The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

Protein glycosylation is a complex form of posttranslational modification and has been shown to be crucial for the function of many proteins. Sialic acid is prominently positioned at the outer end of membrane glycoproteins. It plays a critical role for the regulation of a myriad of cellular functions and it forms a shield around the cell. Furthermore, it constantly interacts with the environment of cells and contributes to histocompatibility. [1] This makes studying sialylation an interesting field of research, but monitoring sialic acid in vivo is challenging. While proteins are routinely labeled by genetic methods, such as expression as GFP fusion proteins, comparable methods are not available for secondary gene products, such as glycans of glycoconjugates. Metabolic oligosaccharide engineering (MOE) is a successful new strategy to visualize the localization of glycans in vitro and in vivo. [2] In this approach, cells are cultivated in the presence of non-natural monosaccharide derivatives that carry a chemical reporter group and are nonetheless accepted by the biosynthetic machinery of a cell. For instance, peracetylated N-azidoacetylmannosamine (Ac 4 ManNAz) is taken up by the cell, deacetylated by cellular esterases, and owing to the promiscuity of the enzymes of sialic acid biosynthesis, is converted into N-azidoacetyl neuraminic acid and incorporated into sialoglycoconjugates. [3] Once presented on the cell surface, the azide-containing sialylated glycan can be visualized through a bioorthogonal ligation reaction. [4] Besides Ac 4 ManNAz, several monosaccharide derivatives of N-acetylgalactosamine, [5] N-acetylglucosamine, [6] and l-fucose [7] are suitable for MOE providing further insights into the role of cellular structures and functions of glycans in the cell.Currently, mainly Staudinger ligation [3] and azide-alkyne [3+2] cycloaddition (copper-catalyzed [8] or strain-promoted, [9] also known as the click reaction) are applied as ligation reactions in MOE. However, both of them rely on the reaction of azides and thus cannot be used for the concurrent detection of two different metabolically incorporated carbohydrates. A labeling strategy that can be carried out in the presence of azides and alkynes would significantly expand the scope of chemical labeling reactions in living cells and is thus highly desirable.Recently, it was shown that the Diels-Alder reaction with inverse electron demand (DARinv) of 1,2,4,5-tetrazines [10] with strained dienophiles, such as trans-cyclooctenes, [11] cyclobutenes, [12] norbornenes, [11d,f, 13] cyclooctynes, [11d,f] and substituted cyclopropenes, [14] fulfills the requirements of a bioorthogonal ligation reaction and furthermore is orthogonal to the azide-alkyne cycloaddition. However, these cyclic alkenes or kinetically stable tetrazines [15] are expected to be too large for being efficiently metabolized by the sialic acid biosynthetic pathway, starting from the corresponding Nacylmannosamine derivative. In search for smaller dienophiles suitable for MOE, we identifi...

Background: Micro-lipid droplets (mLDs) appear in adipocytes upon lipolytic stimulation. LDs may grow by spontaneous, homotypic fusion.Results: Scavenging of fatty acids prevents mLD formation. LDs grow by a slow transfer of lipids between LDs.Conclusion: mLDs form due to fatty acid overflow. LD growth is a controlled process.Significance: Novel mechanistic insights into LD remodeling are provided.

Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.

The objective of this study was to develop a biomimetic, highly porous collagen‐hydroxyapatite (HA) composite scaffold for bone tissue engineering (TE), combining the biological performance and the high porosity of a collagen scaffold with the high mechanical stiffness of a HA scaffold. Pure collagen scaffolds were produced using a lyophilization process and immersed in simulated body fluid (SBF) to provide a biomimetic coating. Pure collagen scaffolds served as a control. The mechanical, material, and structural properties of the scaffolds were analyzed and the biological performance of the scaffolds was evaluated by monitoring the cellular metabolic activity and cell number at 1, 2, and 7 days post seeding. The SBF‐treated scaffolds exhibited a significantly increased stiffness compared to the pure collagen group (4‐fold increase), while a highly interconnected structure (95%) was retained. FTIR indicated that the SBF coating exhibited similar characteristics to pure HA. Micro‐CT showed a homogeneous distribution of HA. Scanning electron microscopy also indicated a mineralization of the collagen combined with a precipitation of HA onto the collagen. The excellent biological performance of the collagen scaffolds was maintained in the collagen‐HA scaffolds as demonstrated from cellular metabolic activity and total cell number. This investigation has successfully developed a biomimetic collagen‐HA composite scaffold. An increase in the mechanical properties combined with an excellent biological performance in vitro was observed, indicating the high potential of the scaffold for bone TE. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009