title: Knockdown of myosin Va isoforms by RNAi as a tool to block melanosome transport in primary human melanocytes.
abstract: The movement of melanosomes, dense melanin-containing organelles, within human melanocytes is actin-dependent and mediated through the formation of a Rab27a-Slac2-a-myosin Va (MyoVa) protein complex. We previously showed that only the melanocyte-specific exon F isoforms of MyoVa are involved in melanosome transport to the dendrite extremities. Here, we investigate siRNA to downregulate the exon F-containing isoforms of MyoVa in primary human melanocytes. Efficient and specific knockdown of the MyoVa exon F isofoms were shown at both mRNA and protein levels. Further, a stable shRNA against the MyoVa exon F isoforms was prepared by using a lentiviral system to improve and confirm the silencing effect in hard-to-transfect melanocyte cells. Immunofluorescence microscopy shows that knockdown of the exon F isoforms results in blockade of intramelanocytic melanosome transport due to the inability to form the Rab27a-Slac2-a-MyoVa tripartite complex. Interestingly, the observed phenotypic effect (that is, perinuclear accumulation of melanosomes) is the same as that seen in melanocytes from patients with human Griscelli syndrome causing abnormal pigmentation. We conclude that our siRNA-based strategy provides a previously unreported tool to block the intracellular melanosome movement in primary human melanocytes and may become an innovative drug to treat hyperpigmentation.
authors.affiliation: Department of Dermatology, Ghent University Hospital, Ghent, Belgium. mireille.vangele@ugent.be
authors.forename: Mireille
authors.initials: M
authors.lastname: Van Gele
authors.forename: Barbara
authors.initials: B
authors.lastname: Geusens
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmitt
authors.forename: Luc
authors.initials: L
authors.lastname: Aguilar
authors.forename: Jo
authors.initials: J
authors.lastname: Lambert
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D000078404:Epidermal Cells; D005091:Exons; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D020460:Melanosomes / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018995:Myosin Heavy Chains / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D024701:Myosin Type V / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D014161:Transduction, Genetic; D020691:rab GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C492410:MLPH protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000075745:rab27 GTP-Binding Proteins; C077511:MYO5A protein, human; D024701:Myosin Type V; C066694:RAB27A protein, human; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins