The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

Balancing the Nanoscale Organization in Multivalent Materials for Functional Inhibition of the Programmed Death-1 Immune Checkpoint.

Dendritic cells (DCs) regulate immune priming by expressing programmed death ligand 1 (PD-L1) and PD-L2, which interact with the inhibitory receptor PD-1 on activated T cells. PD-1 signaling regulates T cell effector functions and limits autoimmunity. Tumor cells can hijack this pathway by overexpressing PD-L1 to suppress antitumor T cell responses. Blocking this inhibitory pathway has been beneficial for the treatment of various cancer types, although only a subset of patients responds. A deepened understanding of the spatial organization and molecular interplay between PD-1 and its ligands may inform the design of more efficacious nanotherapeutics. We visualized the natural molecular PD-L1 organization on DCs by DNA-PAINT microscopy and created a template to engineer DNA-based nanoclusters presenting PD-1 at defined valencies, distances, and patterns. These multivalent nanomaterials were examined for their cellular binding and blocking ability. Our data show that PD-1 nano-organization has profound effects on ligand interaction and that the valency of PD-1 molecules modulates the effectiveness in restoring T cell function. This work highlights the power of spatially controlled functional materials to unravel the importance of multivalent patterns in the PD-1 pathway and presents alternative design strategies for immune-engineering.

Optimized Coiled-Coil Interactions for Multiplexed Peptide-PAINT.

Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions.

Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.

Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation.

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy.

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.

Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane.

Signal transduction by receptor tyrosine kinases (RTKs) involves complex ligand- and time-dependent changes in conformation and modification state. High resolution structures are available for individual receptors dimers, but less is known about receptor clusters that form in plasma membranes composed of many different RTKs with the potential to interact. We report the use of multiplexed super-resolution imaging (Exchange-PAINT) followed by mean-shift clustering and random forest analysis to measure the precise distributions of five receptor tyrosine kinases (RTKs) from the ErbB, IGF-1R and Met families in breast cancer cells. We find that these receptors are intermixed nonhomogenously on the plasma membrane. Stimulation by EGF does not appear to induce a change in the density of EGFR in local clusters but instead results in formation of EGFR-Met and EGFR-ErbB3 associations; non-canonical EGFR-Met interactions are implicated in resistance to anti-cancer drugs but have not been previously detected in drug-naïve cells.

Biophysical characterization of copolymer-protected gene vectors.

A copolymer-protected gene vector (COPROG) is a three-component gene delivery system consisting of a preformed DNA and branched polyethylenimine (bPEI) complex subsequently modified by the addition of a copolymer (P6YE5C) incorporating both poly(ethylene glycol) (PEG) and anionic peptides. Using fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM), we characterized and compared the self-assembly of bPEI/DNA particles and COPROG complexes. In low salt buffer, both bPEI/DNA and COPROG formulations form stable nanoparticles with hydrodynamic radii between 60-120 nm. COPROG particles, as compared to bPEI/DNA, show greatly improved particle stability to both physiological salt as well as low pH conditions. Binding stoichiometry of the three-component COPROG system was investigated by dual-color fluorescence cross-correlation spectroscopy (FCCS). It was found that a significant fraction of P6YE5C copolymer aggregates with excess bPEI forming bPEI/P6YE5C "ghost complexes" with no DNA inside. The ratio of ghost particles to COPROG complexes is about 4:1. In addition, we find a large fraction of excess P6YE5C copolymer, which remains unbound in solution. We observe a 2-4-fold enhanced reporter gene expression with COPROG formulations at various equivalents as compared to bPEI-DNA alone. We believe that both complex stabilization as well as the capture of excess bPEI into ghost particles induced by the copolymer is responsible for the improvement in gene expression.