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• W1981860108 abstract "Melanocytes are pigment producing cells that reside in the basal layer of the epidermis, and form multiple long dendritic processes that transport melanosomes from the melanocyte cell body to the dendritic tips, and then to keratinocytes. Dendrite formation requires actin polymerization in the newly forming dendrite, and dendrite formation in melanocytes is stimulated by hormones and ultraviolet light. The rho-subfamily of monomeric guanosine triphosphate-binding proteins is implicated in remodeling the cellular actin cytoskeleton, resulting in the formation of filopodia, lamellipodia, and stress fibers, as well as in oncogenesis and activation of the Jun/p38 mitogen activated kinase cascade. In this paper we show that rac1 induces the formation of dendrite-like structures when activated mutants are transiently expressed in B16F1 murine melanoma cells and in four human melanoma cell lines. Activated mutants of cdc42 and rhoA induced the formation of filopodia and stress fibers, respectively, in B16F1 cells, but not dendrites. A dominant negative inhibitor of rac1 abrogated the ability of α-melanocyte stimulating hormone, a peptide hormone known to stimulate melanocyte dendrite formation, and ultraviolet light, to induce dendrite formation in B16F1 cells, and α-melanocyte stimulating hormone and ultraviolet light stimulated the localization of rac1 to dendrite cell membranes. These results suggest that rac1 is an important signaling intermediate in dendrite formation in B16F1 cells, and that rac1 mediates the well-known ability of α-melanocyte stimulating hormone and ultraviolet light to induce dendrite formation. Melanocytes are pigment producing cells that reside in the basal layer of the epidermis, and form multiple long dendritic processes that transport melanosomes from the melanocyte cell body to the dendritic tips, and then to keratinocytes. Dendrite formation requires actin polymerization in the newly forming dendrite, and dendrite formation in melanocytes is stimulated by hormones and ultraviolet light. The rho-subfamily of monomeric guanosine triphosphate-binding proteins is implicated in remodeling the cellular actin cytoskeleton, resulting in the formation of filopodia, lamellipodia, and stress fibers, as well as in oncogenesis and activation of the Jun/p38 mitogen activated kinase cascade. In this paper we show that rac1 induces the formation of dendrite-like structures when activated mutants are transiently expressed in B16F1 murine melanoma cells and in four human melanoma cell lines. Activated mutants of cdc42 and rhoA induced the formation of filopodia and stress fibers, respectively, in B16F1 cells, but not dendrites. A dominant negative inhibitor of rac1 abrogated the ability of α-melanocyte stimulating hormone, a peptide hormone known to stimulate melanocyte dendrite formation, and ultraviolet light, to induce dendrite formation in B16F1 cells, and α-melanocyte stimulating hormone and ultraviolet light stimulated the localization of rac1 to dendrite cell membranes. These results suggest that rac1 is an important signaling intermediate in dendrite formation in B16F1 cells, and that rac1 mediates the well-known ability of α-melanocyte stimulating hormone and ultraviolet light to induce dendrite formation. GDP dissociation inhibitors melanocyte stimulating hormone (Nle4-D-Phe7)-α-MSH Melanocytes are neural crest derived cells that form dendrites, specialized cell processes that transport melanosomes to the tips of dendrites for transfer to keratinocytes, in response to hormones and ultraviolet (UV) light. Dendrites are critically important for efficient melanosome transfer, because one melanocyte makes contact with numerous keratinocytes in the epidermis through dendrite cell processes (Fitzpatrick et al., 1967Fitzpatrick T.B. Miyamoto M. Ishikawa K. The evolution of concepts of melanin biology.Arch Dermatol. 1967; 96: 305-323Crossref PubMed Scopus (79) Google Scholar). Although it is known that hormones, including α-melanocyte stimulating hormone (α-MSH), endothelin-1, and nerve growth factor, and UV light induce melanocyte dendrite formation, the molecular mediators are unknown (Hirobe, 1978Hirobe T. Stimulation of dedritogenesis in the epidermal melanocytes of newborn mice by melanocyte-stimulating hormone.J Cell Sci. 1978; 33: 371-383PubMed Google Scholar;Friedmann and Gilchrest, 1987Friedmann P.S. Gilchrest B.A. Ultraviolet radiation directly induces pigment production by cultured human melanocytes.J Cell Physiol. 1987; 133: 88-94Crossref PubMed Scopus (264) Google Scholar;Herlyn et al., 1988Herlyn M. Mancianti M.L. Jambrosic J. Bolen J.B. Koprowski H. Regulatory factors that determine growth and phenotype of normal human melanocytes.Exp Cell Res. 1988; 179: 322-331Crossref PubMed Scopus (77) Google Scholar;Gordon et al., 1989Gordon P.R. Mansur C.P. Gilchrest B.A. Regulation of human melanocyte growth, dendricity, and melanization by keratinocyte derived factors.J Invest Dermatol. 1989; 92: 565-572Abstract Full Text PDF PubMed Google Scholar;Yaar et al., 1991Yaar M. Grossman K. Eller M. Gilchrest B.A. Evidence for nerve growth factor-mediated paracrine effects in human epidermis.J Cell Biol. 1991; 115: 821-828Crossref PubMed Scopus (164) Google Scholar;Hirobe, 1992Hirobe T. Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum-free culture.J Cell Physiol. 1992; 152: 337-345Crossref PubMed Scopus (55) Google Scholar;Ivengar, 1994Ivengar B. UV guided dendritic growth patterns and the networking of melanocytes.Experientia. 1994; 50: 669-672Crossref PubMed Scopus (8) Google Scholar;Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748Crossref PubMed Scopus (116) Google Scholar). Dendrites of vertebrate melanoma cells and melanocytes are composed of both actin filaments and microtubules, which are arranged parallel to the long axis of the dendrite, with microtubules in the center portion of the dendrite, and actin at the periphery (McGuire et al., 1972McGuire J. Moellmann G. McKeon F. Cytochalasin B and pigment translocation.J Cell Biol. 1972; 52: 765-768Crossref Scopus (34) Google Scholar;Moellmann et al., 1973Moellmann G. McGuire J. Lerner A.B. Intracellular dynamics and the fine structure of melanocytes.Yale J Biol Med. 1973; 46: 337-360PubMed Google Scholar;Lacour et al., 1992Lacour J.P. Gordon P.R. Eller M. Bhawan J. Gilchrest B.A. Cytoskeletal events underlying dendrite formation by cultured pigment cells.J Cell Physiol. 1992; 151: 287-299Crossref PubMed Scopus (37) Google Scholar). Confocal microscopy has shown that filamentous actin is particularly enriched immediately under the plasma membrane of dendritic tips and throughout the dendrite, compared with the cell body (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. . Hammer IIIJA. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.J Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar) and the initial process of dendrite formation in human melanocytes in response to UV radiation and to keratinocyte conditioned media has been shown to involve increased formation of microfilaments and actin polymerization (Jimbow et al., 1973Jimbow K. Pathak M.A. Fitzpatrick T.B. Effect of ultraviolet light on the distribution pattern of microfilaments and microtubules and on the nucleus in human melanocytes.Yale J Biol Med. 1973; 46: 411-426PubMed Google Scholar;Lacour et al., 1992Lacour J.P. Gordon P.R. Eller M. Bhawan J. Gilchrest B.A. Cytoskeletal events underlying dendrite formation by cultured pigment cells.J Cell Physiol. 1992; 151: 287-299Crossref PubMed Scopus (37) Google Scholar;Archambault et al., 1995Archambault M. Yaar M. Gilchrest B.A. Keratinocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation.J Invest Dermatol. 1995; 104: 859-867Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Similarly, dendrite extension in B16F1 cells in response to keratinocyte conditioned medium and in melanocytes in response to α-MSH has been shown to require actin assembly (Hirobe, 1978Hirobe T. Stimulation of dedritogenesis in the epidermal melanocytes of newborn mice by melanocyte-stimulating hormone.J Cell Sci. 1978; 33: 371-383PubMed Google Scholar; Lacour et al., 1992Lacour J.P. Gordon P.R. Eller M. Bhawan J. Gilchrest B.A. Cytoskeletal events underlying dendrite formation by cultured pigment cells.J Cell Physiol. 1992; 151: 287-299Crossref PubMed Scopus (37) Google Scholar). Understanding the molecular basis of hormonally induced changes in cell shape has recently been advanced by the discovery that the rho-subfamily of guanosine triphosphate (GTP)-binding proteins orchestrate a variety of cellular shape changes, including lamellipodia, filopodia, and stress fiber formation through their ability to regulate actin assembly in response to growth factors (for review, seeBourne et al., 1991Bourne H.A. Sanders D.A. McCormick F. The GTPase superfamily: conserved structure and molecular mechanism.Nature. 1991; 349: 117-127Crossref PubMed Scopus (2604) Google Scholar;Nobes and Hall, 1995aNobes C.D. Hall A. Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility.Biochem Soc Trans. 1995 a; 23: 456-459Crossref PubMed Scopus (289) Google Scholar). Rac1, rhoA, and cdc42 are members of the rho subfamily of ras-related proteins, all bind and hydrolyze GTP and their activity is controlled by associated regulatory proteins. The active conformation is promoted by guanine nucleotide exchange factors, which promote the exchange of guanosine diphosphate (GDP) for GTP, and the inactive conformation is promoted by GTPase-activating proteins and GDP dissociation inhibitors (GDI). In addition to activating GTP hydrolysis, some GTPase-activating proteins, such as n-chaemerin, may also act as effector molecules in cytoskeletal reorganization (Kozma et al., 1996Kozma R. Sohail A. Best A. Lim L. The GTPase-activating protien n-chimaerin cooperates with rac1 and cdc42hs to induce the formation of lamellipodia and filopodia.Mol Cell Biol. 1996; 16: 5069-5080Crossref PubMed Scopus (131) Google Scholar). RhoA stimulates stress fiber formation in response to lysophosphatidic acid, platelet derived growth factor, fetal calf serum, bombesin, and epidermal growth factor, and rhoA dependent stress fiber formation appears to depend upon tyrosine phosphorylation (Ridley and Hall, 1992Ridley A.J. Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3710) Google Scholar,Ridley and Hall, 1994Ridley A.J. Hall A. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tryosine kinase.Embo J. 1994; 13: 2600-2610Crossref PubMed Scopus (433) Google Scholar;Nobes and Hall, 1995bNobes C.D. Hall A. Rho, Rac, Cdc42 GTPases regulate the assembly of multimoleculer focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995 b; 81: 53-62Abstract Full Text PDF PubMed Scopus (3602) Google Scholar;Flinn and Ridley, 1996Flinn H.M. Ridley A.J. Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin.J Cell Sci. 1996; 109: 1133-1141PubMed Google Scholar). Rac1 and cdc42 have been shown to induce membrane ruffling and lamellipodia formation (rac1) and filopodia formation (cdc42), respectively, when active mutants are microinjected into cells (Nobes and Hall, 1995bNobes C.D. Hall A. Rho, Rac, Cdc42 GTPases regulate the assembly of multimoleculer focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995 b; 81: 53-62Abstract Full Text PDF PubMed Scopus (3602) Google Scholar). Constitutively active rac1 mutants injected into Swiss 3T3 cells induce the formation of focal complexes containing vinculin, focal adhesion kinase, and paxillin at the leading edge of newly formed lamellipodia (Nobes and Hall, 1995bNobes C.D. Hall A. Rho, Rac, Cdc42 GTPases regulate the assembly of multimoleculer focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995 b; 81: 53-62Abstract Full Text PDF PubMed Scopus (3602) Google Scholar). Rac1 has been shown to mediate the effects of a wide variety of hormones on membrane ruffling, as demonstrated by experiments in which dominant negative inhibitors of rac1, when microinjected into Swiss 3T3 cells, inhibited the ability of platelet derived growth factor, phorbol 12-Myristate 13-acetate, epidermal growth factor, bombesin, and insulin to induce membrane ruffling (Ridley et al., 1992Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (2979) Google Scholar). Cdc42 was originally isolated as a yeast mutant involved in defective cell budding and yeast polarity (Adams et al., 1990Adams A.E.M. Johnson D.I. Longnecker R.M. Sloat B.R. Pringle J.R. Cdc42 and cdc43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.J Cell Biol. 1990; 111: 131-142Crossref PubMed Scopus (462) Google Scholar;Johnson and Pringle, 1990Johnson D.I. Pringle J.R. Molecular characterization of cdc42, a Saccharomyces cerevisiae gene involved in the development of cell polarity.J Cell Biol. 1990; 111: 143-152Crossref PubMed Scopus (396) Google Scholar); its human homolog induces the formation of filopodia when active mutants are microinjected into cells (Nobes and Hall, 1995bNobes C.D. Hall A. Rho, Rac, Cdc42 GTPases regulate the assembly of multimoleculer focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995 b; 81: 53-62Abstract Full Text PDF PubMed Scopus (3602) Google Scholar) and cdc42 mediates bradykinin-induced formation of peripheral actin microspikes in Swiss 3T3 cells (Kozma et al., 1995Kozma R. Ahmed S. Best A. Lim A. The Ras-related protein cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.Mol Cell Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (869) Google Scholar). α-MSH is a member of the melanotropic hormones, a family of structurally related peptides derived from one precursor protein, proopiomelanocortin (POMC). There are five different melanocortin receptors (MC1–MC5), a subfamily of G protein-coupled receptors with seven transmembrane domains (Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1415) Google Scholar;Cone et al., 1993Cone R.D. Mountjoy K.G. Robbins L.S. Nadeau J.H. Johnson K.R. Roselli-rehfuss L. Mortrud M.T. Cloning and functional characterization of a family of receptors for the melanotropic peptides.Ann NY Acad Sci. 1993; 680: 342-363Crossref PubMed Scopus (118) Google Scholar). The melanocortin receptors differ in their relative affinities for the various melanotropic peptides and melanocytes and melanoma cells express the MC1 receptor, which has a high affinity for α-MSH and its potent synthetic analog (Nle4-D-Phe7)-α-MSH (NDP-MSH). Binding of these peptide hormones to their cognate MC receptors results in activation of protein kinase A through activation of adenylate cyclase and increased production of cAMP, with subsequent mitogenesis and melanogenesis (Mac Neil et al., 1981Mac Neil S. Johnson S.K. Bleehen S.S. Brown B.L. Tomlinson S. Stimulation of the adenylate cyclase of a B16 melanoma cell line by proopiocortin-related peptides-a structure-activity study.Regulatory Peptides. 1981; 2: 193-200Crossref PubMed Scopus (9) Google Scholar;Hill et al., 1989Hill S.E. Buffey J. Thody A.J. Oliver I. Bleehen S.S. Mac Neil S. Investigation of the regulation in α-melanocyte stimulating hormone responsive and unresponsive cultured B16 melanoma cells.Pig Cell Res. 1989; 2: 161-166Crossref PubMed Scopus (43) Google Scholar;Hunt et al., 1994Hunt G. Donatien P.D. Lunec J. Todd C. Kyne S. Thody A. Cultured human melanocytes respond to MSH peptides and ACTH.Pig Cell Res. 1994; 7: 217-221Crossref PubMed Scopus (74) Google Scholar;Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1415) Google Scholar[BC1];Abdel-malek et al., 1995Abdel-malek Z. Swope V.B. Suzuki I. et al.Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides.Proc Natl Sci USA. 1995; 92: 1789-1793Crossref PubMed Scopus (331) Google Scholar;Swope et al., 1995Swope V.B. Medrano E.E. Smalara D. Abdel-malek Z. Long-term proliferation of human melanocytes is supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor.Exp Cell Res. 1995; 217: 453-459Crossref PubMed Scopus (116) Google Scholar;Suzuki et al., 1996Suzuki I. Cone R.D. Im S. Nordlund J. Abdel-malek Z.A. Binding of melanotropic hormones to the melanocortin recpetor MC1R on human melanocytes stimulates proliferation and melanogenesis.Endocrin. 1996; 137: 1627-1633Crossref PubMed Scopus (236) Google Scholar; for review seeHunt, 1995Hunt G. Melanocyte stimulating hormone: A regulator of human melanocyte physiology.Pathobiol. 1995; 63: 12-21Crossref PubMed Scopus (29) Google Scholar). Melanocyte stimulating hormone, as well as agents that increase cAMP levels, such as isobutylmethylxanthine and dibutyryl cAMP, have been reported to induce dendricity in melanocytes and melanoma cells (Hirobe, 1992Hirobe T. Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum-free culture.J Cell Physiol. 1992; 152: 337-345Crossref PubMed Scopus (55) Google Scholar;Hunt et al., 1994Hunt G. Donatien P.D. Lunec J. Todd C. Kyne S. Thody A. Cultured human melanocytes respond to MSH peptides and ACTH.Pig Cell Res. 1994; 7: 217-221Crossref PubMed Scopus (74) Google Scholar;Abdel-malek et al., 1995Abdel-malek Z. Swope V.B. Suzuki I. et al.Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides.Proc Natl Sci USA. 1995; 92: 1789-1793Crossref PubMed Scopus (331) Google Scholar). Another powerful stimulant of melanocyte dendricity is UV light. Within 24 h of irradiation of cocultures of normal human melanocytes and keratinocytes, melanocytes form long branching dendritic processes, primarily through the upregulation of endothelin-1 production by keratinocytes (Imokawa et al., 1995Imokawa G. Miyagishi M. Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.J Invest Derm. 1995; 105: 32-37Crossref PubMed Scopus (224) Google Scholar), which has been shown to be a powerful inducer of melanocyte dendrite formation (Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748Crossref PubMed Scopus (116) Google Scholar). Melanocyte dendricity can also be induced by UV light in the absence of keratinocytes (Friedmann and Gilchrest, 1987Friedmann P.S. Gilchrest B.A. Ultraviolet radiation directly induces pigment production by cultured human melanocytes.J Cell Physiol. 1987; 133: 88-94Crossref PubMed Scopus (264) Google Scholar), although the mechanisms are unknown. We have used B16F1 murine melanoma cells as a model system to study the role of the rho subfamily of GTP-binding proteins in dendrite formation in response to the α-MSH analog NDP-MSH, and UV light. We show that activated rac1 proteins induce dendrite formation in B16F1 cells, and that a dominant negative inhibitor of rac1 abrogates the ability of NDP-MSH and UV light to induced dendrite formation in B16F1 cells. Further, we show that activated rac1 induces the formation of dendrites in four human melanoma cell lines. These results suggest that rac1 is a primary signaling intermediate for dendrite formation in melanocytic cells. The B16F1 mouse melanoma cell line was obtained from American Type Culture Collection (Rockville, MD). The cells were checked periodically for contamination with mycoplasma by staining with bisBenzimide Hoechst trichloride (Sigma, St. Louis, MO). B16F1 cells were maintained in Dulbecco’s minimal essential media (Gibco BRL, Gaithersberg, MD) supplemented with 2% fetal calf serum (Gibco BRL). The A375P and A375M human melanoma cell lines were a gift of Dr. Ian Hart (Imperial Cancer Research Fund, London, U.K.) and were maintained in minimal essential medium (Gibco BRL) supplemented with 10% fetal calf serum and have been described previously (Marshall et al., 1991Marshall J.F. Nesbitt S.A. Helfrich M.H. Horton M.A. Polakova K. Hart R.I. Integrin expression in human melanoma cell lines: Heterogeneity of vitronectin receptor composition and function.Int J Cancer. 1991; 49: 924-931Crossref PubMed Scopus (74) Google Scholar). The WM-793 and WM-98–1 human primary melanoma cell lines were a generous gift of Dr. Meenhard Herlyn (The Wistar Institute of Anatomy and Biology, Philadelphia, PA), and have been described previously (Albelda et al., 1990Albelda S.M. Mette S.A. Elder D.E. Stewart R. Damjanovich L. Herlyn M. Buck C.A. Integrin distribution in malignant melanoma: Association of the β3 subunit with tumor progression.Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar;Easty et al., 1995Easty D.J. Herlyn M. Bennett D.C. Abnormal protein tyrosine kinase gene expression during melanoma progression and metastasis.Int J Cancer. 1995; 60: 129-136Crossref PubMed Scopus (101) Google Scholar). They were maintained in MCDB-153/L-15 media (4/1; Gibco BRL) supplemented with 10% fetal calf serum. Rabbit polyclonal antibodies to murine tyrosinase-related protein (clone αPEP1) were a generous gift of Dr. Vincent Hearing (National Institutes of Health, Bethesda, MD) and have been previously described (Jimenez et al., 1988Jimenez M. Kameyama K. Maloy W.L. Tomita Y. Hearing V.J. Mammalian tyrosinase: biosynthesis, processing and modulation by melanocyte stimulating hormone.Proc Natl Acad Sci USA. 1988; 85: 3830-3834Crossref PubMed Scopus (121) Google Scholar); polyclonal antibodies to myosin V (clone DIL2) raised against a portion of the myosin V heavy chain (residues 910–1106) were a generous gift of Dr. John Hammer (National Institutes of Health) and have been described previously (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. . Hammer IIIJA. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.J Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar). Monoclonal antibodies to the c-myc epitope (9E10) were purchased from Sigma; polyclonal antibodies to rac1 and control peptides for preabsorption of the antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated goat anti-mouse and anti-rabbit antibodies, phycoerythrin conjugated goat anti-mouse antibodies, and normal goat serum were purchased from Sigma; NDP-MSH and dibutyryl cAMP were purchased from Sigma. NDP-MSH was used because it is more potent and more stable than α-MSH (Sawyer et al., 1982Sawyer T.K. Hruby V.J. Darman P.S. Hadley M.E. (half-Cys4,half-Cys 10) alpha melanocyte stimulating hormone: a cyclic alpha melanotropic exhibiting superagonist biological activity.Proc Natl Acad Sci USA. 1982; 79: 1751-1755Crossref PubMed Scopus (156) Google Scholar). Rhodamine phalloidin was purchased from Molecular Probes (Eugene, OR). Cells on coverslips were fixed in 4% formalin for 10 min at room temperature. Cells were permeabilized by incubation in 0.5% Triton-X 100 in phosphate buffered saline (PBS) for 15 min at room temperature. Non-specific binding of antibody was blocked by incubation of the coverslips in 10% normal goat serum for 10 min followed by incubation with the primary antibody for 1 h at room temperature. After several washes in PBS, secondary antibodies were applied for 30 min at room temperature. For staining of filamentous actin, rhodamine phalloidin (1/40 in PBS) was added along with the secondary antibody. Coverslips were examined using a confocal microscope from Biorad, or with a Nikon immunofluorescence microscope. The source of UV irradiation was a 1000 W Oriel Xenon Arc Solar Simulator with UVC WG320 and Visble cutoff filter UG11, beam turner, and cooling fan. This light source emits electromagnetic radiation from 295 nm (UV-B) to 400 nm and produces 3.75 × 10–3 W per cm2. The output was monitored by an IL1700 radiometer and SED400 sensor probe purchased from International Light (Newburyport, MA). The desired dose of UV light (4.5 J per cm2) was obtained by irradiating the cells for 20 min. During UV irradiation medium was replaced with warm PBS containing Ca2+ and Mg 2+. The PBS was then replaced with medium (Dulbecco’s minimal essential media + 2% fetal calf serum). All transient transfections were generated by transfection of 1 × 105 cells on glass coverslips with 2 μg of each pcEXV-3 eukaryotic expression vectors containing either V12rac1, N17rac1, V12cdc42, N17cdc42, V14rhoA, or A35rac1 engineered such that the protein was tagged with the c-myc epitope. All vectors were kindly provided by Dr. A. Hall (University College, London, U.K.), and are fully described inRidley et al., 1992Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (2979) Google Scholar. The expressed proteins of V12rac1, V12cdc42, and V14rhoA are consitutively active; the expressed proteins of N17rac1 and N17cdc42 function as dominant negative inhibitors; the expressed protein of A35rac1 is inactive. For transfections, cells were placed in OPTI-MEM(R) (Gibco BRL) and incubated with vectors and 10 μl lipofectamine (Gibco BRL) overnight at 37°C. Cells were then fixed and transfected cells were visualized by detection of the c-myc epitope with 9E10 antibodies followed by immunofluorescence microscopy as described above. To determine if treatment of B16F1 cells with NDP-MSH induced the co-localization of actin and melanosomes in dendrites, B16F1 cells were treated with NDP-MSH (10–8 M) for 1 h or 24 h and dual localization of melanosomes and the actin cytoskeleton was performed. Co-localization of actin and melanosomes was assessed by examination of stained coverslips by confocal microscopy (Figure 1a–c). Melanosomes were located in the perinuclear area and throughout the cytoplasm in untreated cells (Figure 1a) and some, but not all melanosomes, co-localized with filamentous actin in the cell cytoplasm as determined by confocal microscopy. Filamentous actin was present at the periphery of the cell, but melanosomes were generally absent from the cell periphery. After 1 h of treatment with NDP-MSH, small dendritic processes appeared in treated cells, and melanosomes were present at the tips of dendrites and co-localized with actin filaments (Figure 2b). After 24 h of treatment with NDP-MSH, long branching dendrites had formed and melanosomes were present at the dendritic tips and along the length of the dendrite (Figure 2c). Dibutyryl cAMP (10–5 M for 1 and 24 h) also induced the formation of actin containing dendrites in B16F1 cells in which actin and melanosomes co-localized (data not shown).Figure 2V12rac1 induces the formation of actin-containing dendritic processes in B16F1 cells. B16F1 cells transiently transfected with V12rac1 were detected by expression of the c-myc epitope (a–f). Low power photomicrographs (a, b;scale bar, 60 μm) show several transfected cells in the field that exhibit dendrites. In comparison, nontransfected cells (arrows) are round and nondendritic. Parts (c)–(f) show the V12rac1 transfected cells under higher power (scale bar, 20 μm). Cells contain thin, branching dendritic structures induced by expression of the V12rac1 protein (arrows). The dendrites were composed of actin, as shown by rhodamine phalloidin staining (g).View Large Image Figure ViewerDownload (PPT) To determine if expression of V12rac1 would induce dendrites in B16F1 cells, cells were transfected with plasmids expressing the constitutively active mutant forms of the rac1 protein (V12rac1) and the morphology and actin cytoskeleton of transfected cells were examined by immunofluorescence microscopy. Cells were also transiently transfected with plasmids expressing constitutively active cdc42 protein (V12cdc42) and with plasmids expressing constitutively active rhoA protein (V14rhoA). Controls consisted of cells transfected with an inactive rac1 protein (A35rac1). Transfected cells were visualized by staining with antibodies to the c-myc epitope and the cytoskeleton was visualized by staining with rhodamine phalloidin (Figure 2, Figure 3), and each experiment was performed at least three separate times. Cells transfected with V12rac1 expression vectors exhibited a complex branching dendritic morphology (Figure 2a–f; c-myc epitope) and the dendritic processes were composed of actin (Figure 2g, rhodamine phalloidin stain of cell pictured in Figure 2f). A low power photomicrograph of a field of V12rac1-transfected cells shows several transfected cells that are highly dendritic (Figure 2a, b); the arrowheads highlight nontransfected cells that are round or polygonal. In general, virtually all transfected cells exhibited a dendritic phenotype compared" @default.
• W1981860108 created "2016-06-24" @default.
• W1981860108 creator A5051726920 @default.
• W1981860108 creator A5085366413 @default.
• W1981860108 date "1998-08-01" @default.
• W1981860108 modified "2023-10-12" @default.
• W1981860108 title "Rac1 Mediates Dendrite Formation in Response to Melanocyte Stimulating Hormone and Ultraviolet Light in a Murine Melanoma Model" @default.
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