The ternary Rab27a-Myrip-Myosin VIIa complex regulates melanosome motility in the retinal pigment epithelium.
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.
Lopes
Vanda S
VS
Molecular and Cellular Medicine, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK.
Ramalho
José S
JS
Owen
Dylan M
DM
Karl
Mike O
MO
Strauss
Olaf
O
Futter
Clare E
CE
Seabra
Miguel C
MC
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015638:Cytochalasin D / Q000494:pharmacology; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D047468:Immunoprecipitation; D020460:Melanosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000081412:Myosin VIIa; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D016372:RNA, Antisense / Q000235:genetics; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D000199:Actins; C521067:MYRIP protein, mouse; C000631456:Myo7a protein, mouse; D000081412:Myosin VIIa; D016372:RNA, Antisense; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D015638:Cytochalasin D; C512962:Rab27a protein, mouse; D009218:Myosins; D004398:Dyneins; D020691:rab GTP-Binding Proteins; D015739:Nocodazole