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Role of Cdc42 in Melanosome Transfer 1443 Approximate Ratio of 1:1 in KGM

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Role of Cdc42 in Melanosome Transfer 1443 Approximate Ratio of 1:1 in KGM Research Article 1441 Filopodia are conduits for melanosome transfer to keratinocytes 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 Cell Science 115, 1441-1451 (2002) © The Company of Biologists Ltd Summary Melanosomes are specialized melanin-synthesizing cultured with keratinocytes induced a highly dendritic organelles critical for photoprotection in the skin. phenotype with extensive contacts between melanocytes Melanosome transfer to keratinocytes, which involves and keratinocytes through filopodia, many of which whole organelle 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 melanocyte 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- Keratinocyte 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, microtubules 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 vacuoles 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 endosomes and lysosomes 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 extracellular space 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 granule 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 Golgi apparatus, 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
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