Kinesin and Kinectin Can Associate with the Melanosomal Surface and Form a Link with Microtubules in Normal Human Melanocytes1 Garnet Vancoillie, Jo.

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Kinesin and Kinectin Can Associate with the Melanosomal Surface and Form a Link with Microtubules in Normal Human Melanocytes1 Garnet Vancoillie, Jo Lambert, Jean Marie Naeyaert Journal of Investigative Dermatology Volume 114, Issue 3, Pages 421-429 (March 2000) DOI: 10.1038/jid.2000.3 Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Kinesin and kinectin mRNA is expressed in human skin cells and tumor cell lines, RT-PCR. mRNA was extracted from cultured cells of a normal individual, or from human tumor cell lines. 400ng of mRNA was reverse transcribed with random hexamers and M-MLV Reverse Transcriptase. PCR amplification was performed with uKHC (a), nKHC (b), or kinectin (c) specific primers (P1-P2, P3-P4, and P5-P6, respectively), resulting in appropriately sized bands. The first lane represents a 100bp ladder with a more intense band of 500bp DNA. NB, human neuroblastoma SK-N-SH; MC, melanocytes; KC, keratinocytes; FB, fibroblasts. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Northern blot analysis reveals similar different length transcripts for uKHC and nKHC in human skin cells and tumor cell lines and confirms kinectin mRNA expression in all cell lines. Two micrograms of mRNA was loaded into each lane. Hybridization was performed with an α32P-labeled fragment of human uKHC (P7-P8) or nKHC (P9-P10) (a) or kinectin (P11-P12) (b) cDNA PCR product. (a) Exposure of 72h reveals messages of 1.2kb, 1.5kb, 5.2kb, 6.7kb, and 14kb. A similar transcription pattern is detected for nKHC. (b) Strong signals are detected at 1.2, 2.3, and 6.5kb in all cell lines. An additional weak signal is detected at 8.5kb. NB, human neuroblastoma SK-N-SH; MC, melanocytes; KC, keratinocytes; FB, fibroblasts; HeLa, wild-type HeLa cell line. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Distribution of antigen recognized by the KHC and kinectin antibodies in human melanocytes. Melanocytes were cultured in coverslips for 24h, fixed, and stained with a mouse monoclonal anti-KHC antibody (a) or a mouse monoclonal anti-kinectin CT-1 antibody (b). (a) KHC is present throughout the cell body, with accentuation in the perinuclear area. Label can also be detected along the dendrites and in the tips. (b) Kinectin is concentrated in the perinuclear area, although the reticular network could be seen extending somewhat into the dendrites. Scale bar: 10μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 KHC colocalizes partially with melanosomes. Human melanocytes were cultured on coverslips for 24h, fixed, and double stained with a mouse monoclonal anti-KHC antibody (FITC signal) (a) and a mouse monoclonal NKIbeteb antibody (texas-red signal) (b). In the overlay image (c) yellow staining indicates colocalization, which can be detected in the perinuclear area, along the dendrites, and in the dendrite tips. Scale bar: 10μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Kinectin colocalizes with a minority of melanosomes. Human melanocytes were cultured on coverslips for 24h, fixed, and double stained with a mouse monoclonal anti-kinectin antibody (FITC signal) (a) and a mouse monoclonal NKIbeteb antibody (texas-red signal) (b). In the overlay yellow dots indicating colocalization are detected in the perinuclear area (c). Scale bar: 10μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Colocalization of KHC with melanosomes in a peripheral dendrite tip. Human melanocytes were cultured on coverslips for 24h, fixed, and double stained with a mouse monoclonal anti-KHC antibody (FITC signal) and a mouse monoclonal NKIbeteb antibody (texas-red signal). In the overlay image yellow dots indicating colocalization can be detected along the dendrite and in the dendrite tip. Scale bar: 10μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 KHC and kinectin can associate with melanosomes. Glutaraldehyde-fixed ultrathin cryosections of cultured normal melanocytes were labeled with anti-KHC (a), or anti-kinectin NT-1 (b), or anti- kinectin CT-1 antibodies followed by incubation in protein A-10nm gold complex. Gold particles are seen in close association with the melanosome membrane in (a), (b), and (c). Hardly any label was seen in the cytoplasm or on melanosomes in the negative control shown in (d). Scale bar: 0.2μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 KHC forms a link between melanosomes and MT, but can be seen on nonmelanized organelles as well. Glutaraldehyde-fixed ultrathin cryosections of normal human melanocytes were labeled with anti-KHC (10nm gold particles) and anti-α-tubulin (5nm gold particles). Kinesin is often seen forming a link between melanosomes and MT (a) but can be detected on other nonmelanized organelles and vesicles as well (b). Scale bar: 0.1μm. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Schematic presentation of the possible cooperation of actin-based and MT-based melanosome transport. Melanosomes could be transported for long distances along MT through the dendrite and afterwards be captured and transferred through the subcortical actin network. SER, smooth endoplasmatic reticulum. Journal of Investigative Dermatology 2000 114, 421-429DOI: (10.1038/jid.2000.3) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions