 in filopodia formation, as however colocalization was not 100%. Cdc42 did not well as data showing increased filopodia formation in colocalize with melanosomes but did colocalize with the melanocytes expressing a constiutively active Cdc42 mutant, transferrin receptor, indicating a component of Cdc42 in are consistent with a role for Cdc42 in mediating filopodia recycling endosomes (not shown). Myosin Va in melanocytes formation in human melanocytes. Although at this point we can colocalizes with melanosomes, the , only speculate on the motors involved in moving melanosomes Golgi apparatus and mitochondria (Nascimento et al., 1997; along the length of the filopodia, the presence of myosin Va at Tabb et al., 1998), and myosin Va has been shown to play an the tips of filopodia and the fact that filopodia contain actin but important role in filopodia extension in neuronal cells, and in not microtubules makes myosin Va a strong candidate. The murine melanocytes myosin Va has been identified at the tips rapid movement and attachment of melanocyte filopodia to of filopodia (Wang et al., 1996; Tsakraklides et al., 1999). keratinocyte membranes is highly analogous to growth cone Myosin Va was also present in a punctate or dot-like pattern at filopodia, which contact nearby axons with subsequent synapse the tips of filopodia and along the length of the filopodia (Fig. formation and synaptic vesicle transmission. In neuronal cells 5c; image 1 and 2). the primary role of growth cone filopodia is to sample the Role of Cdc42 in melanosome transfer 1449 immediate environment and translate environmental cues to the irradiation (Scott and Cassidy, 1998). It is possible that in growth cone, which in turn affects growth cone behavior melanocytes, which, similar to PC10 cells, are neuronally (Rosentreter et al., 1998; Harris, 1999). Recent reports show derived cells, PAK1 is a downstream effector for Rac1- that neuronal filopodia respond to exogenous growth factors mediated dendrite extension. Although PAK1 is heavily such as fibroblast growth factor by doubling their length enriched on melanosomes, activated PAK1, as identified by (Szebenyi et al., 2001). In a similar manner, melanocytes antibodies to phosphorylated PAK1 (a generous gift of Dr respond to keratinocyte-derived growth factors through Chernoff, Fox Chase Cancer Center, Philadelphia, PA) was not dendrite extension and possibly melanosome transfer associated with melanosomes but with the small granule (Imokawa et al., 1995; Hara et al., 1995). It is likely that fraction of melanocytes as demonstrated by western blotting melanocyte filopodia respond to a gradient of keratinocyte- (G.S., unpublished). Therefore activation of PAK1 is unlikely derived growth factors that direct filopodia growth and to occur on the melanosome membrane. attachment and subsequent transfer of melanosomes, although It is likely that melanosome transfer is accomplished through further experiments are needed to establish this. Sabry et al. multiple mechanisms, including phagocytosis of dendrite tips (Sabry et al., 1991) showed that microtubules invade neuronal and possibly exocytosis of the melanosome into the filopodia during growth cone steering events subsequent to extracellular space with uptake by keratinocytes (Yamamoto filopodia attachment to guidepost cells. Therefore, another and Bhawan, 1994). Although we did not directly observe potential function of melanocyte filopodia may be to serve as phagocytosis of melanocyte dendrites in time-lapse movies, a vanguard for dendrite extension. We also frequently observed this may have been due to the relative infrequency of blunt, short projections arising from the shafts of melanocyte melanosome transfer in culture, lack of appropriate stimulus or dendrites, which also functioned as conduits for melanosome both. Although the digital movies presented (jcs.biologists.org/ transfer to keratinocytes. These projections may be analogous supplemental or www.urmc.rochester.edu/derm/scottmovies. to dendritic spines, which, in neuronal cells, are actin- html) provide intriguing evidence of a role for filopodia in containing, short bulbous projections that arise from the sides melanosome transport, we are unable to definitively conclude of neuronal dendrites (Harris, 1999; Kaech et al., 2001). that melanosome transfer occurred because of the optical Activated Cdc42 induced a markedly dendritic morphology properties of the melanocyte and keratinocyte membranes. Our in human melanocytes. Because activation of Rac1 results in initial attempt to circumvent this problem using membrane dendrite extension in melanocytes (Scott and Cassidy, 1998) dyes is suggestive of keratinocyte-melanocyte membrane and because others have shown that inhibition of RhoA results fusion but is not conclusive. We believe that definitive in dendrite extension in melanocytes (Busca et al., 1998), evaluation of melanosome transfer will require in vivo labeling signaling for melanocyte dendrite extension may be analogous of melanosomes with a marker that would allow one to observe to N1E-115 cells in which activated Rac1 inhibits RhoA with movement of melanosomes from the melanocyte to the subsequent dendrite extension (Altun-Gultekin and Wagner, keratinocyte, in combination with high resolution digital 1996; Kozma et al., 1997). A hierarchical, unidirectional movies. This would allow one to assess transferred cascade of activation of Cdc42, Rac and Rho has been melanosomes within keratinocytes both through direct described in a variety of cell types (for a review, see Kjoller visualization, and through biochemical means, in response to and Hall, 1999). In most cell types, Cdc42 activates Rac1, expression of mutants of a variety of candidate proteins, which leads to inhibition of Rho activity (Sander et al., 1999; including PAK1, N-WASP and Cdc42. At the present time we Reid et al., 1999). These studies suggest that in melanocytes are evaluating the ability of a GFP-Pmel17 fusion protein to Cdc42 may be upstream of Rac1 in dendrite formation through label human melanocyte melanosomes. activation of Rac1, which in turn inhibits Rho A. Cdc42 is activated by the inflammatory cytokines tumor necrosis factor- This work was supported by 1R01AR45427(GS). We thank Karen α and interleukin-1 (Wojciak-Stothard et al., 1998; Puls et al., Jensen for her assistance with electron microscopy. 1999), both of which are released by keratinocytes following UV irradiation, a potent stimulus for melanosome transfer References (Pathak et al., 1978; Sturm, 1998; Kondo, 1999). Therefore the Altun-Gultekin, Z. F. and Wagner, J. A. (1996). 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