Different approaches for assaying melanosome transfer.
Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.
Berens
Werner
W
Pigment Cell Biology Section, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Van Den Bossche
Karolien
K
Yoon
Tae-Jin
TJ
Westbroek
Wendy
W
Valencia
Julio C
JC
Out
Coby J
CJ
Marie Naeyaert
Jean
J
Hearing
Vincent J
VJ
Lambert
Jo
J
D000818:Animals; D000951:Antigens, Neoplasm; D002250:Carbon Radioisotopes / Q000032:analysis; D002460:Cell Line; D002469:Cell Separation; D018920:Coculture Techniques; D005434:Flow Cytometry; D005452:Fluoresceins; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018189:Immunomagnetic Separation; D015603:Keratinocytes / Q000378:metabolism*; D058965:MART-1 Antigen; D008543:Melanins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009942:Organometallic Compounds; D010858:Pigmentation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013889:Thiouracil / Q000737:chemistry; D014162:Transfection; D014357:Trypsin
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D000951:Antigens, Neoplasm; D002250:Carbon Radioisotopes; D005452:Fluoresceins; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008543:Melanins; C548668:Mlana protein, mouse; D009363:Neoplasm Proteins; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; C027780:carboxyfluoresceindiacetate; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013889:Thiouracil; D014442:Monophenol Monooxygenase; D014357:Trypsin