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.

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum

“…9A, e and g). These structures were morphologically similar to organized smooth ER (OSER), which forms by expression of particular ER-resident transmembrane proteins with weak homodimerization (32,33). OSER formation in cells expressing SMS1-⌬C22 and SMS1-⌬N68C22 may be due to a decreased affinity for homodimer formation by the C-terminal truncation.…”

The CryoCapsule: Simplifying Correlative Light to Electron Microscopy

“…These cells exhibit a very bright fluorescent signal, easy to detect by light microscopy at low magnification (10x or 40x air extra-long working distance (ELWD) objectives) (Figure 4, Video S1). In these cells, the YFP-langerin accumulates into Organized Smooth Endoplasmic Reticulum (OSER)-like structures [17]. These structures correspond to regions of the ER where the YFP-langerin accumulates by dimerization of the YFP, trapping the fluorescent signal in large layered compartments that also share common morphological features with Birbeck granules [17].…”

“…In these cells, the YFP-langerin accumulates into Organized Smooth Endoplasmic Reticulum (OSER)-like structures [17]. These structures correspond to regions of the ER where the YFP-langerin accumulates by dimerization of the YFP, trapping the fluorescent signal in large layered compartments that also share common morphological features with Birbeck granules [17]. At the EM level, these layered membranous structures sequester the heavy metal salts used to contrast the specimen and are clearly identifiable electron dense and organized regions (Figure 4 G–J) [17].…”

“…To challenge the CryoCapsule at the biological level, we tested three different samples: a fragile in-vitro specimen, the Xenopus laevis mitotic spindle, described as pressure sensitive [16]; melanoma cells (M10 cells) over-expressing a YFP-tagged Langerin that generated a membranous layered structure that is clearly visualized by EM [17]; and a melanocytic cells line (MNT-1 cells) where we followed the endosomal network with fluorescent transferrin bound to endogenous transferrin receptors [18]. …”

Live-Cell CLEM of Subcellular Targets

“…Glass coverslips are the most commonly used substrate for live-cell imaging, since most objective lenses are optimized to their thickness (160-180 mm) and refractive index (1.52-1.54). Alternatively, however, polymer films such as Aclar, Cultfoil, and TOPAS are increasingly being used instead of glass, as they are easier to handle and process for EM (Alpy et al, 2013;Gao et al, 2013;Guizetti, Mäntler, Müller-Reichert, & Gerlich, 2010;Kingsley & Cole, 1988;Lenormand et al, 2013;Müller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007;Spiegelhalter et al, 2010), and are compatible with cryopreservation by high pressure freezing (Jimenez, Humbel, Van Donselaar, Verkleij, & Burger, 2006;Padman, Bach, & Ramm, 2014). While polymer films are usually imaged on top of a carrier glass coverslip, we have recently introduced a method to directly use polymer film as a substrate for optical imaging, thereby reducing the number of refractive interfaces between the sample and objective.…”