The classical complement pathway (CCP) is an essential part of the immune system, activated when complement protein C1 binds to IgG antibody oligomers on the surface of pathogens, infected or malignant cells, culminating in the formation of the membrane attack complex and subsequent cell lysis. IgG oligomers also engage immune effector cells through Fcγ receptors or complement receptors, facilitating antibody‐dependent cellular cytotoxicity and phagocytosis. Understanding the factors that drive IgG oligomerization is thus crucial for improving IgG‐based therapies. Herein, a kinetic model to predict oligomer formation based on IgG concentration, antigen density, IgG subclass, Fc mutants, and oligomerization inhibitors like staphylococcal protein A is developed. The underlying molecular interactions in single molecule force spectroscopy and grating coupled interferometry experiments are characterized. By fitting experimental data from high‐speed atomic force microscopy experiments, key rate constants and thermodynamic parameters, including free energy changes associated with oligomerization and apply the model to predict complement‐mediated lysis in liposomal vesicle‐based assays, are further quantified. The presented mechanistic framework may serve as a basis for optimizing antibody engineering and pharmacokinetic/pharmacodynamic modeling in the context of immunotherapies exploiting the CCP.

The classical complement pathway (CCP), an important branch of the mammalian immune system, is initiated through multivalent binding of complement protein C1 to Immunoglobulin G (IgG) antibody oligomers assembled on the surface of pathogens, infected or malignant cells, culminating in the formation of the membrane attack complex (MAC) and subsequent cell lysis. IgG oligomers can further engage immune effector cells through Fcγ receptors or complement receptors, facilitating antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Detailed knowledge of the factors that drive IgG oligomerization is thus vitally important to establish and improve IgG based therapies. We here focus on the kinetics of antigen-dependent IgG oligomerization and develop a comprehensive model capable of predicting oligomer formation as a function of IgG concentration, antigen density, IgG subclass and Fc point mutants, as well as the presence of Fc-binding and thus oligomerization-inhibiting factors such as staphylococcal protein A (SpA). We characterize the underlying molecular interactions in single molecule force spectroscopy (SMFS) and grating coupled interferometry (GCI) experiments. By fitting experimental data from high-speed atomic force microscopy (HS-AFM) experiments, we further quantify key rate constants and thermodynamic parameters, including free energy changes associated with oligomerization and apply the model to predict complement-mediated lysis in liposomal vesicle-based assays. The presented mechanistic framework may serve as a basis for optimizing antibody engineering and pharmacokinetic/pharmacodynamic modeling in the context of immunotherapies exploiting the CCP. ### Competing Interest Statement The authors declare the following competing financial interest(s): F.J.B., S.B, and A.F.L., are inventors on patent applications related to complement activation by therapeutic antibodies and own Genmab stock. J.P. received Genmab funding.

The Tweety homologues (TTYHs) constitute a family of eukaryotic membrane proteins that, on the basis of structural features, were recently proposed to contribute to lipid transfer between soluble carriers and cellular membranes 1 . However, in the absence of supporting data, this function was hypothetical. Here through pull-down of endogenous proteins, we identify APOE as the interaction partner of human TTYH2. Subcellular fractionation and immunocytochemistry assays showed that both proteins colocalize in endosomal compartments. Characterization of the specific interaction between APOE and TTYH2 through binding assays and structural studies enabled us to identify an epitope in an extended domain of TTYH2 that faces the endosomal lumen. Structures of complexes with APOE-containing lipoprotein particles revealed a binding mode that places lipids in a suitable position to facilitate their diffusion into the membrane. Moreover, in vitro studies revealed that lipid transfer is accelerated by TTYH2. Collectively, our findings indicate that TTYH2 has a role in the unloading of APOE-containing lipoproteins after they are endocytosed. These results define a new protein class that facilitates the extraction of lipids from and their insertion into cellular membranes. Although ubiquitous, this process could be of particular relevance in the brain, where APOE is involved in the transfer of lipids between astrocytes and neurons.

DLC films exhibit high hardness, low friction coefficient and chemical inertness but generally lack sufficient electrical conductivity. To achieve conductive films with substantial thickness, the combination of direct current plasma assisted chemical vapour deposition (DC PACVD) with high coating temperatures has proven to be effective. Nitrogen doping of DLC films, a common method for improving their electrical conduction properties, typically leads to enhanced graphitization and a reduction in hardness and Young's modulus in harder DLC coatings. This study examines how nitrogen doping affects the mechanical and electrical properties of already unusually conductive, soft and thick (> 25 mu m) a-C:H films deposited at elevated temperatures using pulsed direct current PACVD. The a-C:H:N films were grown using C2H2 at 450 degrees C and 550 degrees C with an addition of 0-63 vol.-% N2 to the gas phase and studied subsequently. Nitrogen modification of the a-C:H was highly effective at enhancing mechanical properties in conjunction with electrical conductivity. Hardness and Young's modulus increased by up to 48 % and 95 %, respectively, compared to the undoped films. Relative load bearing capacity improved by up to a factor of 3.7. Specific electrical resistance decreased by more than two orders of magnitude for films deposited at 450 degrees C and by a factor of four for deposition at 550 degrees C, approximating and even surpassing the conductivity of graphite electrodes. Conversely, film thickness and deposition rate decreased significantly due to etching effects compared to the undoped a-C:H.

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (β 2 m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the α 3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α 3 domain of one FHC binds to another FHC in a manner similar to the β 2 m light chain. Significance Statement MHC class I molecules are cell surface transmembrane proteins with key functions in adaptive immunity against viral infections. The spatiotemporal organization of fully assembled MHC I at the cell surface and its function with respect to trans-interactions with T and NK cells has been studied in detail. By contrast, the consequences of peptide and β 2 m dissociation yielding to formation of free heavy chains (FHC) have remained unclear. We have discovered that class I free heavy chains form distinct non-covalent dimers at the cell surface rather than non-specific clustering, and we have identified a dimerization interface mediated by the α 3 domain. We propose that these non-covalent dimers are the basis of distinct signaling and endocytic sorting of MHC I FHC. This is to be explored in further work.

Depositing biomolecule micropatterns on solid substrates via microcontact printing (µCP) usually requires complex chemical substrate modifications to initially create reactive surface groups. Here, we present a simplified activation procedure for untreated solid substrates based on a commercial polymer metal ion coating (AnteoBindTM Biosensor reagent) that allows for direct µCP and the strong attachment of proteins via avidity binding. In proof-of-concept experiments, we identified the optimum working concentrations of the surface coating, characterized the specificity of protein binding and demonstrated the suitability of this approach by subcellular micropatterning experiments in living cells. Altogether, this method represents a significant enhancement and simplification of existing µCP procedures and further increases the accessibility of protein micropatterning for cell biological research questions.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

Bioprinting is a growing, multidisciplinary research area with huge potential. Applications range from printing small organ-tissue equivalents for basic research (e.g. investigation of 3D cell growth) to printing whole organs for implantation in the future. Anyhow, there are a lot of challenges to be solved. An increasing number of researchers, involving students and smaller research groups, are picking up this challenging topic, however, a crucial obstacle in the field is the high cost of a bioprinter. In this publication, we present a super low-cost bioprinter based on DVD-drive components. The printer can be built using tools and components easily accessible for engineers and students and with a budget of less than € 190. Further, our simple but effective system is easily modifiable for specific needs. For curing the bio-ink during printing and to be able to fabricate multilayer structures, a nebulizer unit was used as alternative to a more complex coaxial two-nozzle-system.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Controlling the spatial organization of ligands on cell surfaces is emerging as a powerful tool to assess the relevance of multivalent binding, receptor clustering and molecular cooperativity for cell signaling. A mechanistic understanding of such processes would without doubt accelerate rational approaches in modern pharmacology and cell-based therapies.Here, we have designed a biomimetic interface based on lipid bilayer-anchored DNA origami nanostructures as a tool to study the spatial requirements for receptor-mediated signaling in cell-cell contacts. Additionally, our biointerface is responsive to dynamic receptor rearrangements on the cell surface upon ligand engagement as they typically occur in immunity, host-pathogen interactions as well as neuro-, developmental and cancer biology. Importantly, it allows the experimenter to adjust protein distances with nanoscale precision. We apply our approach to identify the role of nanoscale ligand arrangements for productive T-cell antigen receptor (TCR) signaling. While micron-sized clusters of TCRs and other signaling molecules have long been described to be associated with T-cell activation, recent research has implicated nanoclusters of a few TCRs as signaling-competent units. In the current study we were able to measure the molecular architecture of these signaling-competent units by taking advantage of the distinctive properties of our biointerface. Here, DNA origami function as nanoscale pegboards to position ligands, but act also as spacers to isolate individual ligand units by limiting their approach within clusters. We determine the minimal unit for T-cell triggering as two ligand-engaged TCRs spaced apart. This finding has wide-ranging implications for our mechanistic understanding regarding T-cell antigen recognition and, as a consequence, for the design of T-cell-based immunotherapies.

The fabrication of two- and three-dimensional scaffolds mimicking the extracellular matrix and providing cell stimulation is of high importance in biology and material science. We show two new, biocompatible polymers, which can be 3D structuredviamultiphoton lithography, and determine their mechanical properties. Atomic force microscopy analysis of structures with sub-micron feature sizes reveals Young's modulus values in the 100 MPa range. Assessment of biocompatibility of the new resins was done by cultivating human umbilical vein endothelial cells on two-dimensionally structured substrates for four days. The cell density and presence of apoptotic cells has been quantified.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles—exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles—neither with native miRNA nor artificially loaded with it—did not alter the cellular miRNA level.

The great physiological relevance of glycolipids is being increasingly recognized, and glycolipid interactions have been shown to be central to cell-cell recognition, neuronal plasticity, protein-ligand recognition, and other important processes. However, detailed molecular-level understanding of these processes remains to be fully resolved. Molecular dynamics simulations could reveal the details of the glycolipid interactions, but the results may be influenced by the choice of the employed force field. Here, we have compared the behavior and properties of GM1, a common, biologically important glycolipid, using the CHARMM36, OPLS, GROMOS, and Amber99-GLYCAM06 (in bilayers comprising SLIPIDS and LIPID14 lipids) force fields in bilayers comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids and compared the results to atomic force microscopy and fluorescence resonance energy transfer experiments. We found discrepancies within the GM1 behavior displayed between the investigated force fields. Based on a direct comparison with complementary experimental results derived from fluorescence and AFM measurements, we recommend using the Amber99-GLYCAM force field in bilayers comprising LIPID14 or SLIPIDS lipids followed by CHARMM36 and OPLS force fields in simulations. The GROMOS force field is not recommended for reproducing the properties of the GM1 head group.

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.

The interaction of T cells with antigen presenting cells (APC) plays a central role in the adaptive immune system. However, the molecular mechanisms by which binding of the T cell receptor (TCR) to its ligand triggers a signal within the cell are still debated. Here, we use DNA origami decorated with TCR ligands anchored to a planar glass-supported lipid bilayer to assess the effects of local ligand density, arrangement and mobility on T cell activation. Our experimental setup allows for controlling the nanoscale arrangement of TCR ligands on the DNA origami scaffold as well as the re-organization of ligand and TCR during T cell activation. Further, the spatial distribution of ligands can be tuned independently of ligand concentration. TCRβ-reactive single-chain antibody fragment (scFv) and peptide-loaded major histocompatibility complex II (pMHC) were used as ligands and placed on the DNA origami at engineered capture sites in different layouts and densities. Functionalized DNA origami platforms were characterized using single molecule fluorescence microscopy and high-speed atomic force microscopy (HS-AFM). The activation of T cells interfaced with the APC-mimicking surfaces was determined by measuring intracellular calcium levels; the effects of local ligand density, nanoscale ligand arrangement, platform mobility as well as the nature of the ligand were assessed. Further, the rearrangement of TCR and ligand in the process of T cell activation was monitored by single molecule microscopy.

We show that multiphoton thiol-ene polymerized structures comprise unreacted thiol moieties, which can be used for postpolymerization gold metallization. Some of the thiol groups located at the surface are not involved in thiol-ene reactions and, therefore, can serve as nucleation seeds for the synthesis of similar to 50 nm sized gold nanoislands. Additionally, we show that the nanoislands can be used for immobilization of fluorescent molecules. We observed a significant enhancement of the fluorescence signal on the nanoisland-functionalized polymer structures when compared to the structured polymers without gold. To show a possible application, we bound peroxidase to the gold nanoislands. Peroxidase activity has been verified by cheiniluminescence of the converted luminol substrate.