Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells.

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

Quantitative Single-Molecule Localization Microscopy (qSMLM) of Membrane Proteins Based on Kinetic Analysis of Fluorophore Blinking Cycles.

Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of "blinking" that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of "blinking" allows determining the number of fluorophores in a multi-molecular complex. As such, quantitative information at the molecular level can be extracted, representing a tremendously useful extension of single-molecule super-resolution microscopy. This concept is in particular useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution. Here, we provide an experimental framework for deciphering the stoichiometry of membrane proteins on the basis of SMLM and photoswitching statistics.

Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation.

Ubiquitination of invading Salmonella Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors such as p62/SQSTM1, NDP52/CALCOCO and optineurin (OPTN), which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. Besides this, the functional consequences and mechanistic regulation of differentially linked Ub chains at the host-Salmonella interface have remained unexplored. Here, we show, for the first time, that distinct Ub chains on cytosolic S. Typhimurium serve as a platform triggering further signalling cascades. By using single-molecule localization microscopy, we visualized the balance and nanoscale distribution pattern of linear (M1-linked) Ub chain formation at the surface of cytosolic S. Typhimurium. In addition, we identified the deubiquitinase OTULIN as central regulator of these M1-linked Ub chains on the bacterial coat. OTULIN depletion leads to enhanced formation of linear Ub chains, resulting in local recruitment of NEMO, activation of IKKα/IKKß and ultimately NF-κB, which in turn promotes secretion of pro-inflammatory cytokines and restricts bacterial proliferation. Our results establish a role for the linear Ub coat around cytosolic S. Typhimurium as the local NF-κB signalling platform and provide insights into the function of OTULIN in NF-κB activation during bacterial pathogenesis.

Molecule Counts in Localization Microscopy with Organic Fluorophores.

Single-molecule localization microscopy (SMLM) can be used to count fluorescently labeled molecules even when they are not individually resolved. We demonstrate SMLM molecule counting for nucleic acids labeled with the organic fluorophore Alexa Fluor 647 and imaged under photoswitching conditions. From the observed distributions of the number of fluorophore blinking events, we extract the number of fluorophores per spot using a statistical model. We validate the molecule counting method for single Alexa Fluor 647 fluorophores, and for trimers of Alexa Fluor 647 constructed on a DNA origami structure. This simple counting strategy enables quantitative super-resolution imaging with organic fluorophores.

Model-independent counting of molecules in single-molecule localization microscopy.

Most biomolecular processes rely on tightly controlled stoichiometries, from the formation of molecular assemblies to cellular signaling. Single-molecule localization micro-scopy studies of fluorophore blinking offer a promising route to probe oligomeric states. Here we show that the distribution of the number of blinking events assumes a universal functional form, independent of photophysics, under relatively mild assumptions. The number of photophysical states, the kinetics of interconversion, and the fraction of active fluorophores enter as two or three constants. This essentially model-independent formulation allows us to determine molecule counts from fluorophore blinking statistics. The formulas hold even if the fluorophores have many different yet unresolved dark states, as long as there is only a single fluorescent state, or if there are different yet unresolvable fluorescent states, as long as there is only a single dark state. We demonstrate the practical applicability of this approach by quantifying the oligomerization states of membrane proteins tagged with the mEos2 fluorescent protein. We find that the model parameters, obtained by likelihood maximization, are transferable. With the counting statistics being independent of the detailed photophysics and its parameters being transferable, the method should be robust and broadly applicable to counting colocalized molecules in vivo and in vitro.

One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.

Probing the oligomeric state of abundant molecules, such as membrane proteins in intact cells, is essential, but has not been straightforward. We address this challenge with a simple counting strategy that is capable of reporting the oligomeric state of dense, membrane-bound protein complexes. It is based on single-molecule localization microscopy to super-resolve protein structures in intact cells and basic quantitative evaluation. We validate our method with membrane-bound monomeric CD86 and dimeric cytotoxic T-lymphocyte-associated protein as model proteins and confirm their oligomeric states. We further detect oligomerization of CD80 and vesicular stomatitis virus glycoprotein and propose coexistence of monomers and dimers for CD80 and trimeric assembly of the viral protein at the cell membrane. This approach should prove valuable for researchers striving for reliable molecular counting in cells.

Single-molecule methods to study membrane receptor oligomerization.

Membrane receptors control fundamental cellular processes. Binding of a specific ligand to a receptor initiates communication through the membrane and activation of signaling cascades. This activation process often leads to a spatial rearrangement of receptors in the membrane at the molecular level. Single-molecule techniques contributed significantly to the understanding of receptor organization and rearrangement in membranes. Here, we review four prominent single-molecule techniques that have been applied to membrane receptors, namely, stepwise photobleaching, Förster resonance energy transfer, sub-diffraction localization microscopy and co-tracking. We discuss the requirements, benefits and limitations of each technique, discuss target labeling, present a selection of applications and results and compare the different methodologies.

Receptor-ligand interactions: binding affinities studied by single-molecule and super-resolution microscopy on intact cells.

Protein­ligand interactions play an important role in many biological processes. Notably, membrane receptors are the starting point for a huge variety of cellular signal transduction pathways. Quantifying the binding affinity of a ligand for its transmembrane receptor is of great importance as it provides information on the potency of the ligand. We developed a new experimental procedure to determine binding affinities of ligands for their membrane receptors directly on intact single cells using super-resolution imaging. Dissociation constants were determined by titrating fluorophore-labelled ligand against cells expressing the target protein and applying single-molecule imaging.

A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment.

The localization precision is a crucial and important parameter for single-molecule localization microscopy (SMLM) and directly influences the achievable spatial resolution. It primarily depends on experimental imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to estimate the average experimental localization precision in SMLM, based on the nearest neighbor analysis. By exploring different experimental and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σ SMLM are obtained. Knowing σ SMLM is a prerequisite for consistent visualization or any quantitative structural analysis, e.g., cluster analysis or colocalization studies.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers.

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.