CD3 aptamers promote expansion and persistence of tumor-reactive T cells for adoptive T cell therapy in cancer.

The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity.

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline.

Monomeric agonist peptide/MHCII complexes activate T-cells in an autonomous fashion.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling.

A DNA Origami-Based Biointerface to Interrogate the Spatial Requirements for Sensitized T-Cell Antigen Recognition.

When T cells scan the surface of antigen-presenting cells (APCs), they can detect the presence of just a few antigenic peptide/MHC complexes (pMHCs), in some cases even a single agonist pMHC. These are typically vastly outnumbered by structurally similar yet non-stimulatory endogenous pMHCs. How T cells achieve this enormous sensitivity and selectivity is still not clear, in particular in view of the rather moderate (1-100 μM) affinity that T-cell receptors (TCRs) typically exert for antigenic pMHCs.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions.

Strategies for the Site-Specific Decoration of DNA Origami Nanostructures with Functionally Intact Proteins.

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases.

DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigens.

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering.

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account.

A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein.

A complete understanding of signaling processes at the plasma membrane depends on a quantitative characterization of the interactions of the involved proteins. Fluorescence recovery after photobleaching (FRAP) is a widely used and convenient technique to obtain kinetic parameters on protein interactions in living cells. FRAP experiments to determine unbinding time constants for proteins at the plasma membrane, however, are often hampered by non-specific contributions to the fluorescence recovery signal.

A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns.

Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the feature size of patterns below 1 μm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium.

Determination of the Membrane Environment of CD59 in Living Cells.

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known.

Monomeric TCRs drive T cell antigen recognition.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements.

Protein Micropatterning Assay: Quantitative Analysis of Protein-Protein Interactions.

Characterization, especially quantification, of protein interactions in live cells is usually not an easy endeavor. Here, we describe a straightforward method to identify and quantify the interaction of a membrane protein ("bait") and a fluorescently labeled interaction partner ("prey") (membrane-bound or cytosolic) in live cells using Total Internal Reflection Fluorescence microscopy. The bait protein is immobilized within patterns in the plasma membrane (e.

Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles.

Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells.

With or without rafts? Alternative views on cell membranes.

The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment.

α-Synuclein induces both positive mean curvature and negative Gaussian curvature in membranes.

Using a combination of X-ray scattering, fluorescence correlation spectroscopy, coarse-grained molecular dynamics (MD) simulations and potential of mean force calculations, we have explored the membrane remodeling effects of monomeric α-synuclein (αS). Our initial findings from multiple approaches are that αS (1) causes a significant thinning of the bilayer and (2) stabilizes positive mean curvature, such that the maximum principle curvature matches that of synaptic vesicles, αS-induced tubules, and the synthetic lipid vesicles to which the protein binds most tightly. This suggests that αS binding to synaptic vesicles likely stabilizes their intrinsic curvature.

Allostery in a disordered protein: oxidative modifications to α-synuclein act distally to regulate membrane binding.

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson's disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes.

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin.

Recombinant human erythropoietin (rh-Epo) is well accepted as a hematopoietic drug, but many other pleiotropic properties are currently under investigation. Rh-Epo-induced receptor-mediated signal transductions are accompanied with membrane dynamic processes, which facilitate the activation of individual pathways. However, its direct effect on membrane dynamics is still unknown.

Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles.

At present, a number of transfection techniques are available to introduce foreign DNA into cells, but still minimal intrusion or interference with normal cell physiology, low toxicity, reproducibility, cost efficiency and successful creation of stable transfectants are highly desirable properties for improved transfection techniques.For all previous transfection experiments done in our labs, using serum-free cultivated host cell lines, an efficiency value of approximately 0.1% for selection of stable cell lines has not been exceeded, consequently we developed and improved a transfection system based on defined liposomes, so-called large unilamellar vesicles, consisting of different lipid compositions to facilitate clone selection and increase the probability for creation of recombinant high-production clones.

Oxidized phospholipids in minimally modified low density lipoprotein induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells.

Oxidized phospholipids, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), typically present in minimally modified low density lipoprotein, have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses (inflammation, proliferation, or cell death). It was the aim of this study to understand their impact on intracellular signal transduction pathways that are responsible for these biological effects.