H. Randolph Byers, Mina Yaar, Mark S. Eller, Nicole L

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Role of Cytoplasmic Dynein in Melanosome Transport in Human Melanocytes H. Randolph Byers, Mina Yaar, Mark S. Eller, Nicole L. Jalbert, Barbara A. Gilchrest Journal of Investigative Dermatology Volume 114, Issue 5, Pages 990-997 (May 2000) DOI: 10.1046/j.1523-1747.2000.00957.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic representation of the protein binding regions of the cytoplasmic dynein heavy chain. The organelle linkage protein region represented by the dark shaded region (A) on the amino end is specific to cytoplasmic dyneins. Within this region was the site of PCR amplification. This cDNA probe construction site corresponded to the human brain cytoplasmic dynein cDNA sequence (Genbank Accession T05469 or NCBI GI: 316619). Within this site two antisense oligonucleotides were prepared in order to inhibit cytoplasmic dynein mRNA translation in cultured melanocytes. Sense oligonucleotides were also prepared to serve as controls. The 74 kDa intermediate chain involved in linkage to dynactin–membrane-bound organelles binds within the dark shaded regions of the brain cytoplasmic dynein heavy chain. Antibodies against this intermediate chain were used to characterize the cytoplasmic dynein-linked organelle distribution in human melanocytes. The light shaded region (B) on the carboxyl end represents the microtubule binding region common to both cytoplasmic and axonemal (in flagella and cilia) dyneins. The lightest region between (A) and (B) contains four ATP binding sequences. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Primers for human brain cytoplasmic dynein amplify the appropriately sized reverse transcriptase PCR product using melanocyte (Mc) cDNA. The expected 400 bp band was identified using agarose electrophoresis and a 100 bp incremental standard (std). Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Sequence of amplified 400 bp fragment obtained from human melanocytes exhibits homology with brain cytoplasmic dynein. The original rat brain cytoplasmic dynein sequence (top rows) is compared with human brain cytoplasmic dynein (middle rows) and the human melanocyte cytoplasmic dynein (bottom rows). The light shaded bars indicate sequence differences between rat and human cytoplasmic dynein whereas the open bars indicate new sequence data of human cytoplasmic dynein obtained from sequencing the melanocyte cytoplasmic dynein. The human melanocyte sequence (Genbank Accession: BankIt320710 AF234785) and rat sequence do not have an extra nucleotide (dark bar) reported in the human brain sequence that spuriously encodes a premature stop codon. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Northern blot analysis of total melanocyte (Mc) RNA and the 400 bp cytoplasmic dynein probe detects the presence of melanocyte cytoplasmic dynein. The 32P radiolabeled probe hybridized to cultured Mc RNA at a relative position of 15 kb, the expected size of the cytoplasmic dynein transcript. Control fibroblast (Fb) RNA known to contain cytoplasmic dynein transcripts also shows the probe hybridizes at the expected relative position of 15 kb. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The intermediate chain of cytoplasmic dynein is present in cultured human melanocytes. Immunoblot analysis of lysate from cultured melanocytes (Mc) reveals a single band at a relative mobility (Mr) of 74 kDa corresponding to the 74 kDa intermediate chains of cytoplasmic dynein. Lanes 1 and 2 were loaded with 50 μg and 20 μg respectively of human melanocyte protein. Control fibroblast (Fb) lysate known to contain 74 kDa intermediate chains of cytoplasmic dynein also shows a single band at the expected relative mobility of 74 kDa. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Intermediate chain of cytoplasmic dynein is detected throughout cultured melanocyte cytoplasm in a punctate pattern and is increased in the perinuclear region. (A) Confocal immunofluorescent microscopy reveals punctate intermediate chain staining throughout the cytoplasm with greatest intensity in the perinuclear region (inset). Note that punctate staining is also detected distally in dendrites. (B) Control confocal immunofluorescent staining with secondary antibody alone shows no significant staining of the cytoplasm. Scale bar: 50 μm. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Antisense DNA complementary to cytoplasmic dynein sequence disperses perinuclear melanosomes. Sense DNA treated melanocytes show normal perinuclear distribution of melanosomes at 12 h (a) and 24 h (c). In contrast, antisense DNA treated cells show a perinuclear melanosome-free zone (arrow) with increased peripheral membrane pigment distribution at 12 h (b), and loss of perinuclear melanosomes and marked net anterograde dispersion of melanosomes throughout the cytoplasm at 24 h (d). Scale bar: 5 μm. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Antisense DNA complementary to cytoplasmic dynein sequences redistributes cultured melanocyte pigment from perinuclear to peripheral cytoplasm. Densitometric analysis of pigment distribution in 160 cells shows significant reduction of perinuclear pigment distribution in antisense-compared with sense-treated melanocytes (black bars). Similarly, a significant increase in the peripheral pigment distribution is seen in the same cells (gray bars). *p < 0.001. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Modulation of cytoplasmic dynein intermediate chain by UV irradiation. Western immunodetection of 74 kDa intermediate chain in cultured melanocytes in three donors 24 h after exposure to increasing doses of UV (mJ per cm2 metered at 285 ± 5 nm using solar simulator). Donor 1 (left panel) shows increase in intermediate chain after 5 and 10 mJ per cm2. Donor 2 (middle panel) confirms increased intermediate chain at 10 mJ per cm2. Donors 2 and 3 (right panel) show decrease in intermediate chain at high UV doses (≥30 mJ per cm2). Donor variability is seen at 20 mJ per cm2. Amido-black-stained bottom three panels show protein loading of gels above. Fifty milligrams of melanocyte protein was loaded onto each lane. Journal of Investigative Dermatology 2000 114, 990-997DOI: (10.1046/j.1523-1747.2000.00957.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions