title: Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets.
abstract: Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.
authors.affiliation: Institut Curie, Centre de Recherche, and Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, F-75248 Paris, France.
authors.forename: Ilse
authors.initials: I
authors.lastname: Hurbain
authors.forename: Willie J C
authors.initials: WJ
authors.lastname: Geerts
authors.forename: Thomas
authors.initials: T
authors.lastname: Boudier
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
authors.forename: Arie J
authors.initials: AJ
authors.lastname: Verkleij
authors.forename: Michael S
authors.initials: MS
authors.lastname: Marks
authors.forename: Graç
authors.initials: G
authors.lastname: Raposo
mesh_terms: D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry; D000818:Animals; D045744:Cell Line, Tumor; D020285:Cryoelectron Microscopy; D015638:Cytochalasin D / Q000737:chemistry / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D005615:Freezing; D006801:Humans; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000378:metabolism / Q000648:ultrastructure*; D020460:Melanosomes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D024461:Myosin Type I / Q000737:chemistry / Q000378:metabolism; D011312:Pressure; D017433:Protein Structure, Secondary; D058951:gp100 Melanoma Antigen
chemical_list: D000682:Amyloid; D008543:Melanins; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; C548642:Pmel protein, mouse; D058951:gp100 Melanoma Antigen; D015638:Cytochalasin D; D024461:Myosin Type I