title: Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.
abstract: Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.
authors.affiliation: Unité Mixte de Recherche Santé 725, Institut National de la Santé et de la Recherche Médicale, Strasbourg, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Lenormand
authors.forename: Coralie
authors.initials: C
authors.lastname: Spiegelhalter
authors.forename: Bertrand
authors.initials: B
authors.lastname: Cinquin
authors.forename: Sabine
authors.initials: S
authors.lastname: Bardin
authors.forename: Huguette
authors.initials: H
authors.lastname: Bausinger
authors.forename: Catherine
authors.initials: C
authors.lastname: Angénieux
authors.forename: Anita
authors.initials: A
authors.lastname: Eckly
authors.forename: Fabienne
authors.initials: F
authors.lastname: Proamer
authors.forename: David
authors.initials: D
authors.lastname: Wall
authors.forename: Ben
authors.initials: B
authors.lastname: Lich
authors.forename: Sylvie
authors.initials: S
authors.lastname: Tourne
authors.forename: Daniel
authors.initials: D
authors.lastname: Hanau
authors.forename: Yannick
authors.initials: Y
authors.lastname: Schwab
authors.forename: Jean
authors.initials: J
authors.lastname: Salamero
authors.forename: Henri
authors.initials: H
authors.lastname: de la Salle
mesh_terms: D001692:Biological Transport; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D006801:Humans; D007801:Langerhans Cells / Q000166:cytology*; D037181:Lectins, C-Type / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D037181:Lectins, C-Type; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins