Unité Mixte de Recherche Santé 725, Institut National de la Santé et de la Recherche Médicale, Strasbourg, France.
PLoS One. 2013;8(4):e60813. doi: 10.1371/journal.pone.0060813. Epub 2013 Apr 5.
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.
朗格汉斯细胞中的 Birbeck 颗粒（BGs）是其特征性细胞器，朗格汉斯蛋白（Langerin）对于其生物发生是必需的。我们先前使用一种带有 C 末端腔室 YFP 标签的 Langerin-YFP 融合蛋白，动态解析了伴随 Langerin 运输的分子和细胞过程。为了阐明 Langerin 与其运输效应物的相互作用及其对 BG 生物发生的结构影响，我们生成了一种带有 N 末端胞质 YFP 标签的 YFP-Langerin 嵌合体。这种融合蛋白诱导了 YFP 阳性大斑点的形成。活细胞成像与光漂白后荧光恢复方法相结合的研究表明，这些新形成的隔室中蛋白质的凝聚是静态的。相比之下，在表达 Langerin-YFP 嵌合体的细胞的中心体周围区域中存在的 YFP 阳性结构，显示出与活性膜交换兼容的荧光恢复特征。使用相关的光电子显微镜，我们表明凝聚结构代表了具有典型 BG 五夹板结构的高度组织的膜堆栈。这些细胞器与粗面内质网之间的连续性使我们能够将膜堆栈鉴定为“有序光滑内质网”（OSER）的一种形式，其具有独特的分子和生理特性。使用 A206K 变体的 YFP 证明了细胞质 YFP 分子之间同源相互作用的参与，该变体恢复了在朗格汉斯细胞中观察到的大多数 Langerin 运输和 BG 特征。碳水化合物识别结构域的突变也阻止了 OSER 的形成。因此，“双锁”机制控制 YFP-Langerin 的行为，其中 YFP 标签的不对称同源二聚化和 Langerin 分子的凝集结构域之间的同源相互作用参与其保留和随后形成 BG 样 OSER。这些观察结果证实，只要 Langerin 积累，BG 样结构就会出现，并证实膜运输效应物决定了它们的生理学，并说明了分子相互作用在细胞内膜结构中的重要性。
