Artigo Acesso aberto Revisado por pares

Myosin V Colocalizes with Melanosomes and Subcortical Actin Bundles Not Associated with Stress Fibers in Human Epidermal Melanocytes

1998; Elsevier BV; Volume: 111; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1998.00395.x

ISSN

1523-1747

Autores

Jo Lambert, Yves Vander Haeghen, Garnet Vancoillie, Jean Naeyaert, J. J. M. Onderwater, Henk K. Koerten, A. Mieke Mommaas,

Tópico(s)

Genetic Neurodegenerative Diseases

Resumo

Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells myosin V is expressed and treatment of melanocytes with the cyclic AMP-inducer 3-isobutyl-1-methylxanthine causes an induction of the myosin V message. In all cells myosin V colocalizes with actin bundles, concentrating in the subcortical cell zone. In the melanocyte it is closely associated with melanosomes. Quantitative analysis of myosin V labeling in melanocytes reveals a significantly higher (p < 0.005) presence of myosin V in the periphery of dendrites. These results suggest that myosin V is important in melanosome transport in human melanocytes. Possible roles in the other skin cells remain to be elucidated. Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells myosin V is expressed and treatment of melanocytes with the cyclic AMP-inducer 3-isobutyl-1-methylxanthine causes an induction of the myosin V message. In all cells myosin V colocalizes with actin bundles, concentrating in the subcortical cell zone. In the melanocyte it is closely associated with melanosomes. Quantitative analysis of myosin V labeling in melanocytes reveals a significantly higher (p < 0.005) presence of myosin V in the periphery of dendrites. These results suggest that myosin V is important in melanosome transport in human melanocytes. Possible roles in the other skin cells remain to be elucidated. 3isobutyl1methylxanthine Very little is known about the molecular mechanisms underlying dendrite extension and melanosome transport in human melanocytes. It is expected that cytoskeletal proteins such as actin and microtubule components with their respective motor proteins play a role in these processes. Indeed,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 Cellul Physiol. 1992; 151: 287-299Crossref PubMed Scopus (39) Google Scholar reported that actin microfilaments play a role in dendrite formation and microtubules play a role in dendrite maintenance in cultured human melanocytes. Myosin V is an actin-activated ATPase, a type of motor protein representing the fifth of 13 classes of unconventional myosins (for reviews seeMooseker, 1993Mooseker M. A multitude of myosins.Curr Biol. 1993; 3: 245-248Abstract Full Text PDF PubMed Scopus (33) Google Scholar;Hammer, 1994Hammer J.A. The structure and function of unconventional myosins: a review.J Muscle Res Cell Motility. 1994; 15: 1-10Crossref PubMed Scopus (57) Google Scholar;Mooseker and Cheney, 1995Mooseker M.S. Cheney R.E. Unconventional myosims.Annu Rev Cell Dev Biol. 1995; 11: 633-675Crossref PubMed Scopus (390) Google Scholar;Hasson and Mooseker, 1996Hasson T. Mooseker M.S. Vertebrate unconventional myosins.J Biol Chem. 1996; 271: 16431-16434Crossref PubMed Scopus (46) Google Scholar, 1997b;Titus, 1997aTitus M.A. Motor proteins: Myosin V – the multi-purpose transport motor.Current Biology. 1997; 7: 301-304Abstract Full Text Full Text PDF Google Scholar;Baker and Titus, 1998Baker J.P. Titus M.A. Myosins: matching functions with motors.Current Opin Cell Biol. 1998; 10: 80-86Crossref PubMed Scopus (66) Google Scholar). The first group of myosin V members is encoded by the yeast myo2/myo4 genes (Govindan et al., 1995Govindan L. Bowser R. Novick P. The role of myo2, a yeast class V myosin, in vesicular transport.J Cell Biol. 1995; 128: 1055-1068Crossref PubMed Scopus (302) Google Scholar), the mouse dilute gene (Mercer et al., 1991Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Novel myosin heavy chain encoded by murine dilute coat color locus.Nature. 1991; 349: 709-713Crossref PubMed Scopus (458) Google Scholar), the chicken p190 gene (Espreafico et al., 1992Espreafico E.M. Cheney R.E. Matteoli M. Nascimento A.A.C. De Camilli P.V. Larson R.E. Mooseker M.S. Primary structure and cellular localization of chicken brain myosin-V (p190), an uconventional myosin with calmodulin light chains.J Cell Biol. 1992; 119: 1541-1557Crossref PubMed Scopus (307) Google Scholar), and the human myoxin gene (Engle and Kennett, 1994Engle L.J. Kennett R.H. Cloning, analysis, and chromosomal localization of myosin (MYH12), the human homologue to the mouse dilute gene.Genomics. 1994; 19: 407-416Crossref PubMed Scopus (33) Google Scholar;Moore et al., 1995Moore K.J. Testa J.R. Francke U. Milatovich A. Copeland N.G. Jenkins N.A. Cloning and regional assignment of the human myosin heavy chain 12 (MYH12) gene chromosome band 15q21.Cytogenet Cell Genet. 1995; 69: 53-58Crossref PubMed Scopus (9) Google Scholar). Recently, myr 6, a second group of the myosin V family, was cloned and characterized in rat (Zhao et al., 1996Zhao L.-P. Koslovsky J.S. Reinhard J. Bähler M. Witt A.E. Provance Jr, Dw Mercer J.A. Cloning and characterization of myr 6, an unconventional myosin of the dilute/myosin V family.Proc Natl Acad Sci USA. 1996; 93: 10826-10831Crossref PubMed Scopus (76) Google Scholar). Structurally, myosin V is a two-headed, nonfilamentous protein consisting of an N-terminal head domain containing actin-binding and ATP-hydrolysis sites, a neck domain with six IQ motifs that bind calmodulin/light chains and a tail domain consisting of a coiled-coil stalk and a carboxy-terminal globular domain that is thought to bind myosin to organelles (Cheney et al., 1993Cheney R.E. O'shea M.K. Heuser J.E. et al.Brain myosin-V is a two-headed unconventional myosin with motor activity.Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (380) Google Scholar;Nascimento et al., 1996Nascimento A.A.C. Cheney R.E. Tauhata S.B.F. Larson R.E. Mooseker M.S. Enzymatic characterization and functional domain mapping of brain myosin-V.J Biol Chem. 1996; 271: 17561-17569Crossref PubMed Scopus (108) Google Scholar). Yeast myo2 gene mutations result in large, unbudding cells that pack vesicles in their cytoplasm. The originally described mouse dilute locus mutations termed dilute-viral (dv) cause a dilution of coat color (Silvers, 1979Silvers W.K. Dilute and Leaden, the, p. -locus, ruby-eye and ruby-eye-2.in: Silvers W.K. Coat Colours of Mice. A Model for Mammalian Gene Action and Interaction. Spriner-Verlag, New York1979: 83-104Google Scholar). Other more severe dilute alleles (dilute-lethal, dL) also produce neurologic defects with seizures, opisthotonus, and death at early age (Mercer et al., 1991Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Novel myosin heavy chain encoded by murine dilute coat color locus.Nature. 1991; 349: 709-713Crossref PubMed Scopus (458) Google Scholar). The human autosomal recessive Griscelli–Pruniéras syndrome (Griscelli et al., 1978Griscelli C. Durandy A. Guy-grand D. Daguillard F. Herzog C. Pruniéras M. A syndrome associating partial albinism and immunodeficiency.Am J Med. 1978; 65: 691-702Abstract Full Text PDF PubMed Scopus (301) Google Scholar) is characterized by partial albinism with silvery-blond hair discoloration, primary immunodeficiency, and severe neurologic disorders, with death occurring in the first decades of life. Recently, mutations in the myosin V gene of two Griscelli patients were reported byPastural et al., 1997Pastural E. Barrat F.J. Dufourcq-lagelouse R. et al.Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene.Nature Genetics. 1997; 16: 289-292Crossref PubMed Scopus (366) Google Scholar. Initially, the pigment disorder seen in humans and mice was thought to be due to underdeveloped melanocyte dendrites, but recent findings (Provance et al., 1996Provance Jr, Dw Wei M. Ipe V. Mercer J.A. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution.Proc Natl Acad Sci USA. 1996; 93: 14554-14558Crossref PubMed Scopus (150) Google Scholar;Wei et al., 1997Wei Q. Wu X. Hammer J.A. The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport.J Muscle Res Cell M. 1997; 18: 517-527Crossref PubMed Scopus (67) Google Scholar) have shown that dilute melanocytes do not lack dendrites but display defective melanosome transport into these dendrites. These phenotypic findings have led to the hypothesis that class V myosins play a role in organelle transport or membrane dynamics.Govindan et al., 1995Govindan L. Bowser R. Novick P. The role of myo2, a yeast class V myosin, in vesicular transport.J Cell Biol. 1995; 128: 1055-1068Crossref PubMed Scopus (302) Google Scholar suggest that yeast myo2 plays a role in transporting secretory vesicles from the mother cell along actin cables into the bud. Myosin V is abundantly present in neurons (Mercer et al., 1991Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Novel myosin heavy chain encoded by murine dilute coat color locus.Nature. 1991; 349: 709-713Crossref PubMed Scopus (458) Google Scholar), where it may participate in organelle transport along actin filament bundles in rat neuroneal growth cones (Evans and Bridgman, 1995Evans L.L. Bridgman P.C. Particles move along actin filament bundles in nerve growth cones.Proc Natl Acad Sci USA. 1995; 92: 10954-10958Crossref PubMed Scopus (43) Google Scholar;Evans et al., 1997Evans L.L. Hammer J. Bridgman P.C. Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons.J Cell Sci. 1997; 110: 439-449Crossref PubMed Google Scholar;Prekeris and Terrian, 1997Prekeris R. Terrian D.M. Brain myosin V is synaptic vesicle-associated motor protein: evidence for a Ca2+–dependent interaction with the synaptobrevin-synaptophysin complex.J Cell Biol. 1997; 137: 1589-1601Crossref PubMed Scopus (218) Google Scholar).Wang et al., 1996Wang F.-S. Wolenski J.S. Cheney R.E. Mooseker M.S. Jay D.G. Function of myosin-V in filopodial extension of neuronal growth cones.Science. 1996; 273: 660-663Crossref PubMed Scopus (168) Google Scholar suggest a role for myosin V in filopodial extension in growth cones of chick dorsal root ganglion neurons, whereasEvans et al., 1997Evans L.L. Hammer J. Bridgman P.C. Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons.J Cell Sci. 1997; 110: 439-449Crossref PubMed Google Scholar observed no necessary role of myosin V in regulating filopodial dynamics in nerve growth cones, because neurite outgrowth in dilute-lethal mice is normal. As for pigment cells,Provance et al., 1996Provance Jr, Dw Wei M. Ipe V. Mercer J.A. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution.Proc Natl Acad Sci USA. 1996; 93: 14554-14558Crossref PubMed Scopus (150) Google Scholar showed that myosin V can be immunoprecipitated from isolated melanosomes in mice, a finding that is highly suggestive for actin-based locomotion of melanosomes, confirmed by light immunofluorescence microscopy and immunoelectron microscopy byWu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. 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. In this study of the role of myosin V in human melanocytes, we examine the role of myosin V in dendrite formation and melanosome transport by looking at myosin V expression and subcellular localization. Moreover, we investigate its expression in the other cutaneous cells, keratinocytes, and dermal fibroblasts. We find myosin V to be expressed in all studied skin cells and, in melanocytes, to be associated with melanosomes. Epidermal melanocyte primary cultures were obtained from neonatal foreskins and established in M199 medium (Gibco BRL, Merelbeke, Belgium) supplemented with 2% fetal bovine serum, 10–9 M choleratoxin, 10 ng basic fibroblast growth factor per ml, 10 μg insulin per ml, 1.4 μM hydrocortisone, and 10 μg transferrin per ml as described earlier (Naeyaert et al., 1991Naeyaert J.M. Eller M. Gordon P.R. Park H.-Y. Gilchrest B.A. Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.Br J Dermatol. 1991; 125: 297-303Crossref PubMed Scopus (103) Google Scholar). Post-primary cultures were cultured in low calcium (0.03 mM) M199 medium supplemented with the same factors and 10% fetal bovine serum. For the experiments, third passage melanocytes were cultured for at least 4 d in a medium free of the cAMP-enhancer choleratoxin as described byDonatien et al., 1993Donatien P. Surlève-bazeille J.E. Thody A.J. Taïeb A. Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes.Arch Dermatol Res. 1993; 285: 385-392Crossref PubMed Scopus (68) Google Scholar. Briefly, this melanocyte growth medium is composed of two-thirds MCDB153 and one-third Iscove's modified Dulbecco's medium supplemented with whole bovine pituitary extract (47 μg per ml), epidermal growth factor (3.3 ng per ml), insulin (3.3 μg per ml), hydrocortisone (0.94 μM), and 3% fetal bovine serum. For the induction experiments, 100 μM 3-isobutyl-1-methylxanthine (IBMX) was added to these cultures for 48 h. All chemicals and growth factors noted here are from Sigma (Bornem, Belgium), except whole bovine pituitary extract which is available at Gibco BRL (Gent, Belgium). Cultures of human keratinocytes were initiated from adult skin biopsies and cultured in growth factor- and serum-supplemented Dulbecco's modified Eagle's medium/Ham's F12 to near confluency as previously described (Beele et al., 1991Beele H. Naeyaert J.M. Goeteyn M. De Mil M. Kint A. Repeated cultured epidermal allografts in the treatment of chronic leg ulcers of various origins.Dermatologica. 1991; 183: 31-35Crossref PubMed Scopus (59) Google Scholar). Fibroblasts were established as explants from dermal portions of skin biopsies and cultured in Optimem I supplemented with 2% Ultroser G and 1% glutamine (Gibco BRL, Merelbeke, Belgium). Total cellular RNA was harvested from subconfluent cell cultures in Tri-reagent (Sigma) and isolated according to the manufacturer's instructions. Poly(A)+ RNA was obtained using PolyA Tract mRNA Isolation System III (Promega, Leiden, The Netherlands). Purity of the obtained total RNA and mRNA samples was checked by measuring the absorbance ratio at 260/280 nm, which was >1.7 in all samples. Total RNA was DNAse treated (DNAse I, Promega, Leiden, The Netherlands) prior to use in reverse transcriptase-polymerase chain reaction (RT-PCR) to eliminate amplification of genomic DNA. Approximately 1 μg of total RNA was reverse transcribed with random primers and amplified using the GeneAmp RNA-PCR kit (Perkin Elmer, Brussels, Belgium). Two oligonucleotides (P1, 5′-TGCACCTGCTTATGGAGAC-3′; P2, 5′-TTCGCTTCCTCTTCACTTAGT-3′) were chosen defining a 1006 bp cDNA in the neck region of the human myosin V cDNA (Engle and Kennett, 1994Engle L.J. Kennett R.H. Cloning, analysis, and chromosomal localization of myosin (MYH12), the human homologue to the mouse dilute gene.Genomics. 1994; 19: 407-416Crossref PubMed Scopus (33) Google Scholar; Genbank accession numbers L19401 or Y07759) using Oligo 5.0 software. Considering the high probability of sequence overlap with other myosin sequences (Hasson et al., 1996Hasson T. Skowron J.F. Gilbert D.J. et al.Mapping of unconventional myosins in mouse and human.Genomics. 1996; 36: 431-439Crossref PubMed Scopus (72) Google Scholar), the oligonucleotides were carefully checked for false priming sites in the following published myosin coding sequences (Genbank): human myosin IB and IE (X98411 and X98507), human myosin IC gene (U14391), human myosin VI (U90236), human myosin VIIa (U39226), and human myosin IXb (U42391). False priming sites in human frequent sequences were excluded as well. PCR amplification conditions were 95°C for 1 min, 60.5°C for 1 min, and 72°C for 1.5 min during 35 cycles after an initial denaturation at 95°C for 2 min and terminating the reaction with 7 min at 72°C. cDNA bands were visualized on a 1.5% TAE-agarose ethidium bromide stained gel. The resulting 1006 bp cDNA was eluted from the gel (QiaEx gel extraction kit, Westburg, The Netherlands) and after purification used as a hybridization probe in northern blot. Northern blot was performed as previously described (Wintzen et al., 1996Wintzen M. Yaar M. Burbach P.H. Gilchrest B.A. Proopiomelanocortin gene product regulation in keratinocytes.J Invest Dermatol. 1996; 106: 673-678Crossref PubMed Scopus (128) Google Scholar). Briefly, 2 μg of poly(A)+ RNA was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde. RNA was transferred overnight onto a nylon membrane (Hybond-N+, Amersham, Gent, Belgium) and immobilized by UV irradiation in a UV-Stratalinker (Stratagene, La Jolla, CA). Prehybridization of the blot was in a solution containing 50% deionized formamide, 5×Denhardt's solution, 0.02% sodium dodecyl sulfate, and 0.2 mg salmon sperm DNA per ml at 45°C during 2 h. The 1006 bp myosin V cDNA probe was labeled with [32P]-dCTP using Oligolabeling kit (Pharmacia, Roosendaal, The Netherlands) and hybridized to the blot overnight at 45°C in a solution containing 50% deionized formamide, 2.5 × Denhardt's solution, 0.02% sodium dodecyl sulfate, 5 × SSPE, and 0.02 mg salmon sperm DNA per ml together with a radiolabeled G3PDH control probe. Washes were performed at room temperature once for 5 min and three times for 20 min in 0.3 M NaCl, 0.03 M sodium citrate, 0.1% sodium dodecyl sulfate, and subsequently twice for 20 min at 45°C and twice at 55°C in 15 mM NaCl, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate. Blots were exposed to a phosphor screen (Molecular Dynamics, Krefeld, Germany) during 48 h and the digitized images were evaluated densitometrically using Phosphor Imager Imagequant software. Cells were grown on coverslips and fixed for 20 min at room temperature with 3% paraformaldehyde in phosphate-buffered saline (PBS). For permeabilization, cells were treated for 5 min with 0.2% Triton X-100 in PBS. Double staining for myosin V proteins and actin was performed by incubating for 1 h at room temperature with a rabbit polyclonal anti-myosin V antibody DIL-2 (1:400) (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. 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). After three washes in TBS, the slides were incubated in a biotinylated donkey-anti-rabbit antibody (Amersham) 1:50 dilution for 1 h at room temperature, again washed, and finally incubated in a mixture of fluorescein isothiocyanate-conjugated phalloidin (Sigma) and streptavidin-texas red for 30 min at room temperature. For double staining of myosin V and melanosomes, slides were first incubated for 1 h at room temperature in a 1:40 mouse monoclonal NKI-beteb (Monosan, Uden, The Netherlands) and a 1:400 DIL-2 dilution, followed by a second incubation step of 1 h at room temperature with an fluorescein isothiocyanate-linked swine-anti-rabbit antibody (1:20) and a biotinylated sheep anti-mouse antibody (1:50) (both of Prosan, Gent, Belgium) and a final third incubation step with streptavidin-texas red. Slides were coverslipped in Prosan fluorescence mounting fluid and confocal images (1 μm sections) were obtained with a Zeiss laser confocal microscope. For negative controls incubation with an irrelevant isotype-matched antibody was performed. The (polyclonal) antibody DIL-2 used in our immunocytochemical experiments was extensively tested on its specific interaction with a myosin V isoform (raised against myosin V heavy chain residues 910–1106) encoded by the dilute locus by its generators (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. 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). Confluent cell cultures were fixed for 1 h at room temperature in 0.1% glutaraldehyde in 0.14 M cacodylate buffer, pH 7.3, and processed for immunogold labeling as described elsewhere (Mommaas et al., 1992Mommaas A.M. Wijsman M.C. Mulder A.A. Van Praag M.C.G. Vermeer B.J. Koning F. HLA class II expression on human epidermal Langerhans cells in situ: upregulation during the elicitation of allergic contact dermatitis.Human Immunol. 1992; 34: 99-106Crossref PubMed Scopus (31) Google Scholar). Briefly, cells were pelleted and embedded in 10% gelatin, cut into 1 mm3 cubes, cryoprotected in 2.3 M sucrose for 25 min and snapfrozen in liquid nitrogen. Ultrathin cryosections were prepared and incubated with the polyclonal rabbit anti-myosin V antibody DIL-2 (cfr. supra) diluted 1:250, followed by 10 nm protein A-gold. For double labeling with actin bundles a mouse anti-human actin bundles monoclonal antibody (dilution 1:20) from Chemicon (Biognost, Wevelgem, Belgium) was used and visualized with 5 nm protein A-gold after an intermediate incubation step with a secondary rabbit anti-mouse immunoglobulin (Dako, The Netherlands). For negative controls the primary antibody step was omitted and incubation with an irrelevant isotype-matched antibody was performed. After immunolabeling, sections were embedded and contrasted in methylcellulose/uranyl acetate and viewed with a Philips EM 410 electron microscope. For quantitation of myosin V immunolabeling, immunoelectron microscopy photographs of whole melanocytes (n = 6) were made at a final magnification of 55,000×. Four different cell regions were considered Figure 1: periphery of the dendrite, center of the dendrite, perinuclear area, and periphery of the perinuclear area. Periphery of the dendrite and of the perinuclear area was recognized as a more darkly stained peripheral area of cytoplasm that represents the 150 nm thick actin subcortical meshwork. The perinuclear area was defined as the vesicle-rich area surrounding the nucleus. These cell zones were scored quantitatively by automatical analysis of the number of myosin V colloidal gold particles per μm2 using Kontron KS400 image processing software. Nuclear labeling was taken as background. Results obtained for various experimental groups were compared using the nonparametric Friedman test. p values <0.05 were considered to be statistically significant. The three examined skin cells show the appropriately sized myosin V 1006 bp cDNA band after RT-PCR Figure 2. Northern blot analysis Figure 3a reveals the complex transcription pattern of myosin V in all cells with 7, 8, and 12 kb messages as described in the literature (Mercer et al., 1991Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Novel myosin heavy chain encoded by murine dilute coat color locus.Nature. 1991; 349: 709-713Crossref PubMed Scopus (458) Google Scholar).Figure 3Northern blot analysis of different skin cell types reveals different myosin V transcripts and induction of myosin V in melanocytes under the influence of IBMX. (a) Melanocytes (lane 1), keratinocytes (lane 2), and dermal fibroblasts (lane 3) have the 7, 8, and 12 kb messages probed with a [32P]-labeled fragment of human myosin V cDNA PCR product. (b) Two micrograms mRNA was isolated out of control melanocytes versus IBMX-treated melanocytes and analyzed by northern blot analysis hybridized for myosin V and G3PDH. On this representative blot, quantitation of density of the myosin V bands using a phosphorimager revealed a 5-fold induction of myosin V after correction for loading differences relative to the G3PDH mRNA intensity.View Large Image Figure ViewerDownload (PPT) Melanocytes grown in melanocyte growth medium display a bipolar to tripolar slender cell body. IBMX (100 μM final concentration) changed the morphology of the bipolar to tripolar melanocytes towards cells with a higher number of dendrites (four or more) per cell body within 48 h after addition (not shown). Dendrites were also generally longer and sometimes highly branched in the treated melanocytes. Quantitative analysis of the northern blot Figure 3b of control bipolar melanocytes versus highly dendritic melanocytes showed that the myosin V mRNA level increases up to 5-fold in melanocytes treated with IBMX for 48 h above control melanocytes. In all cells studied, myosin V immunofluorescent images reveal a punctate-staining pattern throughout the cytoplasm with clear intensification in the perinuclear area and, in melanocytes Figure 4, throughout the dendrites. Double labeling of myosin V with actin Figure 5 shows that in the melanocyte myosin V colocalizes with subcortical actin. In the central cytoplasm no well-structured colocalization of myosin V with actin bundles (e.g., with the focal contacts and stress fibers) can be observed. Moreover, myosin V staining is accentuated at the tip of each melanocytic dendrite Figures 4, 5.Figure 5Double immunofluorescent staining for myosin V and actin. Human melanocytes were cultured on coverslips for 24 h, fixed, and double stained with a polyclonal anti-myosin V antibody (texas red signal) and phalloidin (fluorescein signal). In this confocal photomicrograph, yellow staining (arrows) indicates the presence of myosin V in the actin-rich subcortical meshwork and near actin bundles in the perinuclear area. Scale bar: 10 μm.View Large Image Figure ViewerDownload (PPT) For better resolution the subcellular localization of myosin V was also determined with immunoelectron microscopy. In all cultured skin cells, actin immunoreactivity (5 nm gold particles) is prominent in the subcortical region where actin is also morphologically recognizable as thin filamentous bundles Figure 6a. In the melanocytic dendrite actin bundles are mainly localized in the periphery. Actin is less organized and less present in the rest of the cytoplasm, e.g., in the organelle-rich perinuclear area. In all these actin-rich areas there is a striking overlap with myosin V staining (10 nm gold particles) and in the melanocytes, colocalization of myosin V and actin is mainly seen in the periphery of the dendrites Figure 6b. In control sections, only a few scattered gold particles were seen Figure 6c. Quantitative analysis of myosin V labeling in the melanocyte revealed a significantly higher (p < 0.005; see Table 1) presence of myosin V in the subcortical cell zone, i.e., in the periphery of the dendrite and the peripheral perinuclear zone as compared with the center of the dendrite and the perinuclear area.Table 1Myosin V is present in a significantly higher number in the subcortical region of the melanocytecpg per μm2aThe background corrected number of myosin V colloidal gold particles per μm2 (cpg per μm2) in four different melanocytic cytoplasmic regions.MelanocytePerinuclear areaPeripheral perinuclear areaCenter of the dendritePeriphery of the dendriteMelanocyte 11.611.56.88.8Melanocyte 24.612.31.112.1Melanocyte 313.419.611.013.9Melanocyte 414.320.723.329.0Melanocyte 56.013.011.212.9Melanocyte 67.011.56.29.0Mean ± SEMbn = 6.7.8 ± 2.014.8 ± 1.7cp < 0.005.9.9 ± 3.014.3 ± 3.1cp < 0.005.Median6.512.78.912.5a The background corrected number of myosin V colloidal gold particles per μm2 (cpg per μm2) in four different melanocytic cytoplasmic regions.b n = 6.c p < 0.005. Open table in a new tab In addition to the colocalization with actin, in all cells myosin V is occasionally seen in close association with cytoplasmic membranous organelles that are not mitochondria. In the melanocyte, double staining results using myosin V antibody and NKI-beteb, a (pre)melanosomal marker, indicate colocalization (yellow color) of myosin V with melanosomes in the perinuclear area and in the dendrites Figure 7. At the electron microscopic level, late stage melanosomes are easily recognized as membrane-lined melanin-containing vesicles. Several melanosomes located in the dendrites of melanocytes colocalize with an actin bundle and myosin V, with myosin V seen as an intermediate molecule between the melanosome and an actin bundle Figure 8a. Melanosomes also label with myosin V without evidence for actin bundles in their neighborhood. Myosin V specific gold particles are also seen on nonmelanized organelles in the perinuclear area Figure 8b. It remains to be investigated whether these are early melanosomes (stage I melan

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