title: The endosomal proteome of macrophage and dendritic cells.
abstract: The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types.
authors.affiliation: Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, QC, Canada.
authors.forename: Sophie
authors.initials: S
authors.lastname: Duclos
authors.forename: Giovanna
authors.initials: G
authors.lastname: Clavarino
authors.forename: Gilles
authors.initials: G
authors.lastname: Rousserie
authors.forename: Guillaume
authors.initials: G
authors.lastname: Goyette
authors.forename: Jonathan
authors.initials: J
authors.lastname: Boulais
authors.forename: Voahirana
authors.initials: V
authors.lastname: Camossetto
authors.forename: Evelina
authors.initials: E
authors.lastname: Gatti
authors.forename: Sylvie
authors.initials: S
authors.lastname: LaBoissière
authors.forename: Philippe
authors.initials: P
authors.lastname: Pierre
authors.forename: Michel
authors.initials: M
authors.lastname: Desjardins
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000276:immunology / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000276:immunology; D011992:Endosomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008247:Lysosomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008863:Microspheres; D010588:Phagosomes / Q000276:immunology / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000145:classification / Q000276:immunology / Q000378:metabolism*; D020543:Proteome / Q000032:analysis / Q000145:classification / Q000276:immunology / Q000378:metabolism*; D013395:Sucrose / Q000737:chemistry
chemical_list: D008070:Lipopolysaccharides; D011506:Proteins; D020543:Proteome; D013395:Sucrose