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Role of Cytoplasmic Dynein in Melanosome Transport in Human Melanocytes

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Role of Cytoplasmic Dynein in Melanosome Transport in Human Melanocytes Role of Cytoplasmic Dynein in Melanosome Transport in Human Melanocytes H. Randolph Byers, Mina Yaar, Mark S. Eller, Nicole L. Jalbert, and Barbara A. Gilchrest Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, U.S.A. Cytoplasmic dynein is a microtubule-associated transport of melanosomes from the cell body into the retrograde-directed motor molecule for transport of dendrites, whereas melanosome distribution was not membrane-bound organelles. To determine whether affected in sense-treated melanocytes. To determine cytoplasmic dynein is expressed in melanocytes, we whether ultraviolet irradiation modi®es cytoplasmic performed reverse transcriptase polymerase chain dynein expression, melanocyte cultures were reaction using melanocyte cDNA and primers com- exposed to increasing doses of solar-simulated irra- plementary to human brain cytoplasmic dynein diation, equivalent to a mild to moderate sunburn heavy chain. A polymerase chain reaction product of exposure for intact skin. Within 24 h, doses of 5 and the expected molecular size was generated and the 10 mJ per cm2 induced cytoplasmic dynein protein, identity was con®rmed by sequence analysis. whereas doses of 30 mJ per cm2 or more were asso- Western blotting of total melanocyte proteins reacted ciated with decreased levels of cytoplasmic dynein with an anti-intermediate chain cytoplasmic dynein compared with sham-irradiated controls. Our data antibody identi®ed the appropriate 74 kDa band. To show that cytoplasmic dynein participates in retro- determine whether cytoplasmic dynein plays a role grade melanosomal transport in human melanocytes in melanosome transport, duplicate cultures were and suggest that the altered melanosomal distribu- treated with cytoplasmic dynein antisense or sense tion in skin after sun exposure is due, at least in (control) oligodeoxynucleotides and the cells were part, to decreased cytoplasmic dynein levels result- observed by high-resolution time-lapse microscopy, ing in augmented anterograde transport. Key words: which allows visualization of melanosomal aggre- cytoplasmic dynein/melanocytes/melanosome/ultraviolet gates and individual melanosomes. Antisense-treated irradiation. J Invest Dermatol 114:990±997, 2000 melanocytes demonstrated a strong anterograde kin color and ultraviolet (UV) photoprotection are largely an important motor in actin-®lament-based transport of organelles dependent on melanin content and distribution. Melano- (Mehta et al, 1999). Mutations of myosin V in dilute mice (Mercer cytes synthesize melanin within membrane-bound et al, 1991) and in patients with Griscelli PrunieÂras syndrome organelles called melanosomes that are transported from (Griscelli et al, 1978; Pastural et al, 1997) result in altered skin and Sthe perinuclear region, their site of origin, into the hair due to reduced melanosome transport into melanocyte melanocyte dendrites and eventually into the adjacent keratinocytes dendrites (Provance et al, 1996; Wu et al, 1997, 1998; (Mottaz and Zelickson, 1967; Cohen and Szabo, 1968; Klaus, Lambert et al, 1998). The majority of the kinesin family motor 1969; Wolff et al, 1974; Jimbow et al, 1976). As with all membrane- proteins are plus-end directed microtubule-activated ATPases that bound organelles, the transport of melanosomes is mediated by the are important in fast axoplasmic or anterograde transport of net sumof centripetal and centrifugal cytoskeletal forces acting organelles in neurons (Brady et al, 1982; Brady, 1985; Schnapp et al, upon the organelle. Intracellular transport of organelles in all cells is 1985; Vale et al, 1985; Bloom et al, 1988). Kinesin functions in all directed by two major polarized cytoskeletal macromolecular cells to transport lysosomes, mitochondria, and other cargo polymers, actin ®laments and microtubules, composed of actin membrane-bound organelles (Hirokawa, 1998), and recent work monomers and a:b-tubulin dimers respectively. Organelles are from our laboratory suggests a role for kinesin in melanosome shuttled along actin ®laments and microtubules by certain members transport in melanocytes (Hara et al, 2000). Cytoplasmic dynein, of three major families of adenosine triphosphatase (ATPase) another microtubule-activated motor protein, is believed to chemokinetic motor proteins: dyneins (Hirokawa, 1998), kinesins transport organelles such as endosomes and multivesicular bodies (Hirokawa, 1998), and myosins (Mermall et al, 1998). Myosin V is in the opposite direction to kinesin in neurons (retrograde transport) (Lye et al, 1987; Paschal et al, 1987; Schnapp and Reese, Manuscript received November 2, 1999; revised January 21, 2000 1989; Schroer et al, 1989). The transport of organelles in all cells is accepted for publication February 1, 2000 thus believed to be controlled in part by the net balance of the Reprint requests to: Dr. H. Randolph Byers, Department of myosin V actin-®lament-based transport, the kinesin micro- Dermatology, Boston University School of Medicine, 609 Albany St, tubule-based motors, and the opposing cytoplasmic dynein Boston, MA 02118. Email: [email protected] microtubule-based motor. The focus of our current investigation Abbreviations: Dyh1, DHC1, cytoplasmic dynein heavy chain 1; Dyh2, DHC2, cytoplasmic dynein heavy chain 2; DHC3, cytoplasmic dynein is to determine the role of cytoplasmic dynein in melanosome heavy chain 3. transport in melanocytes. 0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 990 VOL. 114, NO. 5 MAY 2000 CYTOPLASMIC DYNEIN AND MELANOSOME TRANSPORT 991 Cytoplasmic dynein is a multimeric high-molecular mass Diego, CA). Digital images were taken every 5 s to every 30 s for a duration complex of two heavy chains, three intermediate chains, and four of several minutes at 0, 5, 10, 24, and 48 h following sense or antisense light intermediate chains. The ATPase mechanochemical transduc- oligonucleotide treatment or 24 h after solar-simulated UV exposure. The tion residues are found in two cytoplasmic dynein heavy chains of 5±30 s intervals permitted study of bidirectional individual melanosome migration and the hour intervals permitted evaluation of net displacement approximately 530 kDa each (Holzbaur and Vallee, 1994; Schroer, of melanosomes. Video frames were captured with an LG-3 scienti®c frame 1994). Molecular cloning and sequencing of the most abundant, grabber card (Scion, Frederick, MD) and analyzed for transport rates and ubiquitous cytoplasmic dynein heavy chain (Koonce et al, 1992; pigment granule distribution using IP lab spectrum software (Scanalytics, Mikami et al, 1993; Zhang et al, 1993) has determined it to be Fairfax, VA). relatively conserved from amoeba to mammalian brain. Partial sequence data and biochemical evidence has led to the identi®ca- Northern blot analysis Rat brain cytoplasmic dynein heavy chain has tion of at least three other cytoplasmic heavy chain dyneins (Tanaka been cloned and sequenced by two independent investigators (Mikami et al, et al, 1995; Vaisberg et al, 1996; Criswell and Asai, 1998). Recent 1993; Zhang et al, 1993). A 400 nucleotide partial sequence of the cytoplasmic dynein heavy chain from human fetal brain was obtained from evidence indicates that the 74 kDa intermediate chains and the light Genbank (Accession T05469 or NCBI GI: 316619). This nucleotide intermediate chains (approximately 55 kDa) participate in the sequence exhibits overlap with the cloned rat brain or cytoplasmic dynein binding to the protein complex dynactin, a macromolecular heavy chain 1 (DHC1) isoformbut not the cytoplasmicdynein heavy chain complex linked to the membranes of transported organelles (Gill 2 (DHC2) or cytoplasmic dynein heavy chain 3 (DHC3) isoforms et al, 1991; Schafer et al, 1994; Karki and Holzbaur, 1995; Vaughan (Vaisberg et al, 1996). Total RNA was isolated fromcultured human and Vallee, 1995). Cytoplasmic dynein functions as a chemo- melanocytes using the Tri-reagent (Gibco-BRL), and RNA concentrations mechanical motor that ratchets along the a- and b-tubulin dimers were determined as described previously by absorbance at 260 nm within microtubules toward the minus-end, pulling along the (Goukassian et al, 1999). The purity of the RNA was determined by the dynactin linkage mechanism that is attached to the membrane- ratio of 260/280 readings and was consistently over 1.8 (Chomczynski and Sacchi, 1987). The cDNA was generated using the Molony leukemia virus bound organelle. reverse transcriptase (Pharmacia LKB Biotech, Piscataway, NJ). Degenerate Based on research in a variety of eukaryotic cells, transport of primers ¯anking a 600 bp region in the non-microtubule binding domain membrane-bound pigment granules or melanosomes into melano- and non-conserved region near the 5¢ end in the rat brain sequence were cyte dendrites is hypothesized to be controlled in part by the two prepared. Polymerase chain reaction (PCR) was then performed with opposing motors dynein and kinesin prior to pigment transfer to human melanocyte cDNA. Primers were also constructed framing the keratinocytes. For example, Western blots of melanosomes isolated 400 bp sequence of human fetal brain cytoplasmic dynein and PCR was from ®sh melanophores show enrichment of cytoplasmic dynein performed with human melanocyte cDNA. The ampli®ed products and kinesin-like proteins compared with whole lysates of cultured obtained were puri®ed and sequenced to con®rmidentity. Then the melanophores (Rogers et al, 1997); and microinjection of anti- cDNA was
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