Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier.

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

Coronaviruses Use ACE2 Monomers as Entry-Receptors.

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors µm-2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

Site-specific protein labeling strategies for super-resolution microscopy.

Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions.

Optimized genetic code expansion technology for time-dependent induction of adhesion GPCR-ligand engagement.

The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair enables site-specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co-labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein-coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5-CD55 ligation and replenishment of mature receptor-ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand-dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions.

Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection.

Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus-cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus-cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus-host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.